WO2015164800A2

WO2015164800A2 - Compositions and methods for modulating polarization of monocytes

Info

Publication number: WO2015164800A2
Application number: PCT/US2015/027614
Authority: WO
Inventors: Dinender Singla
Original assignee: University of Central Florida Research Foundation Inc
Current assignee: University of Central Florida Research Foundation Inc
Priority date: 2014-04-24
Filing date: 2015-04-24
Publication date: 2015-10-29
Anticipated expiration: 2016-10-24
Also published as: WO2015164800A3

Abstract

In an aspect, the present dislcosure relates to compositions comprising FGF-9 and methods of using disclosed compositions. In an aspect, the present dislcosure relates to compositions comprising one or more of FGF-9, FGF-8, and BMP-7 and methods of using disclosed compositions. In an aspect, the present dislcosure relates to compositions for and methods of modulating polarization of monocytes. This abstract is intended as a scanning tool for purposes of searching in the particular art and is not intended to be limiting of the present present dislcosure.

Description

COMPOSITIONS AND METHODS FOR MODULATING

POLARIZATION OF MONOCYTES

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0001] This invention was made with government support under ROl HL090646 and HL117241 and R01HL094467 awarded by the National Institutes of Health. The government has certain rights in the invention.

BACKGROUND OF THE INVENTION

[0002] Diabetes development, progression, and pathogenesis are complex and multifactorial involving abnormal hyperglycemia, oxidative stress, and inflammation (Glass et al, 2010; King et al, 2004; Lee et al, 2010; and Trinanes et al., 2012). Myocardial infarction (MI) in the diabetic subject leads to heart failure. It is also a non-resolving inflammatory process as monocytes continue to enter in the infarcted heart leading to heart failure at all stages of the disease progression. Inflammation is associated with the infiltration of monocytes into organs such as the injured heart, and is also associated with the release of pro-inflammatory cytokines such as TNF-alpha, IL-6, and MCP-1 in animal studies (King et al, 2004; Lee et al, 2010; Trans et al, 2012; Anzai et al, 2012; Jain et al., 2007; Jain et al, 2009; and Yan et al, 2013). Infarcted hearts have monocyte/macrophage recruitment and the number of Ml macrophages is higher than the number of M2 macrophages. It is thought that macrophage polarization is present in cardiac remodeling and/or in the development and progression of heart failure (Anzai et al., 2012; Yan et al, 2013; Leor et al, 2006). Following infiltration into cardiac injury (such as the development of diabetic cardiomyopathy and heart failure), monocytes can differentiate into two major classical (Ml) or alternative (M2) macrophages.

[0003] However, despite advances in the understanding of the pathology of myocardial infarction in the diabetic heart, there is still a need for compositions and methods that efficiently prevent or ameliorate damage to the heart following MI.

BRIEF SUMMARY OF THE INVENTION

[0004] Disclosed herein is method of modulating polarization of a population of monocytes in a subject, comprising administering to the subject a composition comprising an effective amount of one or more of FGF-9, FGF-8, BMP-7, M-CSF or a combination thereof, thereby modulating the polarization of a population of monocytes.

[0005] Disclosed herein is a method of modulating polarization of a population of monocytes in a subject having a skin disease or skin disorder, comprising administering to the subject a composition comprising an effective amount of FGF-9, FGF-8, BMP-7, M-CSF or a combination thereof, thereby modulating the polarization of a population of monocytes.

[0006] Disclosed herein is a method of ameliorating cellular injury in a subject, comprising administering to the subject a composition comprising an effective amount of FGF-9, FGF-8, BMP-7, M-CSF or a combination thereof, thereby modulating the polarization of infiltrating monocytes in the subject; and reducing the severity of the cellular injury in the subject.

[0007] Disclosed herein is a method of ameliorating cellular injury in a subject having a skin disease or skin disorder, comprising administering to the subject a composition comprising an effective amount of FGF-9, FGF-8, BMP-7, M-CSF or a combination thereof, thereby modulating the polarization of infiltrating monocytes in the subject; and reducing the severity of the skin disease or skin disorder in the subject.

[0008] Disclosed herein is a method of modulating polarization of a population of monocytes, comprising administering a composition comprising an effective amount of FGF- 9, FGF-8, BMP-7, M-CSF or a combination thereof, to a population of monocytes; and modulating the polarization of the population of monocytes.

[0009] Disclosed herein is a method of modulating cellular phenotype of macrophages, comprising administering a composition comprising an effective amount of FGF-9, FGF-8, BMP-7, M-CSF or a combination thereof, to a population of macrophages; and modulating the cellular phenotype of the population of macrophages such that the macrophages exhibit characteristics of M2 macrophages.

[0010] Disclosed herein is a method of ameliorating cellular injury in a subject in need thereof, comprising administering a composition comprising an effective amount of FGF-9, FGF-8, BMP-7, M-CSF or a combination thereof, to the subject; modulating the polarization of infiltrating monocytes in the subject; and increasing the expression of anti-inflammatory cytokines in the subject.

[001 1] Disclosed herein is a method of attenuating fibrosis in a subject in need thereof, comprising administering a composition comprising an effective amount of FGF-9, FGF-8, BMP-7, M-CSF or a combination thereof, to the subject; modulating the polarization of infiltrating monocytes in the subject; and increasing the expression or activity of one or more anti-inflammatory cytokines in the subject. [0012] Disclosed herein is a method of modulating FGF receptor expression in monocytes, comprising administering a composition comprising an effective amount of FGF-9, FGF-8, BMP-7, M-CSF or a combination thereof, to a population of monocytes; and increasing the expression of a FGF receptor in the monocytes.

[0013] Disclosed herein are compositions comprising FGF-9, FGF-8, BMP-7, M-CSF or a combination thereof. A composition may comprise FGF-9. A composition may comprise FGF-8. A composition may comprise BMP-7. A composition may comprise M-CSF. A composition may comprise one or more of FGF-9, FGF-8, BMP-7, M-CSF or a combination thereof.

[0014] Disclosed herein is a pharmaceutical composition, comprising (i) FGF-9; (ii) M-CSF, an agent that modulates the polarization of monocytes, or a combination thereof; and (iii) a pharmaceutically acceptable carrier.

[0015] Disclosed herein is a pharmaceutical composition, comprising FGF-9, FGF-8, BMP-

7, or a combination thereof; and a pharmaceutically acceptable carrier.

[0016] Disclosed herein is a pharmaceutical composition, comprising (i) FGF-9; (ii) FGF-8,

BMP-7, or a combination thereof; and (iii) a pharmaceutically acceptable carrier.

[0017] Disclosed herein is a pharmaceutical composition comprising FGF-9, FGF-8, BMP-7,

M-CSF or a combination thereof, and a pharmaceutically acceptable carrier.

BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The accompanying figures, which are incorporated in and constitute a part of this specification, illustrate several aspects and together with the description serve to explain the principles of the disclosure.

[0019] FIG. 1A-FIG. ID are graphs (1A and 1C) and photomicrographs (IB and 1 D) showing that expression of p-FGFRl increased following treatment with FGF-9. FIG. 1A shows percentage of CD14 (monocytes) cells expressing p-FGFRl . FIG. IB shows representative photomicrographs of p-FGFRl (a, e, i, m), CD14 (b, f, j, n), DAPI (c, g, k, o), and merged images (d, h, 1, p). FIG. 1C shows percentage of Arginase-1 (M2 macrophages) cells expressing p-FGFRl. FIG. ID shows representative photomicrographs of p-FGFRl (a, e, i, m), Arginase-1 (b, f, j, n), DAPI (c, g, k, o), and merged images (d, h, 1, p). Boxes indicate enlarged merged images. (*p < 0.05 vs. Control, #p < 0.05 vs. ACM, $ p < 0.05 vs. ACM + F-9).

[0020] FIG. 2A-FIG. 2C are graphs (2A and 2B) and photomicrographs (2C) showing the percentage of monocytes following treatment of HM and THP-1 cells with FGF-2 and FGF- 9. FIG. 2A shows CD 14 (monocytes) positive cells from HM. FIG. 2B shows CD 14 positive cell from THP-1 cells. FIG. 2C shows representative photomicrographs of CD 14 (a, d, g, j), DAPI (b, e, h, k), and merged images (c, f, I, 1). Boxes indicate enlarged merged images. (*p < 0.05 vs. Control).

[0021] FIG. 3A-3C are graphs (3A and 3B) and photomicrographs (3C) showing the percentage of Ml macrophages following treatment of HM and THP-1 cells with FGF-2 and FGF-9. FIG. 3A shows percentage of iNOS (Ml macrophages) from HM. FIG. 3B shows percentage of iNOS positive cells from THP-1 cells. FIG. 3C shows representative photomicrographs of iNOS (a, d, g, j), DAPI (b, e, h, k), and merged images (c, f, i, 1). Boxes indicate enlarged merged images. (*p < 0.05 vs. Control, #p < 0.05 vs. FGF-9).

[0022] FIG. 4A-FIG. 4E are graphs (4A, 4C, 4D and 4E) and photomicrographs (4B) showing the percentage of M2 macrophages following treatment of THP- 1 cells and HM with FGF-2 and FGF-9. FIG. 4A shows CD206 positive cells from THP-1 cells. FIG. 4C shows arginase 1 (M2 macrophages) positive cells from THP-1 cells. FIG. 4B shows representative photomicrographs of CD206 (a, d, g, j), DAPI (b, e, h, k), and merged images (c, f, I, 1). Boxes indicate enlarged merged images. FIG. 4D (CD206+) and FIG. 4E (arginase 1 positive) show M2 macrophages from primary HM. (*p < 0.05 vs. Control, ACM and FGF- 2)·

[0023] FIG. 5A-FIG. 5E are graphs showing an ELISA analysis of TNF-alpha, MCP-1, and IL-6, which are cytokines expressed by Ml macrophages, and an ELISA analysis of IL-10 and IL-1 receptor antagonist (IL-lra), which are cytokines expressed by M2 macrophages, using THP-1 cells. *p < 0.05 vs. Control, #p < 0.05 vs. ACM).

[0024] FIG. 6A-FIG. 6D are graphs showing an ELISA analysis of TNF-alpha, MCP-1, and IL-6, which are cytokines expressed by Ml macrophages, and ELISA analysis of IL-10 and IL-1 receptor antagonist (IL-lra), which are cytokines expressed by M2 macrophages, using HM cells. (*p < 0.05 vs. Control, #p < 0.05 vs. ACM + FGF-9).

[0025] FIG. 7A-FIG. 7B are a graph (7B) and photomicrographs (7 A) showing that FGF-9 treatment following an MI decreased infarct size. FIG. 7A displays sections of the hearts per group and FIG. 7B shows quantitative analysis of the infarct size. (*p < 0.05 vs. Sham, #p < 0.05 vs. MI).

[0026] FIG. 8A-FIG. 8B are a graph (8B) and photomicrographs (8A) showing that treatment with FGF-9 decreased interstitial fibrosis following MI. FIG. 8A shows representative photomicrographs of the interstitial fibrosis per group. FIG. 8B shows quantitative analysis of the interstitial fibrosis. (*p < 0.05 vs. Sham; #p < 0.05 vs. MI). [0027] FIG. 9A-FIG. 9B are a graph (9B) and photomicrographs (9A) showing representative photomicrographs of CD 14 immunostaining (a, f, k), sarcomeric a-actin (b, g, 1), DAPI (c, h, m), merged images (d, i, n), and enlarged merged images (e, j, o). FIG. 9B shows CD 14 (monocytes) positive cells. (*p < 0.05 vs. Control, #p < 0.05 vs. ACM).

[0028] FIG. 1 OA-FIG. 10B are a graph (10B) and photomicrographs (10A) showing representative photomicrographs of iNOS immunostaining (a, f, k), sarcomeric a-actin (b, g, 1), DAPI (c, h, m), merged images (d, i, n), and enlarged merged images (e, j, o) (FIG. 10A). FIG. 10B shows iNOS (Ml macrophages) positive cells. (*p < 0.05 vs. Control, #p < 0.05 vs. ACM).

[0029] FIG. 11A-FIG. 11B are a graph (11B) and photomicrographs (11A) showing representative photomicrographs are shown of CD206 (a, f, k), sarcomeric a-actin (b, g, 1), DAPI (c, h, m), merged images (d, I, n), and enlarged merged images (e, j, o) (FIG. 11A). FIG. 1 IB shows CD206 (M2 macrophages) positive cells. (*p < 0.05 vs. Control, #p < 0.05 vs. ACM).

[0030] FIG. 12A-FIG. 12D are graphs 1(2B and 12D) and photomicrographs (12A and 12C) showing CD 14 and CD206 staining and quantification. FIG. 12A shows representative photomicrographs of CD 14 (monocytes) (a, f), p-FGFRl (b, g), sarcomeric a-actin (c, h), DAPI (d, i), and merged images (e, j). FIG. 12B shows CD 14 positive cells expressing p- FGFR1. FIG. 12C shows representative photomicrographs of CD206 (M2 macrophages) (a, f), p-FGFRl (b, g), sarcomeric a-actin (c, h), DAPI (d, i), and merged images (e, j). FIG. 12D shows CD206 positive cells expressing p-FGFRl. (*p < 0.05 vs. Control, #p < 0.05 vs. ACM).

[0031] FIG. 13A-FIG. 13E are graphs showing that treatment with FGF-9 decreased proinflammatory cytokines while increasing anti-inflammatory cytokine expression. Serum was collected from each group and prepared for ELISA analysis of TNF-alpha (FIG. 13 A), MCP- 1 (FIG. 13B), and IL-6 (FIG. 13C), which are three cytokines expressed by Ml macrophages, and IL-10 (FIG. 13D) and IL-1 receptor antagonist (IL-lra) (FIG. 13E), which are two cytokines expressed by M2 macrophages. Histograms show quantitative analysis. (*p < 0.05 vs. Control, #p < 0.05 vs. MI).

[0032] FIG. 14A-FIG. 14C shows Western blot analysis of p-PI3K, p-YAP, and Ras following treatment with FGF-9 after an MI. Top panel shows representative bands for Ras and β-actin (FIG. 14A), for p-PI3K and PI3K (FIG. 14B), and for p-YAP and YAP (FIG. 14C). Histogram shows densitometric analysis of RAS (FIG. 14A), p-PI3K (FIG. 14B), and p-YAP (FIG. 14C). (*p < 0.05 vs. Sham, #p < 0.05 vs. MI). [0033] FIG. 15 is a graph showing that cardiac function improved following treatment with FGF-9. Histogram shows quantitative analysis of percentage of fractional shortening (FS). (*p < 0.05 vs. Sham, #p < 0.05 vs. MI).

[0034] FIG. 16A-FIG. 16E are graphs (16B, 16D and 16E) and photomicrographs (16 A and 16C) showing that BMP-7 ameliorated inflammation by regulating cytokine expression. FIG. 16A shows representative photomicrographs of tissue sections stained for CD 14, a marker for monocytes (a, f, k), sarcomeric a-actin (b, g, 1), DAPI (c, h, m), and merged images (d, i, n). White box indicates enlarged merged images (e, j, o). (Scale bar = 100 μιη, n = 4-5 per group). FIG. 16B shows quantitative analysis of percent monocytes. FIG. 16C shows representative photomicrographs of tissue sections stained for TNF-a, a pro-inflammatory cytokine, (a, f, k), sarcomeric a-actin (b, g, 1), DAPI (c, h, m), and merged images (d, i, n). Box indicates enlarged merged images (e, j, o). (Scale bar = 100 μιη, n = 4-5 per group). FIG. 16D shows quantitative analysis of the TNF-a immunostaining. FIG. 16E shows quantitative analysis of the TNF-a ELISA performed on serum samples (n = 5-7 samples per group; A.U. = arbitrary units; *p < 0.05 vs. control and #p < 0.05 vs. PD).

[0035] FIG. 17A-FIG. 17C are graphs (17B and 17C) and photomicrographs (17A) showing that BMP-7 inhibited pro-inflammatory cytokine, IL-6, in PD hearts. FIG. 17A shows representative photomicrographs of tissue sections stained for IL-6 (a, f, k), sarcomeric a- actin (b, g, 1), total DAPI (c, h, m), and merged images (d, i, n). White boxes indicate enlarged merged images (e, j, o). (Scale bar = 100 μιη. n = 7-8 per group). FIG. 17B shows quantitative analysis of the IL-6 immunostaining. FIG. 17C shows quantitative analysis of the IL-6 ELISA performed on serum samples, (n = 4-6 per group; A.U. = arbitrary units; *p < 0.05 vs. control and #p < 0.05 vs. PD).

[0036] FIG. 18A-FIG. 18C are graphs (18B and 18C) and photomicrographs (18A) showing that BMP-7 stimulated MnSOD expression and mitigated oxidative stress in PD hearts. FIG. 18A shows representative photomicrographs of heart sections stained with DHE (a, d, g), DAPI (b, e, h), and merged images (c, f, i). (Scale bar = 100 μιη; n = 6 animals). FIG. 18B shows quantitative analysis of DHE immunostaining. FIG. 18C shows representative bands and quantitative western blot analysis of MnSOD expression, (n = 5 animals per group; A.U. = arbitrary units; *p < 0.05 vs. control and #p < 0.05 vs. PD).

[0037] FIG. 19A-FIG. 19E are a graphs (19B), photomicrographs (19A and 19C) and Western blot analysis (19 D and 19E) showing that BMP-7 attenuated apoptosis in the PD heart by regulating PTEN and AKT. FIG. 19A shows representative photomicrographs of heart sections demonstrating TUNEL positive cells (apoptotic nuclei) (a, d, g), DAPI (b, e, h), and merged images (c, f, i). (Scale bar = 100 μιη; n = 5-6 samples per group). FIG. 19B shows quantitative analysis of percentage apoptotic nuclei. FIG. 19C shows representative photomicrographs illustrating apoptosis occurs in cardiomyocytes with TU EL positive cells (a), sarcomeric a-actin (b), caspase-3 (c), DAPI (d), merged image (e), and enlarged merged image (f). FIG. 19D shows representative bands and quantitative analysis of p-PTEN western blot, (n = 5-6 samples per group). FIG. 19E shows representative bands and quantitative western blot analysis of p-AKT expression, (n = 5-6 samples per group; A.U. = arbitrary units; *p < 0.05 vs. control and #p < 0.05 vs. PD).

[0038] FIG. 20A-FIG. 20C are graphs (20B and 20C) and photomicrographs (20A) showing that BMP-7 blunted cardiac fibrosis in the pre-diabetic heart. FIG. 20A shows representative photomicrographs of Masson's Trichrome staining performed on heart sections to assess interstitial (a, b, c) and vascular (d, e, f) fibrosis, (n = 7-9 samples per group). FIG. 20B shows quantitative analysis of interstitial fibrosis. FIG. 20C shows quantitative analysis of vascular fibrosis. (VF/VA = vascular fibrosis/vascular area; *p < 0.05 vs. control and #p < 0.05 vs. PD).

[0039] FIG. 21A-FIG. 2 IF are graphs (2 IB, 21 C, 2 IE and 2 IF) and photomicrographs (21 A and 2 ID) showing that BMP-7 stimulates increased M2 polarization and anti-inflammatory cytokine secretion. FIG. 21 A shows representative photomicrographs of heart tissues stained for CD206, a marker for M2 macrophages (a, f, k), sarcomeric a-actin (b, g, 1), DAPI (c, h, m), and merged images (d, i, n). White boxes indicate enlarged merged images (e, j, o). (Scale bar = 100 μιη; n = 4 animals per group). FIG. 20B shows quantitative analysis of percent M2 macrophages. FIG. 20C shows quantitative analysis of the IL-IRA ELISA performed on serum samples, (n = 5-7 samples). FIG. 20D shows representative photomicrographs of heart tissues stained for IL-10, an anti-inflammatory cytokine (a, f, k), sarcomeric a-actin (b, g, 1), DAPI (c, h, m), and merged images (d, i, n). White boxes indicate enlarged merged images (e, j, o). (Scale bar = 100 μιη; n = 5 samples per group). FIG. 2 IE shows quantitative analysis of IL-10 expression per group. FIG. 2 IF shows quantitative analysis of the IL-10 ELISA performed on serum samples, (n = 4-5 samples; A.U. = arbitrary units; *p < 0.05 vs. control and #p < 0.05 vs. PD).

[0040] FIG. 22A-FIG. 22G are graphs (22A -22BG and a chart (22A) showing that BMP-7 improves overall cardiac function in the PD heart. Cardiac function was measured in all mice through transthoracic echocardiography (n = 6-9 animals). FIG. 22A is a table that provides the mean + SEM values for LVIDd, LVIDs, EDV, and ESV. FIG. 22B-FIG. 22G show representative histograms demonstrating quantitative analysis of LVIDd (FIG. 22B), LVIDs (FIG. 22C), FS% (FIG. 22D), EDV (FIG. 22E), ESV (FIG. 22F), and EF% (FIG. 22G) for control and experimental groups. (*p < 0.05 vs. control and #p < 0.05 vs. PD).

[0041] Figures 23A and 23Bare a graph (23B) and photomicrographs (23 A) that BMP-7 inhibits plaque formation following PLCA ligation. A: Representative photomicrographs of Masson's trichrome stained arterial sections from all control and experimental groups. B: Histogram of quantitative data indicates plaque formation is inhibited following BMP-7 treatment. *p<0.05 vs. sham and #p<0.05 vs. PLCA.

[0042] Figures 24A - 24D are graphs (24B and 24D) and photomicrographs (24A and 24 C) that BMP-7 inhibits monocyte infiltration in ATH Apo E-/- mouse model. 24A: Representative photomicrographs of carotid artery sections depicting CD14+ve cells, a marker for monocytes, (a, e, and i), DAPI, for nuclei, (b, f, and j), merged images (c, g, and k), and enhanced merged images (d, h, and 1). 24B shows quantitative data indicating monocytic infiltration is significantly diminished following BMP-7 treatment. Scale bars in k and 1 = 100 μιη and 25 μιη, respectively. 24C: Representative photomicrographs of carotid artery sections depicting iNOS+ve cells, a marker for Ml macrophages, (a, e, and i), DAPI, for nuclei, (b, f, and j), merged images (c, g, and k), and enhanced merged images (d, h, and 1). 24D shows quantitative analysis indicating Ml macrophage differentiation is unremarkable between PLCA and PLCA+BMP-7 groups. Scale bars in g and h = 100 μιη and 25 μιη, respectively. *p<0.05 vs. sham and #p<0.05 vs. PLCA.

[0043] Figures 25A, 25B, 25C, and 25D are graphs showing pro-inflammatory cytokine secretion is significantly diminished following BMP-7 treatment. A: Quantitative analysis of secreted circulatory MCP-1 expression via ELISA B: Circulating TNF-a is significantly down regulated post-PLCA ligation with BMP-7 treatment. C: Augmented TNF-a expression in PLCA and PLCA+BMP-7 groups is confirmed by RT-PCR. D: Transcribed IL-6 is significantly enhanced in the PLCA group whereas IL-6 mRNA is significantly diminished following BMP-7 treatment. A. U = arbitrary units. *p<0.05 vs. sham and #p<0.05 vs. PLCA.

[0044] Figure 26A and 26B are photomicrographs (26A) and a graph (26B) showing post- PLCA ligation, BMP-7 enhances M2 macrophage differentiation. (26A) Representative images of LCA sections shown in a-1 depict CD206 positive M2 macrophages (a, e, and i), total nuclei (b, f, and j), merged images (c, g, and k), and enhanced merged images (d, h, and 1). Scale bars in k and 1 = 100 μιη and 25 μιη, respectively. Data analysis indicates BMP-7 treatment significantly enhances M2 macrophage differentiation (26B). [0045] Figures 27A, 27B, 27C, and 27D are graphs showing that BMP-7 enhances circulating anti-inflammatory cytokine secretion. A: Quantitative analysis of secreted circulatory IL-IRA expression via ELISA B: Circulating IL-10 is significantly upregulated post-PLCA ligation with BMP-7 treatment. C: Histogram represents immunohistochemistry analysis of Arginase 1 in LCA sections. D: Transcribed Arginase 1 is significantly enhanced in the PLCA+BMP-7 group. A. U = arbitrary units. *p<0.05 vs. sham and #p<0.05 vs. PLCA.

[0046] Figure 28 is a graph showing BMP-7 serum levels significantly diminished post- PLCA ligation. Following PLCA ligation, circulating BMP-7 expression was significantly decreased whereas exogenous treatment with BMP-7 significantly enhanced blood serum BMP-7 levels. A. U = arbitrary units. *p<0.05 vs. sham and #p<0.05 vs. PLCA.

[0047] Figures 29A - 29D are graphs (29B and 29 D) and photomicrographs (29A and 29C) showing that BMP-7 enhances BMP-7 receptor expression on monocytes and M2 macrophages. A: Representative images of LCAs stained with anti-BMP-7R shown (a, b, c, and d), anti-CD 14 (monocytes) shown (e, f, g, and h), DAPI shown (i, j, k, and 1) and merged images (m, n, o and p) are depicted in Figure 7A. Scale bars in n and o = 100 μιη and 25 μιη, respectively. Data analysis indicates BMP-7 treatment significantly enhances BMP-7R on monocytes (left histogram). B: Representative photomicrographs depict LCAs stained with anti-BMP-7R shown (a, b, c, and d), anti-CD206 (M2 macrophages) shown (e, f, g, and h), DAPI shown (i, j, k, and 1) and merged images (m, n, o and p). Scale bars in n and o = 100 μιη and 25 μιη, respectively. Data analysis indicates BMP-7 treatment also significantly enhances BMP-7R on M2 macrophages (left histogram). *p<0.05 vs. sham and #p<0.05 vs. PLCA.

[0048] Figure 30 is a graph showing that BMP-7 treatment enhances blood flow post-PLCA surgery. Blood flow, measured as systolic velocity, was quantitated two weeks post sham or PLCA ligation. Systolic velocity was significantly decreased post-PLCA ligation but was significantly enhanced when additionally treated with BMP-7. *p<0.05 vs. sham and #p<0.05 vs. PLCA.

[0049] Figures 31 A, 3 IB, and 31C are graphs showing that bone morphogenetic protein (BMP)-7 attenuates prediabetic (PD) hyperglycemia. A and B: representative blood glucose data at 7 days (A) and 21 days (B) after the last streptozotocin injection. C: representative graph of glucose tolerance test results at 0, 30, 60, 90, and 120 min postglucose administration, n 7-10 animals/group. *P 0.05 vs. the control group; #P <0.05 vs. the PD group. [0050] Figures 32A-32E are graphs (32B and 32-D-E) and photomicrographs (32A and 32C) showing that BMP-7 ameliorates inflammation by regulating cytokine expression. A: representative photomicrographs of tissue sections stained for CD 14, a marker for monocytes (a, f, and k), sarcomeric β-actin (b, g, and 1), 4=,6-diamidino-2-phenylindole (DAPI) (c, h, and m), and merged images (d, i, and n). White boxes are enlarged in merged images in e, j, and o. Scale bar 100 μιη. n 4-5 per group. B: quantitative analysis of percent monocytes. C: representative photomicrographs of tissue sections stained for TNF-a, a proinflammatory cytokine (a, f, and k), sarcomeric β-actin (b, g, and 1), DAPI (c, h, and m), and merged images (d, i, and n). White boxes are enlarged in merged images in e, j, and o. Scale bar ΙΟΟμιη. n 4- 5 per group. D: quantitative analysis of TNF-a immunostaining. A.U., arbitrary units. E: quantitative analysis of the TNF-a ELISA performed on serum samples, n 5-7 samples/group. *P < 0.05 vs. the control group; #P< 0.05 vs. the PD group.

[0051] Figures 33A, 33B, and 33C are graphs (33B and 33C) and photomicrographs (33A) showing that BMP-7 inhibits the proinflammatory cytokine IL-6 in PD hearts. A: representative photomicrographs of tissue sections stained for IL-6 (a, f, and k), sarcomeric β- actin (b, g, and 1), total DAPI (c, h, and m), and merged images (d, i, and n). White boxes are enlarged in merged images in e, j, and o. Scale bar 100 μιη. n = 7-8 per group. B: quantitative analysis of IL-6 immunostaining. C: quantitative analysis of the IL-6 ELISA performed on serum samples, n 4-6 per group. *P < 0.05 vs. the control group; #P < 0.05 vs. the PD group.

[0052] Figures 34A-34F are graphs (34B-34C and 34E-34F) and photomicrographs (34A and 34D) showing that BMP-7 stimulates increased M2 polarization and anti-inflammatory cytokine secretion. 34A: representative photomicrographs of heart tissues stained for CD206, a marker for M2 macrophages (a, f, and k), sarcomeric β-actin (b, g, and 1), DAPI (c, h, and m), and merged images (d, i, and n). White boxes are enlarged in merged images in e, j, and o. Scale bar 100 μιη. n 4 animals/group. 34B: quantitative analysis of percent M2 macrophages. 34C: quantitative analysis of the IL-1 receptor antagonist (IL-IRA) ELISA performed on serum samples, n = 5-7 samples/group. 34D: representative photomicrographs of heart tissues stained for IL-10, an anti-inflammatory cytokine (a, f, and k), sarcomeric β- actin (b, g, and 1), DAPI (c, h, and m), and merged images (d, i, and n). White boxes are enlarged in merged images in e, j, and o. Scale bar 100 μιη. n = 5 samples/group. 34E: quantitative analysis of IL-10 expression per group. 34F: quantitative analysis of the IL-10 ELISA performed on serum samples, n 4-5 samples/group. *P <0.05 vs. the control group; #P < 0.05 vs. the PD group. [0053] Figures 35A, 35B, and 35C are graphs (35B and 35C) and photomicrographs (35A) showing that BMP-7 stimulates MnSOD expression and mitigates oxidative stress in PD hearts. 35A: representative photomicrographs of heart sections stained with dihydroethidium (DHE) (a, d, and g), DAPI (b, e, and h), and merged images (c, f, and i). Scale bar 100 μιη. n = 6 animals/group. 35B: quantitative analysis of DHE immunostaining. 35C: representative bands and quantitative Western blot analysis of MnSOD expression, n = 5 animals/group. *P< 0.05 vs. the control group; #P < 0.05 vs. the PD group.

[0054] Figures 36A-36E are a graph (36B), photomicrographs (36A and 36C), and western blot analysis (36D and 36E) showing that BMP-7 attenuates apoptosis in the PD heart by regulating phosphatase and tensin homolog (PTEN) and Akt. 36A: representative photomicrographs of heart sections demonstrating TU EL-positive cells (apoptotic nuclei) stained (a, d, and g), DAPI (b, e, and h), and merged images (c, f, and i). Scale bar 100 μιη. n = 5-6 samples/group. 36B: quantitative analysis of percent apoptotic nuclei.36 C: representative photomicrographs demonstrating that apoptosis occurs in cardiomyocytes with TUNEL-positive cells (a), sarcomeric β-actin in green (b), caspase-3 (c), DAPI (d), merged image (e), and enlarged merged image (f). 36D: representative bands and quantitative Western blot analysis of phosphorylated (p-)PTEN and total PTEN expresssion. n =5-6 samples/group. 36E: representative bands and quantitative Western blot analysis of p-Akt and total β-actin expression, n = 5-6 samples/group. *P < 0.05 vs. the control group; #P< 0.05 vs. the PD group.

[0055] Figures 37A, 37B, and 37C are graphs (37B and 37C) and photomicrographs (37A) showing that BMP-7 blunts cardiac fibrosis in the PD heart. 37A: representative photomicrographs of Masson's trichrome staining performed on heart sections to assess interstitial (a-c) and vascular (d-f) fibrosis, n = 7-9 samples/group. 37B: quantitative analysis of interstitial fibrosis. 37C: quantitative analysis of vascular fibrosis. VF/VA, vascular fibrosis/vascular area. *P < 0.05 vs. the control group; #P< 0.05 vs. the PD group.

[0056] Figures 38A and 38B are a chart (38A) and graphs (38B) showing exogenous BMP-7 improves cardiac function in PD mice. Raw echocardiogram data obtained are shown in Fig. 38A. The data indicated impaired LV function in the PD group as demonstrated by deviated LV internal dimension at diastole, LV internal dimension at systole, end-diastolic volume, end-systolic volume, ejection fraction, and fractional shortening compared with the control group (P < 0.001; Fig. 38B). More importantly, a significant improvement in cardiac function was achieved after BMP-7 administration as indicated by augmented fractional shortening and ejection fraction relative to the PD group (P <0.05; Fig. 38B). [0057] Additional advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or can be learned by practice of the invention. The advantages of the invention will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed.

START

DETAILED DESCRIPTION

[0058] The present invention can be understood more readily by reference to the following detailed description of the disclosure and the Examples included therein.

[0059] Before the present compounds, compositions, articles, systems, devices, and/or methods are disclosed and described, it is to be understood that they are not limited to specific synthetic methods unless otherwise specified, or to particular reagents unless otherwise specified, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, example methods and materials are now described.

[0060] All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. A. Compositions

[0061] Disclosed herein compositions comprising active molecules, which may be used in methods disclosed herein. Disclosed herein are compositions comprising FGF-9, FGF-8, BMP-7, M-CSF or a combination thereof. A composition may comprise FGF-9. A composition may comprise FGF-8. A composition may comprise BMP-7. A composition may comprise M-CSF. A composition may comprise one or more of FGF-9, FGF-8, BMP-7, M-CSF or a combination thereof. The compositions disclosed herein can comprise an effective amount of one of more of FGF-9, FGF-8, BMP-7, M-CSF or a combination thereof.

[0062] Disclosed herein is a pharmaceutical composition comprising (i) FGF-9; (ii) M-CSF, an agent that modulates the polarization of monocytes, or a combination thereof; and (iii) a pharmaceutically acceptable carrier. In an aspect, a composition can act at a FGF receptor. In an aspect, a FGF receptor can be FGFR1.

[0063] In an aspect, a disclosed composition can modulate the polarization of a population of monocytes. In an aspect, modulating the polarization of a population of monocytes can comprise increasing the polarization of the monocytes into M2 macrophages. In an aspect, modulating the polarization of a population of monocytes can comprise decreasing the polarization of monocytes into Ml macrophages. In an aspect, modulating the polarization of a population of monocytes can comprise increasing the polarization of the monocytes into M2 macrophages and decreasing the polarization of monocytes into Ml macrophages.

[0064] In an aspect, a disclosed composition can change the phenotype of Ml macrophages into M2 macrophages.

[0065] In an aspect, a population of monocytes can be infiltrating monocytes. In an aspect, a population of monocytes can be in a subject. In an aspect, a subject can have experienced a myocardial infarction, or a subject can have diabetes, or a subject can have pre-diabetes, or a subject can have an inflammatory disease or disorder, or a subject can have a combination thereof. In an aspect, a subject can have a skin disease or disorder. For example, in an aspect, a subject can have diabetes and can have experienced a myocardial infarction, or a subject can have pre-diabetes and can have experienced a myocardial infarction.

[0066] In an aspect, a disclosed composition can increase the expression of one or more antiinflammatory cytokines. In an aspect, anti-inflammatory cytokines can comprise IL-10 and IL-lra. In an aspect, a composition can decrease the expression of one or more proinflammatory cytokines. In an aspect, pro-inflammatory cytokines can comprise interleukin- 6, monocyte chemotactic protein- 1, and tumor necrosis factor alpha.

[0067] In an aspect, a disclosed composition can ameliorate cellular injury. In an aspect, ameliorating cellular injury can comprise reducing the size of the myocardial infarction, reducing the amount of fibrosis in the infarcted tissue, reducing the amount of apoptosis in the infarcted tissue, or a combination thereof.

[0068] In an aspect, a disclosed composition can enhance cardiac function. In an aspect, enhancing cardiac function can comprise (i) improving left ventricular function, (ii) improving fractional shortening, (iii) improving ejection fraction, (iv) reducing end-diastolic volume, (v) decreasing left ventricular mass, and (v) normalizing of heart geometry, or (vi) a combination thereof.

[0069] Disclosed herein is a pharmaceutical composition comprising (i) FGF-9; (ii) FGF-8, BMP-7, or a combination thereof; and optionally, (iii) a pharmaceutically acceptable carrier. In an aspect, a composition can comprise FGF-9, FGF-8, and a pharmaceutically acceptable carrier. For example, in an aspect, a pharmaceutical composition can comprise FGF-9, BMP- 7, and a pharmaceutically acceptable carrier. In an aspect, a pharmaceutical composition can comprise FGF-9, FGF-8, BMP-7, and a pharmaceutically acceptable carrier.

[0070] In an aspect, a disclosed composition can act at a FGF receptor. In an aspect, a FGF receptor can be FGFRl . In an aspect, a composition can modulate the polarization of a population of monocytes. In an aspect, modulating the polarization of a population of monocytes can comprise increasing the polarization of the monocytes into M2 macrophages. In an aspect, modulating the polarization of a population of monocytes can comprise decreasing the polarization of monocytes into Ml macrophages. In an aspect, modulating the polarization of a population of monocytes can comprise increasing the polarization of the monocytes into M2 macrophages and decreasing the polarization of monocytes into Ml macrophages. In an aspect, a composition can change the phenotype of Ml macrophages into M2 macrophages.

[0071] In an aspect, a population of monocytes can be infiltrating monocytes. In an aspect, a population of monocytes can be in a subject. In an aspect, a subject can have experienced a myocardial infarction, or a subject can have diabetes, or a subject can have pre-diabetes, or a subject can have an inflammatory disease or disorder, or a subject can have a combination thereof. For example, in an aspect, a subject can have diabetes and can have experienced a myocardial infarction, or a subject can have pre-diabetes and can have experienced a myocardial infarction.

[0072] In an aspect, a subject can have a skin disease or skin disorder. In an aspect, a skin disease or skin disorder can comprise an inflammatory reaction.

[0073] In an aspect, a disclosed composition can increase the expression of one or more antiinflammatory cytokines. In an aspect, anti-inflammatory cytokines can comprise IL-10 and IL-lra. In an aspect, a composition can decrease the expression of one or more proinflammatory cytokines. In an aspect, pro-inflammatory cytokines can comprise interleukin- 6, monocyte chemotactic protein- 1, and tumor necrosis factor alpha.

[0074] In an aspect, a disclosed composition can ameliorate cellular injury. In an aspect, ameliorating cellular injury can comprise reducing the size of the myocardial infarction, reducing the amount of fibrosis in the infarcted tissue, reducing the amount of apoptosis in the infarcted tissue, or a combination thereof.

[0075] In an aspect, a disclosed composition can ameliorate one or more signs or symptoms associated with a skin disease or disorder. [0076] In an aspect, a disclosed composition can enhance cardiac function. In an aspect, enhancing cardiac function can comprise (i) improving left ventricular function, (ii) improving fractional shortening, (iii) improving ejection fraction, (iv) reducing end-diastolic volume, (v) decreasing left ventricular mass, and (v) normalizing of heart geometry, or (vi) a combination thereof.

[0077] Disclosed herein is a composition comprising FGF-9, FGF-8, BMP-7, or a combination thereof; and and for a pharmaceutical composition, further comprise a pharmaceutically acceptable carrier.

B. Methods

1. METHODS OF MODULATING POLARIZATION OF A POPULATION OF MONOCYTES

[0078] Disclosed herein is a method of modulating polarization of a population of monocytes in a subject, comprising administering to a subject a composition disclosed herein, for example, a composition comprising an effective amount of FGF-9, thereby modulating the polarization of a population of monocytes.

[0079] In an aspect, modulating the polarization of a population of monocytes can comprise increasing the polarization of the monocytes into M2 macrophages. In an aspect, modulating the polarization of a population of monocytes can comprise decreasing the polarization of monocytes into Ml macrophages. In an aspect, modulating the polarization of a population of monocytes can comprise increasing the polarization of the monocytes into M2 macrophages and decreasing the polarization of monocytes into Ml macrophages. In an aspect, a disclosed composition comprising FGF-9 can change the phenotype of Ml macrophages into M2 macrophages.

[0080] In an aspect, a subject can have experienced a myocardial infarction, or a subject can have diabetes, or a subject can have pre-diabetes, or a subject can have an inflammatory disease or disorder, or a subject can have a combination thereof. For example, in an aspect, a subject can have diabetes and can have experienced a myocardial infarction, or a subject can have pre-diabetes and can have experienced a myocardial infarction.

[0081] In an aspect, a composition disclosed herein, for example, a composition comprising FGF-9 can be intravenously administered to a subject. In an aspect, a composition can be administered one or more times. In an aspect, administration of a composition disclosed herein, for example, a composition comprising FGF-9 can be repeated hourly, daily, weekly, bi-weekly, monthly, bi-monthly, annually, every other year, or at any other interval deemed appropriate by the skilled person in the art. In an aspect of a disclosed method, a composition disclosed herein, for example, a composition comprising FGF-9 can be administered continuously.

[0082] In an aspect, a disclosed method of modulating polarization of a population of monocytes can comprise administering to a subject a second composition disclosed herein, for example, a composition comprising an effective amount of M-CSF, an agent that modulates the polarization of monocytes, or a combination thereof. In an aspect, a disclosed method of modulating polarization of a population of monocytes can comprise administering to a subject a second composition disclosed herein, for example, a composition comprising an effective amount of FGF-8, BMP-7, or a combination thereof. In an aspect, a second composition can be intravenously administered to a subject. In an aspect, a second composition can be administered one or more times. In an aspect, a second composition can polarize monocytes independently of a composition comprising FGF-9.

[0083] In an aspect, administration of a disclosed second composition can be repeated hourly, daily, weekly, bi-weekly, monthly, bi-monthly, annually, every other year, or at any other interval deemed appropriate by the skilled person in the art. In an aspect of a disclosed method, a second composition can be administered continuously.

[0084] In an aspect, a disclosed method of modulating polarization of a population of monocytes can comprise co-administering a composition disclosed herein, for example, a composition comprising FGF-9 and a second composition. In an aspect, a disclosed method can comprise separately administering a composition comprising FGF-9 and a second composition. In an aspect, a disclosed method can comprise administering a composition comprising FGF-9 and a second composition one or more times.

[0085] In an aspect of a disclosed method, a composition disclosed herein, for example, a composition comprising FGF-9 can further comprise an effective amount of M-CSF, an agent that modulates the polarization of monocytes. For example, in an aspect, a composition disclosed herein, for example, a composition can comprise (i) FGF-9 and M-CSF, or (ii) FGF-9 and an agent that modulates the polarization of monocytes, or (iii) FGF-9, M-CSF, and an agent that modulates the polarization of monocytes.

[0086] In an aspect of a disclosed method, a composition disclosed herein, for example, a composition comprising FGF-9 can further comprise an effective amount of FGF-8, BMP-7, or a combination thereof. For example, in an aspect, a disclosed composition can comprise (i) FGF-9 and FGF-8, (ii) FGF-9 and BMP-7, or (iii) FGF-9, FGF-8, and BMP-7. [0087] In an aspect, a disclosed method of modulating polarization of a population of monocytes, a population of monocytes can express a FGF receptor. In an aspect, a FGF receptor can be FGFR1.

[0088] In an aspect, a disclosed method of modulating polarization of a population of monocytes can comprise altering one or more administering steps. In an aspect, administering a composition comprising FGF-9 or a second composition can be altered, for example, by changing the route of administration, or changing the dose of a composition, or changing the timing of administration, or changing the frequency of the administration, or a combination thereof.

[0089] Disclosed herein is a method of modulating polarization of a population of monocytes in a subject having a skin disease or skin disorder, comprising administering to a subject a composition disclosed herein, for example, a composition comprising an effective amount of FGF-9, thereby modulating the polarization of a population of monocytes.

[0090] In an aspect, a skin disease or skin disorder can comprise an inflammatory reaction. In an aspect, a disclosed composition can ameliorate one or more signs or symptoms associated with a skin disease or disorder.

[0091] In an aspect, modulating the polarization of a population of monocytes can comprise increasing the polarization of the monocytes into M2 macrophages. In an aspect, modulating the polarization of a population of monocytes can comprise decreasing the polarization of monocytes into Ml macrophages. In an aspect, modulating the polarization of a population of monocytes can comprise increasing the polarization of the monocytes into M2 macrophages and decreasing the polarization of monocytes into Ml macrophages. In an aspect, a composition comprising FGF-9 can change the phenotype of Ml macrophages into M2 macrophages.

[0092] In an aspect, a subject can have experienced a myocardial infarction, or a subject can have diabetes, or a subject can have pre-diabetes, or a subject can have an inflammatory disease or disorder, or a subject can have a combination thereof. For example, in an aspect, a subject can have diabetes and can have experienced a myocardial infarction, or a subject can have pre-diabetes and can have experienced a myocardial infarction.

[0093] In an aspect, a composition disclosed herein, for example, a composition comprising FGF-9 can be intravenously administered to a subject. In an aspect, a composition disclosed herein, for example, a composition comprising FGF-9 can be topically administered to a subject. In an aspect, topical administration can comprise applying a composition to a subject's skin. In an aspect, a composition can be administered one or more times. [0094] In an aspect, administration of a composition disclosed herein, for example, a composition comprising FGF-9 can be repeated hourly, daily, weekly, bi-weekly, monthly, bi-monthly, annually, every other year, or at any other interval deemed appropriate by the skilled person in the art. In an aspect of a disclosed method, a composition comprising FGF-9 can be administered continuously.

[0095] In an aspect, a disclosed method of modulating polarization of a population of monocytes can comprise administering to a subject a second composition comprising an effective amount of M-CSF, an agent that modulates the polarization of monocytes, or a combination thereof. In an aspect, a disclosed method of modulating polarization of a population of monocytes can comprise administering to a subject a second composition comprising an effective amount of FGF-8, BMP-7, or a combination thereof. In an aspect, a second composition can be intravenously administered to a subject. In an aspect, a second composition can be topically administered to a subject. In an aspect, topical administration can comprise applying a composition to a subject's skin. In an aspect, a composition can be administered one or more times.

[0096] In an aspect, administration of a disclosed second composition can be repeated hourly, daily, weekly, bi-weekly, monthly, bi-monthly, annually, every other year, or at any other interval deemed appropriate by the skilled person in the art. In an aspect of a disclosed method, a second composition can be administered continuously. In an aspect, a second composition can polarize monocytes independently of a composition comprising FGF-9.

[0097] In an aspect, a disclosed method of modulating polarization of a population of monocytes can comprise co-administering a composition disclosed herein, for example, a composition comprising FGF-9 and a second composition. In an aspect, a disclosed method can comprise separately administering a composition disclosed herein, for example, a composition comprising FGF-9 and a second composition. In an aspect, a disclosed method can comprise administering a composition comprising FGF-9 and a second composition one or more times. In an aspect of a disclosed method, a composition can comprise (i) FGF-9, (i) FGF-9 and FGF-8, (ii) FGF-9 and BMP-7, or (iii) FGF-9, FGF-8, and BMP-7.

[0098] In an aspect of a disclosed method, a composition disclosed herein, for example, a composition comprising FGF-9 can further comprise an effective amount of M-CSF, an agent that modulates the polarization of monocytes, or a combination thereof. For example, in an aspect, a disclosed composition can comprise (i) FGF-9 and M-CSF, or (ii) FGF-9 and an agent that modulates the polarization of monocytes, or (iii) FGF-9, M-CSF, and an agent that modulates the polarization of monocytes. In an aspect, a population of monocytes can express a FGF receptor. In an aspect, a FGF receptor can be FGFR1.

[0099] In an aspect, a disclosed method of modulating polarization of a population of monocytes can comprise altering one or more administering steps. In an aspect, administering a composition disclosed herein, for example, a composition comprising FGF-9 or a second composition can be altered, for example, by changing the route of administration, or changing the dose of a composition, or changing the timing of administration, or changing the frequency of the administration, or a combination thereof.

[0100] Disclosed herein is a method of modulating polarization of a population of monocytes in a subject, comprising administering to a subject a composition comprising an effective amount of one or more of FGF-9, FGF-8, BMP-7, or a combination thereof, thereby modulating the polarization of a population of monocytes.

[0101] Disclosed herein is a method of modulating polarization of a population of monocytes, comprising (i) administering FGF-9 to a population of monocytes; and (ii) modulating the polarization of the population of monocytes.

[0102] Disclosed herein is a method of modulating polarization of a population of monocytes, comprising (i) administering to a population of monocytes one or more of FGF-9, FGF-8, BMP-7, or a combination thereof; and (ii) modulating the polarization of the population of monocytes.

[0103] Also disclosed are methods of modulating polarization of a population of monocytes in a subject, comprising administering to the subject a composition comprising an effective amount of BMP-7, thereby modulating the polarization of a population of monocytes.

[0104] Disclosed are methods of modulating polarization of a population of monocytes in a subject, comprising administering to the subject a composition comprising an effective amount of BMP-7, thereby modulating the polarization of a population of monocytes, wherein the subject is pre-diabetic, has diabetes, or has atherosclerosis.

[0105] Disclosed are methods of modulating polarization of a population of monocytes in a subject, comprising administering to the subject a composition comprising an effective amount of BMP-7, thereby modulating the polarization of a population of monocytes, wherein modulating the polarization of the population of monocytes comprises increasing the polarization of the monocytes into M2 macrophages.

[0106] Disclosed are methods of modulating polarization of a population of monocytes in a subject, comprising administering to the subject a composition comprising an effective amount of BMP-7, thereby modulating the polarization of a population of monocytes, wherein modulating the polarization of the population of monocytes comprises decreasing the polarization of monocytes into Ml macrophages.

[0107] Disclosed are methods of modulating polarization of a population of monocytes in a subject, comprising administering to the subject a composition comprising an effective amount of BMP-7, thereby modulating the polarization of a population of monocytes, wherein the composition comprising BMP-7 changes the phenotype of Ml macrophages into M2 macrophages.

2. METHODS OF AMELIORATING CELLULAR INJURY IN A SUBJECT

[0108] Disclosed herein is a method of ameliorating cellular injury in a subject, comprising administering to a subject a composition disclosed herein, for example, a composition comprising an effective amount of FGF-9, thereby modulating the polarization of infiltrating monocytes in the subject; and reducing the severity of the cellular injury in the subject.

[0109] In an aspect, modulating the polarization of the infiltrating monocytes can comprise increasing the polarization of the monocytes into M2 macrophages. In an aspect, modulating the polarization of the infiltrating monocytes can comprise decreasing the polarization of monocytes into Ml macrophages. In an aspect, modulating the polarization of the infiltrating monocytes can comprise increasing the polarization of the monocytes into M2 macrophages and decreasing the polarization of monocytes into Ml macrophages.

[0110] In an aspect, reducing the severity of the cellular injury can comprise reducing the size of the myocardial infarction, reducing the amount of fibrosis, reducing the amount of apoptosis, or a combination thereof.

[011 1] In an aspect, a subject can have experienced a myocardial infarction, or a subject can have diabetes, or a subject can have pre-diabetes, or a combination thereof. For example, in an aspect, a subject can have diabetes and can have experienced a myocardial infarction, or a subject can have pre-diabetes and can have experienced a myocardial infarction.

[0112] In an aspect, a disclosed composition disclosed herein, for example, a composition comprising FGF-9 can be intravenously administered to a subject. In an aspect, a composition can be administered one or more times. In an aspect, administration of a composition disclosed herein, for example, a composition comprising FGF-9 can be repeated hourly, daily, weekly, bi-weekly, monthly, bi-monthly, annually, every other year, or at any other interval deemed appropriate by the skilled person in the art. In an aspect of a disclosed method, a composition comprising FGF-9 can be administered continuously.

[0113] In an aspect, a disclosed method of ameliorating cellular injury in a subject can comprise administering to a subject a second composition comprising an effective amount of M-CSF, an agent that modulates the polarization of monocytes, or a combination thereof. In an aspect, a disclosed method of ameliorating cellular injury in a subject can comprise administering to a subject a second composition comprising an effective amount of FGF-8, BMP-7, or a combination thereof. In an aspect, a second composition can be intravenously administered to a subject. In an aspect, a second composition can be administered one or more times.

[0114] In an aspect, administration of a disclosed second composition can be repeated hourly, daily, weekly, bi-weekly, monthly, bi-monthly, annually, every other year, or at any other interval deemed appropriate by the skilled person in the art. In an aspect of a disclosed method, a second composition can be administered continuously. In an aspect, a second composition can polarize monocytes independently of a composition disclosed herein, for example, a composition comprising FGF-9.

[0115] In an aspect, a disclosed method of ameliorating cellular injury in a subject can comprise co-administering a composition disclosed herein, for example, a composition comprising FGF-9 and a second composition. In an aspect, a disclosed method can comprise separately administering a composition disclosed herein, for example, a composition comprising FGF-9 and a second composition. In an aspect, a disclosed method can comprise administering a composition disclosed herein, for example, a composition comprising FGF-9 and a second composition one or more times.

[0116] In an aspect of a disclosed method, a composition disclosed herein, for example, a composition comprising FGF-9 can further comprise an effective amount of M-CSF, an agent that modulates the polarization of monocytes, or a combination thereof. For example, in an aspect, a disclosed composition can comprise (i) FGF-9 and M-CSF, or (ii) FGF-9 and an agent that modulates the polarization of monocytes, or (iii) FGF-9, M-CSF, and an agent that modulates the polarization of monocytes.

[0117] In an aspect of a disclosed method, a composition disclosed herein, for example, a composition comprising FGF-9 can further comprise an effective amount of FGF-8, BMP-7, or a combination thereof. For example, in an aspect, a disclosed composition can comprise (i) FGF-9 and FGF-8, (ii) FGF-9 and BMP-7, or (iii) FGF-9, FGF-8, and BMP-7.

[0118] In an aspect, following the administration of a composition disclosed herein, for example, a composition comprising FGF-9, blood velocity can be improved, fibrosis can be reduced, or a combination thereof. In an aspect, following the administration of the composition disclosed herein, for example, a composition comprising FGF-9, cardiac function can be enhanced. In an aspect, enhanced cardiac function can comprise (i) improving left ventricular function, (ii) improving fractional shortening, (iii) improving ejection fraction, (iv) reducing end-diastolic volume, (v) decreasing left ventricular mass, and (v) normalizing of heart geometry, or (vi) a combination thereof.

[0119] In an aspect, a disclosed method ameliorating cellular injury in a subject can comprise altering one or more administering steps. In an aspect, administering the composition disclosed herein, for example, a composition comprising FGF-9 or a second composition can be altered, for example, by changing the route of administration, or changing the dose of the composition, or changing the timing of administration, or changing the frequency of the administration, or a combination thereof.

[0120] Disclosed herein is a method of ameliorating cellular injury in a subject having a skin disease or skin disorder, comprising administering to a subject an effective amount of a composition disclosed herein, for example, a composition comprising an effective amount of FGF-9, thereby modulating the polarization of infiltrating monocytes in a subject; and reducing the severity of the skin disease or skin disorder in a subject.

[0121] In an aspect, modulating the polarization of the population of monocytes can comprise increasing the polarization of the monocytes into M2 macrophages. In an aspect, modulating the polarization of a population of monocytes can comprise decreasing the polarization of monocytes into Ml macrophages. In an aspect, modulating the polarization of a population of monocytes can comprise increasing the polarization of the monocytes into M2 macrophages and decreasing the polarization of monocytes into Ml macrophages.

[0122] In an aspect, a skin disease or skin disorder can comprise an inflammatory reaction.

[0123] In an aspect, a subject can have experienced a myocardial infarction, or a subject can have diabetes, or a subject can have pre-diabetes, or a combination thereof. For example, in an aspect, a subject can have diabetes and can have experienced a myocardial infarction, or a subject can have pre-diabetes and can have experienced a myocardial infarction.

[0124] In an aspect, a composition disclosed herein, for example, a composition comprising FGF-9 can be intravenously administered to a subject. In an aspect, a composition disclosed herein, for example, a composition comprising FGF-9 can be topically administered to a subject. In an aspect, topical administration can comprise applying the composition to a subject's skin. In an aspect, a composition can be administered one or more times.

[0125] In an aspect, administration of a composition disclosed herein, for example, a composition comprising FGF-9 can be repeated hourly, daily, weekly, bi-weekly, monthly, bi-monthly, annually, every other year, or at any other interval deemed appropriate by the skilled person in the art. In an aspect of a disclosed method, a composition disclosed herein, for example, a composition comprising FGF-9 can be administered continuously.

[0126] In an aspect, a disclosed method of ameliorating cellular injury can comprise administering to a subject a second composition comprising an effective amount of M-CSF, an agent that modulates the polarization of monocytes, or a combination thereof. In an aspect, a disclosed method of ameliorating cellular injury can comprise administering to a subject a second composition comprising an effective amount of FGF-8, BMP-7, or a combination thereof. In an aspect, a second composition can be intravenously administered to a subject. In an aspect, a second composition can be topically administered to a subject. In an aspect, topical administration can comprise applying a composition to a subject's skin. In an aspect, a composition can be administered one or more times.

[0127] In an aspect, administration of the a disclosed second composition can be repeated hourly, daily, weekly, bi-weekly, monthly, bi-monthly, annually, every other year, or at any other interval deemed appropriate by the skilled person in the art. In an aspect of a disclosed method, a second composition can be administered continuously.

[0128] In an aspect, a second composition can polarize monocytes independently of a composition comprising FGF-9.

[0129] In an aspect, a disclosed method ameliorating cellular injury in a subject can comprise co-administering a composition disclosed herein, for example, a composition comprising FGF-9 and a second composition. In an aspect, a disclosed method can comprise separately administering a composition disclosed herein, for example, a composition comprising FGF-9 and a second composition. In an aspect, a disclosed method can comprise administering a composition disclosed herein, for example, a composition comprising FGF-9 and a second composition one or more times.

[0130] In an aspect of a disclosed method of ameliorating cellular injury, following the administration of a composition disclosed herein, for example, a composition comprising FGF-9, blood velocity can be improved, fibrosis can be reduced, or a combination thereof. In an aspect of a disclosed method of ameliorating cellular injury, following the administration of a composition disclosed herein, for example, a composition comprising FGF-9, cardiac function can be enhanced. In an aspect, enhanced cardiac function can comprise (i) improving left ventricular function, (ii) improving fractional shortening, (iii) improving ejection fraction, (iv) reducing end-diastolic volume, (v) decreasing left ventricular mass, and (v) normalizing of heart geometry, or (vi) a combination thereof. [0131] In an aspect, a disclosed composition can ameliorate one or more signs or symptoms associated with a skin disease or disorder.

[0132] In an aspect, a disclosed method of ameliorating cellular injury in a subject can comprise altering one or more administering steps. In an aspect, administering a composition disclosed herein, for example, a composition comprising FGF-9 or a second composition can be altered, for example, by changing the route of administration, or changing the dose of a composition, or changing the timing of administration, or changing the frequency of the administration, or a combination thereof.

[0133] Disclosed herein is a method of ameliorating cellular injury in a subject, comprising administering to a subject a composition disclosed herein, for example, a composition comprising an effective amount of one or more of FGF-9, FGF-8, BMP-7, or a combination thereof, thereby modulating the polarization of infiltrating monocytes in the subject; and reducing the severity of the cellular injury in the subject.

[0134] Disclosed herein is a method of ameliorating cellular injury in a subject in need thereof, comprising (i) administering an effective amount of a composition disclosed herein, for example, a composition comrpising FGF-9 to the subject; (ii) modulating the polarization of infiltrating monocytes in the subject; and (iii) increasing the expression of antiinflammatory cytokines in the subject.

[0135] Disclosed herein is a method of ameliorating cellular injury in a subject in need thereof, comprising (i) administering to the subject an effective amount of a composition disclosed herein, for example, a composition comprising one or more of FGF-9, FGF-8, BMP-7, or a combination thereof; (ii) modulating the polarization of infiltrating monocytes in the subject; and (iii) increasing the expression of anti-inflammatory cytokines in the subject. 3. METHODS OF MODULATING CELLULAR PHENOTYPE OF MACROPHAGES

[0136] Disclosed herein is a method of modulating cellular phenotype of macrophages, comprising (i) administering an effective amount of a composition disclosed herein, for example, a composition comprising FGF-9 to a population of macrophages; and (ii) modulating the cellular phenotype of the population of macrophages such that the macrophages exhibit characteristics of M2 macrophages.

[0137] Disclosed herein is a method of modulating cellular phenotype of macrophages, comprising (i) administering to a population of macrophages an effective amount of a composition disclosed herein, for example, a composition comprising one or more of FGF-9, FGF-8, BMP-7, or a combination thereof; and (ii) modulating the cellular phenotype of the population of macrophages such that the macrophages exhibit characteristics of M2 macrophages.

[0138] In an aspect, a subject can have experienced a myocardial infarction, or a subject can have diabetes, or a subject can have pre-diabetes, or a subject can have an inflammatory disease or disorder, or a subject can have a combination thereof. For example, in an aspect, a subject can have diabetes and can have experienced a myocardial infarction, or a subject can have pre-diabetes and can have experienced a myocardial infarction.

[0139] In an aspect, a composition disclosed herein, for example, a composition comprising FGF-9 can be intravenously administered to a subject. In an aspect, a composition can be administered one or more times. In an aspect, administration of a composition comprising FGF-9 can be repeated hourly, daily, weekly, bi-weekly, monthly, bi-monthly, annually, every other year, or at any other interval deemed appropriate by the skilled person in the art. In an aspect of a disclosed method, a composition comprising FGF-9 can be administered continuously.

[0140] In an aspect, administration of a second composition can be repeated hourly, daily, weekly, bi-weekly, monthly, bi-monthly, annually, every other year, or at any other interval deemed appropriate by the skilled person in the art. In an aspect of a disclosed method, a second composition can be administered continuously.

[0141] In an aspect, an effective amount of a composition disclosed herein, for example, a composition comprising FGF-9 can be intravenously administered to a subject. In an aspect, a composition disclosed herein, for example, a composition comprising FGF-9 can be topically administered to a subject. In an aspect, topical administration can comprise applying the composition to a subject's skin. In an aspect, a composition disclosed herein, for example, a composition comprising FGF-9 can be administered one or more times. In an aspect, administration of a composition disclosed herein, for example, a composition comprising FGF-9 can be repeated hourly, daily, weekly, bi-weekly, monthly, bi-monthly, annually, every other year, or at any other interval deemed appropriate by the skilled person in the art. In an aspect of a disclosed method, FGF-9 can be administered continuously.

[0142] In an aspect, a disclosed method of ameliorating cellular injury in a subject can comprise altering one or more administering steps. In an aspect, administering a composition disclosed herein, for example, a composition comprising FGF-9 or a second composition can be altered, for example, by changing the route of administration, or changing the dose of a composition, or changing the timing of administration, or changing the frequency of the administration, or a combination thereof. 4. METHODS OF ATTENUATING FIBROSIS IN A SUBJECT

[0143] Disclosed herein is a method of attenuating fibrosis in a subject in need thereof, comprising (i) administering an effective amount of a composition disclosed herein, for example, a composition comprising FGF-9 to a subject; (ii) modulating the polarization of infiltrating monocytes in the subject; and (iii) increasing the expression or activity of one or more anti-inflammatory cytokines in the subject.

[0144] Disclosed herein is a method of attenuating fibrosis in a subject in need thereof, comprising (i) administering to the a subject an effective amount of a composition disclosed herein, for example, a composition comprising one or more of FGF-9, FGF-8, BMP-7, or a combination thereof; (ii) modulating the polarization of infiltrating monocytes in the subject; and (iii) increasing the expression or activity of one or more anti-inflammatory cytokines in the subject.

[0145] In an aspect, modulating the polarization of the infiltrating monocytes can comprise increasing the polarization of the monocytes into M2 macrophages. In an aspect, modulating the polarization of the infiltrating monocytes can comprise decreasing the polarization of monocytes into Ml macrophages. In an aspect, modulating the polarization of the infiltrating monocytes can comprise increasing the polarization of the monocytes into M2 macrophages and decreasing the polarization of monocytes into Ml macrophages.

[0146] In an aspect, a subject can have experienced a myocardial infarction, or a subject can have diabetes, or a subject can have pre-diabetes, or a combination thereof. For example, in an aspect, a subject can have diabetes and can have experienced a myocardial infarction, or a subject can have pre-diabetes and can have experienced a myocardial infarction. In an aspect, a subject can have a skin disease or disorder or the subject can have an inflammatory disease or disorder. In an aspect, a disclosed composition can ameliorate one or more signs or symptoms associated with a skin disease or disorder.

[0147] In an aspect, a composition disclosed herein, for example, a composition comprising FGF-9 can be intravenously administered to a subject. In an aspect, a composition can be administered one or more times. In an aspect, a composition disclosed herein, for example, a composition comprising FGF-9 can be repeated hourly, daily, weekly, bi-weekly, monthly, bi-monthly, annually, every other year, or at any other interval deemed appropriate by the skilled person in the art. In an aspect of a disclosed method, a composition disclosed herein, for example, a composition comprising FGF-9 can be administered continuously.

[0148] In an aspect, following the administration of a composition disclosed herein, for example, a composition comprising FGF-9, blood velocity can be improved, fibrosis can be reduced, or a combination thereof. In an aspect, following the administration of a composition disclosed herein, for example, a composition comprising FGF-9, cardiac function can be enhanced. In an aspect, enhanced cardiac function can comprise (i) improving left ventricular function, (ii) improving fractional shortening, (iii) improving ejection fraction, (iv) reducing end-diastolic volume, (v) decreasing left ventricular mass, and (v) normalizing of heart geometry, or (vi) a combination thereof.

5. METHODS OF MODULATING FGF RECEPTORS EXPRESSION IN MONOCYTES

[0149] Disclosed herein is a method of modulating FGF receptor expression in monocytes, comprising (i) administering a composition disclosed herein, for example, a composition comprising FGF-9 to a population of monocytes; and (ii) increasing the expression of a FGF receptor in the monocytes.

[0150] Disclosed herein is a method of modulating FGF receptor expression in monocytes, comprising (i) administering to a population of monocytes an effective amount of a composition disclosed herein, for example, a composition comprising one or more of FGF-9, FGF-8, BMP-7, or a combination thereof; and (ii) increasing the expression of a FGF receptor in the monocytes.

[0151]

6. METHODS OF TREATING ATHEROSCLEROSIS

[0152] Disclosed are methods of treating a subject with atherosclerosis with a composition disclosed herein, for example, a method comprising administering a composition comprising an effective amount of BMP-7.

[0153] Disclosed are methods of treating a subject with atherosclerosis comprising administering a composition comprising an effective amount of BMP-7, wherein plaque formation is reduced.

[0154] Disclosed are methods of treating a subject with atherosclerosis comprising administering a composition comprising an effective amount of BMP-7, wherein monocyte infiltration into arteries is reduced.

[0155] Disclosed are methods of treating a subject with atherosclerosis comprising administering a composition comprising an effective amount of BMP-7, wherein one or more pro-inflammatory cytokines are reduced. The pro-inflammatory cytokine can be TNF-a, MCP-1, IL-6, or a combination thereof.

[0156] Disclosed are methods of treating a subject with atherosclerosis comprising administering a composition comprising an effective amount of BMP-7, wherein one or more anti-inflammatory cytokines are reduced. The anti-inflammatory cytokine can be IL-10, IL- lRA, Arginase 1, or a combination thereof.

[0157] Disclosed are methods of treating a subject with atherosclerosis comprising administering a composition comprising an effective amount of BMP-7, wherein the BMP-7 causes an increase in M2 macrophages. An increase in M2 macrophages can be due to an increase in monocyte to M2 macrophage polarization.

7. METHODS OF INCREASING BMP-7 RECEPTOR

[0158] Disclosed are methods of increasing BMP-7 receptor (BMP-7R) on M2 macrophages comprising administering a composition comprising an effective amount of BMP-7.

[0159]

A. DEFINITIONS

[0160] As used in the specification and the appended claims, the singular forms "a," "an" and "the" include plural referents unless the context clearly dictates otherwise.

[0161] Ranges can be expressed herein as from "about" one particular value, and/or to "about" another particular value. When such a range is expressed, a further aspect includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent "about," it will be understood that the particular value forms a further aspect. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as "about" that particular value in addition to the value itself. For example, if the value "10" is disclosed, then "about 10" is also disclosed. It is also understood that each unit between two particular units are also disclosed. For example, if 10 and 15 are disclosed, then 1 1, 12, 13, and 14 are also disclosed.

[0162] References in the specification and concluding claims to parts by weight of a particular element or component in a composition denotes the weight relationship between the element or component and any other elements or components in the composition or article for which a part by weight is expressed. Thus, in a compound containing 2 parts by weight of component X and 5 parts by weight component Y, X and Y are present at a weight ratio of 2:5, and are present in such ratio regardless of whether additional components are contained in the compound.

[0163] As used herein, the terms "optional" or "optionally" means that the subsequently described event or circumstance can or cannot occur, and that the description includes instances where said event or circumstance occurs and instances where it does not. For example, in an aspect, a disclosed method can optionally comprise one or more additional steps, such as, for example, repeating an administering step or altering an administering step.

[0164] As used herein, the term "subject" refers to the target of administration, e.g., an animal. The term "subject" also includes domesticated animals (e.g., cats, dogs, etc.), livestock (e.g., cattle, horses, pigs, sheep, goats, etc.), and laboratory animals (e.g., mouse, rabbit, rat, guinea pig, fruit fly, etc.). Thus, the subject of the herein disclosed methods can be a vertebrate, such as a mammal, a fish, a bird, a reptile, or an amphibian. Alternatively, the subject of the herein disclosed methods can be a human, non-human primate, horse, pig, rabbit, dog, sheep, goat, cow, cat, guinea pig, or rodent. The term does not denote a particular age or sex. Thus, adult and newborn subjects, as well as fetuses, whether male or female, are intended to be covered. In an aspect, a subject can be a human patient.

[0165] A "patient" refers to a subject afflicted with one or more diseases or disorders, such as, for example, diabetes, or pre-diabetes, or a skin disease or skin disorder. A patient can refer to a subject that has experienced a myocardial infarction. In an aspect, a patient can have an inflammatory disorder. In an aspect, a patient can have one or more diseases or disorder and can have experienced a myocardial infarction.

[0166] As used herein, the term "treatment" refers to the medical management of a patient with the intent to cure, ameliorate, stabilize, or prevent a disease, pathological condition, or disorder (such as, for example, a skin disease or disorder, an inflammatory disease or disorder, or a heart disease or disorder (i.e., a myocardial infarction). This term includes active treatment, that is, treatment directed specifically toward the improvement of a disease, pathological condition, or disorder, and also includes causal treatment, that is, treatment directed toward removal of the cause of the associated disease, pathological condition, or disorder. In addition, this term includes palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder; preventative treatment, that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder; and supportive treatment, that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological condition, or disorder. In various aspects, the term covers any treatment of a subject, including a mammal (e.g., a human), and includes: (i) preventing the disease from occurring in a subject that can be predisposed to the disease but has not yet been diagnosed as having it; (ii) inhibiting the disease, i.e., arresting its development; or (iii) relieving the disease, i.e., causing regression of the disease.

[0167] As used herein, the term "prevent" or "preventing" refers to precluding, averting, obviating, forestalling, stopping, or hindering something from happening, especially by advance action. It is understood that where reduce, inhibit or prevent are used herein, unless specifically indicated otherwise, the use of the other two words is also expressly disclosed. In an aspect, preventing monocyte infiltration is intended. In an aspect, preventing monocyte polarization into Ml macrophages is intended.

[0168] As used herein, the term "diagnosed" means having been subjected to a physical examination by a person of skill, for example, a physician, and found to have a condition that can be diagnosed or treated by the compounds, compositions, or methods disclosed herein. For example, "diagnosed with a skin disease or disorder" means having been subjected to a physical examination by a person of skill, for example, a physician, and found to have a condition that can be diagnosed or can be treated by a compound or composition that can prevent or inhibit an inflammatory reaction mediated by Ml macrophages or infiltrating monocytes. For example, "diagnosed with an inflammatory disease or disorder" means having been subjected to a physical examination by a person of skill, for example, a physician, and found to have a condition that can be diagnosed or can be treated by a compound or composition that can prevent or inhibit an inflammatory reaction mediated by Ml macrophages or infiltrating monocytes.

[0169] As used herein, the phrase "identified to be in need of treatment for a disorder," or the like, refers to selection of a subject based upon need for treatment of the disorder. For example, a subject can be identified as having a need for treatment of a disorder (e.g., diabetes, or pre-diabetes, or a skin disease or disorder, or an inflammatory disease or disorder, or heart disease or disorder) based upon an earlier diagnosis by a person of skill and thereafter subjected to treatment for the disorder. It is contemplated that the identification can, in one aspect, be performed by a person different from the person making the diagnosis. It is also contemplated, in a further aspect, that the administration can be performed by one who performed the diagnosis.

[0170] As used herein, the terms "administering" and "administration" refer to any method of providing a disclosed composition, complex, or a pharmaceutical preparation to a subject. Such methods are well known to those skilled in the art and include, but are not limited to: oral administration, transdermal administration, administration by inhalation, nasal administration, topical administration, intravaginal administration, ophthalmic administration, intraaural administration, intracerebral administration, rectal administration, sublingual administration, buccal administration, and parenteral administration, including injectable such as intravenous administration, intra-arterial administration, intramuscular administration, and subcutaneous administration. Administration can be continuous or intermittent. In various aspects, a preparation can be administered therapeutically; that is, administered to treat an existing disease or condition. In further various aspects, a preparation can be administered prophylactically; that is, administered for prevention of a disease or condition. In an aspect, the skilled person can determine an efficacious dose, an efficacious schedule, and an efficacious route of administration for a disclosed composition or a disclosed complex so as to treat a subject or inhibit or prevent an inflammatory reaction. In an aspect, the skilled person can also alter, change, or modify an aspect of an administering step so as to improve efficacy of a disclosed complex or disclosed composition.

[0171] As used herein, "altering one or more administering steps" can comprise altering the administration of one or more disclosed compositions or disclosed complexes. In an aspect, administering a composition comprising one or more of FGF-9, FGF-8, BMP-7, or a second agent that polarized monocytes can be altered, for example, by changing the route of administration, or changing the dose of a disclosed composition, or changing the timing of administration, or changing the frequency of the administration, or a combination thereof. In an aspect, administering the complex comprising a copolymer carrier and one or more oligonucleotides can be altered, for example, by changing the route of administration, or changing the dose of a disclosed composition, or changing the timing of administration, or changing the frequency of the administration, or a combination thereof. In an aspect, altering one or more administering steps can comprise altering the administering of a disclosed composition.

[0172] The term "contacting" as used herein refers to bringing a disclosed composition, compound, or complex together with an intended target (such as, e.g., a cell or population of cells, a receptor, an antigen, or other biological entity) in such a manner that the disclosed composition, compound, or complex can affect the activity of the intended target (e.g., receptor, transcription factor, cell, population of cells, etc.), either directly (i.e., by interacting with the target itself), or indirectly (i.e., by interacting with another molecule, co-factor, factor, or protein on which the activity of the target is dependent). In an aspect, a disclosed composition or complex can be contacted with a cell or population of cells, such as, for example, a population of monocytes, such as infiltrating monocytes. [0173] The term "pharmaceutically acceptable" describes a material that is not biologically or otherwise undesirable, i.e., without causing an unacceptable level of undesirable biological effects or interacting in a deleterious manner. As used herein, the term "pharmaceutically acceptable carrier" refers to sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, as well as sterile powders for reconstitution into sterile injectable solutions or dispersions just prior to use. Examples of suitable aqueous and nonaqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol and the like), carboxymethylcellulose and suitable mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters such as ethyl oleate. These compositions can also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms can be ensured by the inclusion of various antibacterial and antifungal agents such as paraben, chlorobutanol, phenol, sorbic acid and the like. It can also be desirable to include isotonic agents such as sugars, sodium chloride and the like. Prolonged absorption of the injectable pharmaceutical form can be brought about by the inclusion of agents, such as aluminum monostearate and gelatin, which delay absorption. Injectable depot forms are made by forming microencapsule matrices of the drug in biodegradable polymers such as polylactide-polyglycolide, poly(orthoesters) and poly(anhydrides). Depending upon the ratio of drug to polymer and the nature of the particular polymer employed, the rate of drug release can be controlled. Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions which are compatible with body tissues. The injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable media just prior to use. Suitable inert carriers can include sugars such as lactose.

[0174] As used herein, "a skin disease or disorder" can refer to a pathology that affects the epidermis, dermis, and/or subcutaneous tissue. A skin disease or disorder can have an inflammatory component. A skin disease or disorder can include, but is not limited to, the following: eczema, psoriasis, lichen planus, bullous pephigoid, vasculitis, granuloma annulare, and acne.

[0175] "Peptide" as used herein refers to any peptide, oligopeptide, polypeptide, gene product, expression product, or protein. For example, a peptide can be an enzyme. A peptide is comprised of consecutive amino acids. Polypeptides encompass naturally occurring or synthetic molecule, and may contain modified amino acids. Polypeptides can be modified by either natural processes, such as post-translational processing, or by chemical modification techniques which are well known in the art. Modifications can occur anywhere in the polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini. The same type of modification can be present in the same or varying degrees at several sites in a given polypeptide.

[0176] In general, the biological activity or biological action of a gene / nucleic acid or peptide refers to any function exhibited or performed by the gene / nucleic acid or peptide that is ascribed to the naturally occurring form of the gene / nucleic acid or peptide as measured or observed in vivo (i.e., in the natural physiological environment of the gene / nucleic acid or peptide) or in vitro (i.e., under laboratory conditions).

[0177] The term "enzyme" as used herein refers to any peptide that catalyzes a chemical reaction of other substances without itself being destroyed or altered upon completion of the reaction. Typically, a peptide having enzymatic activity catalyzes the formation of one or more products from one or more substrates. Such peptides can have any type of enzymatic activity including, without limitation, the enzymatic activity or enzymatic activities associated with enzymes such as those disclosed herein.

[0178] Cells can be obtained from commercial sources such as the American Type Culture Collection (ATCC) and can be prokaryotic or eukaryotic. Cells can be grown in liquid media culture or on tissue culture plates. The growth conditions will be dependent upon the specific cells used and such conditions would be known to one of skill in the art. Transfection and growth of cells are described in Maniatis et al.

[0179] As used herein, the term "determining" can refer to measuring or ascertaining a quantity or an amount or a change in expression and/or in activity level or in prevalence. For example, determining can refer to measuring or ascertaining the quantity or amount of a nucleotide or transcript or polypeptide or pro-inflammatory cytokine or anti-inflammatory cytokine. For example, determining can refer to measuring or ascertaining the prevalence of a particular cell type in a sample or in a subject such as, for example, the number of monocytes or macrophages (i.e., Ml macrophages, M2 macrophages, etc.). Methods and techniques used to determining the amount or quantity or prevalence of a disclosed transcript or polypeptide or cell in a sample as used herein can refer to the steps that the skilled person would take to measure or ascertain some quantifiable value of the transcript or polypeptide or cell in the sample. The art is familiar with the ways to measure an amount of a disclosed nucleotides, transcripts, polypeptides, cells, etc. In an aspect, "determining" can refer to a rate of some type of activity, such as, for example, the rate of polarization of monocytes into Ml macrophage or M2 macrophages.

[0180] As used herein, "pro-inflammatory cytokines" can include but are not limited to cytokines characterized as being inducible and belonging to different families, including IL- 1, IL-6, and TNFa. In an aspect, pro-inflammatory cytokines can include subsequently discovered molecules of the TNF family with related properties that promote inflammatory host reactions such as secreted lymphotoxin a (also known as TNF ), the heterotrimeric membrane-bound lymphotoxin αβ, LIGHT, CD40 ligand, Fas ligand, CD30 ligand, CD27 ligand, 4- IBB ligand, and the Ox40 ligand. Other pro-inflammatory cytokines can include IFN, IL-2, both products chiefly of TH1 cells as, well as IFN.

[0181] As used herein, "anti-inflammatory cytokines" can include, but are not limited to, interleukin (IL)-l receptor antagonist (IL-lra), IL-4, IL-6, IL-10, IL-11, and IL-13, or other cytokines known to those of skill in the art.

[0182] As used herein, the term "level" refers to the amount of a target molecule or a target cell in a sample, e.g., a sample from a subject. The amount of the molecule or the cell can be determined by any method known in the art and will depend in part on the nature of the molecule (i.e., gene, mRNA, cDNA, protein, enzyme, etc.) or the nature of the cell type (macrophage, monocyte, etc.). The art is familiar with quantification methods for nucleotides (e.g., genes, cDNA, mRNA, etc.) as well as proteins, polypeptides, enzymes, etc. The art is familiar with quantification methods for various cell types. It is understood that the amount or level of a molecule or the amount or level of a cell type in a sample need not be determined in absolute terms, but can be determined in relative terms (e.g., when compare to a control or a sham or an untreated sample).

[0183] By "modulate" is meant to alter, i.e., by an increase or a decrease. As used herein, a "modulator" can mean a composition that can either increase or decrease the expression level or activity level of a gene or gene product such as a peptide, or that can either increase or decrease the prevalence of a specific cell type such as a monocyte, a Ml macrophage, or a M2 macrophage. Modulation in expression or activity does not have to be complete. For example, expression or activity can be modulated by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 100% or any percentage in between as compared to a control cell wherein the expression or activity of a gene or gene product has not been modulated by a composition, such as, for example, a composition comprising BMP-7 or M-CSF or a second agent that polarize monocytes or a combination thereof. Similarly, modulation of the prevalence of a specific cell type does not have to be complete. For example, prevalence of a specific cell type can be modulated by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 100% or any percentage in between as compared to a control cell or tissue or subject, wherein the cell or tissue or subject has not been modulated by a composition, such as, for example, a composition comprising one or more of FGF-9, FIGF-8, BMP-7, a second agent that polarize monocytes, M-CSF, or a combination thereof. In an aspect, "modulate" can refer to increasing or decreasing the rate of some type of activity, such as, for example, the increasing or decreasing the rate of polarization of monocytes into Ml macrophage or M2 macrophages.

[0184] As used herein, the terms "effective amount" and "amount effective" can refer to an amount that is sufficient to achieve the desired result such as, for example, the polarization of monocytes to Ml macrophages and/or M2 macrophages. As used herein, the terms "effective amount" and "amount effective" can refer to an amount that is sufficient to achieve the desired an effect on an undesired condition. For example, a "therapeutically effective amount" refers to an amount that is sufficient to achieve the desired therapeutic result or to have an effect on undesired symptoms, but is generally insufficient to cause adverse side effects. The specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration; the route of administration; the rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed and like factors well known in the medical arts. For example, it is well within the skill of the art to start doses of a compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. If desired, then the effective daily dose can be divided into multiple doses for purposes of administration. Consequently, single dose compositions can contain such amounts or submultiples thereof to make up the daily dose. The dosage can be adjusted by the individual physician in the event of any contraindications. Dosage can vary, and can be administered in one or more dose administrations daily, for one or several days. Guidance can be found in the literature for appropriate dosages for given classes of pharmaceutical products. In further various aspects, a preparation can be administered in a "prophylactically effective amount"; that is, an amount effective for prevention of a disease or condition.

[0185] In an aspect, "polarization" can refer to the functional and spatial asymmetry that plays a role for cells such as immune cells, which undergo chemotaxis and sense extracellular stimulus gradients. For example, distinct subsets of effector T-cells that bridge the innate and adaptive immune system are said to be polarized in the sense that these cells are characterized by the expression of distinct subsets of cytokines and receptors. For examples see: Bel cells, Be2 cells, IL17-Th2 cells, Thl cells, Th2 cells, Th3 cells, Th5 cells, Th6 cells, Th9 cells, Thl7 cells, Th22 cells. For a comprehensive gene expression profile of Thl and Th2 cells, see Nagai et al, 2001.

[0186] In an aspect, "differentiation" can refer to cellular differentiation, which can be used to describe the process by which a less specialized cell becomes a more specialized cell type. For example, differentiation can occur numerous times during the development of a multicellular organism as the organism changes from a simple zygote to a complex system of tissues and cell types. Differentiation can be considered a common process in adults as well: adult stem cells divide and create fully differentiated daughter cells during tissue repair and during normal cell turnover. Differentiation can dramatically change a cell's size, shape, membrane potential, metabolic activity, and responsiveness to signals.

[0187] Disclosed are the components to be used to prepare a composition of the invention as well as the compositions themselves to be used within the methods disclosed herein. These and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed that while specific reference of each various individual and collective combinations and permutation of these compounds cannot be explicitly disclosed, each is specifically contemplated and described herein. For example, if a particular compound is disclosed and discussed and a number of modifications that can be made to a number of molecules including the compounds are discussed, specifically contemplated is each and every combination and permutation of the compound and the modifications that are possible unless specifically indicated to the contrary. Thus, if a class of molecules A, B, and C are disclosed as well as a class of molecules D, E, and F and an example of a combination molecule, A-D is disclosed, then even if each is not individually recited each is individually and collectively contemplated meaning combinations, A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F are considered disclosed. Likewise, any subset or combination of these is also disclosed. Thus, for example, the sub-group of A-E, B-F, and C- E would be considered disclosed. This concept applies to all aspects of this application including, but not limited to, steps in methods of making and using the compositions of the invention. Thus, if there are a variety of additional steps that can be performed it is understood that each of these additional steps can be performed with any specific embodiment or combination of embodiments of the methods of the invention. [0188] The present invention can be understood more readily by reference to the following detailed description of the invention and the Examples included therein.

[0189] All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided herein can be different from the actual publication dates, which can require independent confirmation.

[0190]

B. EXAMPLES

[0191] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how the compounds, compositions, articles, devices and/or methods claimed herein are made and evaluated, and are intended to be purely exemplary of the invention and are not intended to limit the scope of what the inventors regard as their invention. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

[0192] Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.), but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in °C or is at ambient temperature, and pressure is at or near atmospheric.

1. Preliminary Experiments

[0193] The experiments described herein examined whether (i) increasing levels of FGF-9 correlated with the increased presence of M2 macrophage phenotype in MI-db/db mice, (ii) whether the loss of FGF-9 receptor (FGF-9R) function inhibited M2 polarized macrophages, (iii) whether FGF-9 independently differentiated monocytes into M2 polarized macrophages, and (iv) whether FGF-9 induced the differentiation of monocytes into to M2 macrophage was mediated through PI3K-YAP (HIPPO) pathway and MAP Kinase activation. The experiments presented herein identified fibroblast growth factor (FGF-9), but not FGF-2, as a growth factor that has at least one type of receptor located on monocytes. Therefore, FGF-9 polarized monocytes into M2 macrophages. M-CSF also induced differentiation of monocytes into M2 macrophages.

INDUCED HEART FAILURE MODEL IN DB/DB MICE

[0194] Type 2 diabetic db/db mice develop myocardial infarction (MI) and heart failure (Varma et al, 2012; Yan et al, 2011). The experiments presented herein utilized db/db mice to generate MI by ligating the left coronary artery (Yan et al, 2011). In this model, MI with or without diabetes, induced apoptosis, fibrosis, and heart failure, which were associated with decreased heart function established by echocardiography (Yan et al, 2011 ; Glass et al, 2011 ; Singla et al, 2007, 201 1(a), 2011(b). Transplanted stem cells reversed pathological changes in the MI-db/db heart and improved heart function (Yan et al, 2011).

FGF-9 IN INFLAMMATION AND CARDIOVASCULAR DISEASES

[0195] FGFs are polypeptide growth factors associated with organogenesis, tissue repair, nervous system control, physiological development, metabolism, and regulation of myocardial cell type proliferation and differentiation (Pirvola et a., 2004; Gay et al, 2013; Geske et al, 2008; Hung et al, 2007; Jiang et al, 2004; Lavine et al, 2005; Wang et al, 2013; White et al, 2007). FGFs have four different receptors, which bind to different isoforms of FGFs. FGF receptors are expressed in many cell types (Lavine et al, 2005; Wang et al, 2013; White et al, 2007; Zhang et al, 2006). Data shows that FGF-9 signals through a FGF-receptor 1 in the heart (Lavine et al, 2005). FGF-9 contributes to vasculogenesis and neovascularization in the heart, regulates inner ear morphogenesis, stimulates epithelial cells and induces hair follicle neogenesis after wounding (Pirvola et al, 2004; Gay et al, 2013; Geske et al, 2008; Hung et al., 2007; Tomanek et al, 2010).

[0196] Whether cardiac protection provided by FGF-2 and FGF-9 was mediated through the activation of infiltrated monocytes into M2 macrophage polarization was explored. The data presented herein demonstrated that FGF-9 (but not FGF-2) decreased pro-inflammatory cytokines released by Ml macrophages by directly interfering with the Ml macrophage and/or by converting the Ml macrophages into M2 macrophages. The data presented herein also indicated that, in db/db mice, FGF-receptor type 1 was predominantly present on monocytes after infiltration into the post-MI heart.

FGF-9 STIMULATED MONOCYTE DIFFERENTIATION INTO M2 MACROPHAGES

[0197] FGF-9 treatment polarized monocytes into M2 macrophages. Monocytes were treated with FGF-9 (50 ng/mL) or FGF-2 (50 ng/niL) were used and the presence of FGF receptor 1 (FGFR1) on the monocyte cell surface was determined. Immunostaining and confocal data indicated that THP-1 cells (which were confirmed with monocyte marker CD 14), also expressed FGFRl identified (FIG. IB). Quantitative data showed that the number of THP-1 cells with FGFRl staining was significantly increased following FGF-9 treatment, but not with FGF-2 treatment (FIG. 1A). Furthermore, M2 macrophages were confirmed with specific marker arginase 1 (FIG. 1C, FIG. ID). The number of M2 macrophages expressing FGFRl increased following FGF-9 treatment (FIG. 1C, FIG. ID). These data show that both monocytes and M2 macrophage contained the FGFRl receptor (FGF-R1) and that the receptor bound to FGF-9 but not FGF-2.

[0198] To determine the effects of different treatments on monocyte polarization in the cell culture, H9c2 cells were treated with H2O2 to generate apoptotic conditioned medium (ACM). ACM was used to generate an inflammatory induced cell culture model in THP-1 and primary human monocytes (HM) (Rocher et al, 2012). The THP-1 and primary HM cells were treated with ACM to generate stressed conditions and to determine the effects of FGF-9 and FGF-2. The data indicated that the total number of monocytes was significantly reduced when compared with controls in both cell types (i.e., THP-1 and HM cells) (FIG. 2A-FIG. 2B).

[0199] Next, the loss of monocytes caused by each treatment was determined. FIG. 3 demonstrates that ACM and FGF-2 treatment polarized monocytes into Ml macrophages as determined by iNOS specific staining for Ml macrophages (FIG. 3A-FIG.3B). Furthermore, the presence of M2 macrophages was determined using two markers specific for M2 macrophages, arginase 1 and CD206 antibody (FIG. 3C). FIG. 4A-FIG. 4E demonstrate that FGF-9 treatment significantly enhanced M2 macrophage polarization when compared with ACM and FGF-2 treatment groups. These data indicate that FGF-9 polarized monocytes into M2 macrophages, but that FGF-2 did not (FIG. 3 and FIG. 4). The effect of polarized monocytes/macrophages on the expression of pro-inflammatory and anti-inflammatory cytokines was evaluated. In THP-1 cells, the levels of pro-inflammatory cytokines (TNF-a, MCP-1, and IL-6) were significantly increased in the ACM group when compared with controls. However, when the THP-1 cells were treated with FGF-9, the FGF-9 treatment significantly reduced the levels of pro-inflammatory cytokines (FIG. 5A-FIG. 5C). FGF-9 treatment also significantly increased the levels of the anti-inflammatory cytokines IL-10 and IL-lra. (FIG. 5D-FIG. 5E, *p < 0.05).

[0200] In HM cells, there was a significant increase in the levels of the pro-inflammatory cytokines TNF-a (FIG. 6A) and MCP-1 (FIG. 6B) in the ACM group. FGF-9 treatment significantly reduced the levels of pro-inflammatory cytokines in HM cells. The levels of the anti-inflammatory cytokines IL-10 (FIG. 6D) and IL-lra (FIG. 6C) were reduced in ACM groups. However, when the HM cells were treated with FGF-9, the decrease in antiinflammatory cytokines was reversed. Collectively, the data generated using these two cell type cell culture models indicated that FGF-9 polarized monocytes and/or Ml macrophages into M2 macrophages and affected the release of anti-inflammatory and pro-inflammatory cytokines.

EFFECTS OF FGF-9 ON INFARCT SIZE, CARDIAC FIBROSIS, AND HEART FUNCTION

[0201] The effect of MI on db/db mice was examined. Mice (db/db mice), 8 to 10 weeks old, were purchased and segregated into three study groups: Sham (n = 6), MI (n = 6), and MI + FGF-9 (n = 6). MI was performed as described herein (Yan et al, 201 1; Singla et al, 2007, 2011a, and 201 1b). Following surgery, animals were injected intramyocardially with either mouse recombinant FGF-9 (100 ng/kg) or saline. At two weeks post-MI, heart function was recorded using echocardiography. Hearts were then isolated and embedded in paraffin blocks using a standard protocol (Singla et al, 2007, 201 la, 201 lb, Jiang et al, 2004). Sections were prepared to verify the presence of infarct size and fibrosis (FIG. 7A and FIG. 8A). Cross sections were stained with Hematoxylin and Eosin and the infarct size was measured under a bright field light (Singla et al, 2007). Immunohistochemical staining showed formation of the infarct size and level of fibrosis in the db/db group following MI, which was significantly greater than infarct size and level of fibrosis of the sham controls. FGF-9 treatment significantly reduced the formation of infarct size and the level of fibrosis (FIG. 7B and FIG. 8B).

EFFECTS OF FGF-9 ON POST-MI MONOCYTE INFILTRATION

[0202] Following MI, mouse-specific CD 14 antibodies were used to identify infiltrating monocytes. FIG. 9A shows confocal microscopy images specific for monocyte identification post-MI. Sarcomeric a-actin was used to stain cardiac myocytes in order to demosntrate that monocytes/macrophages infiltrated into the infarcted myocardium. Quantitative data indicated a significant increase in monocytes in the MI-db/db group (FIG. 9B). However, this increase in the number of monocytes was inhibited by FGF-9 treatment.

EFFECTS OF FGF-9 ON MONOCYTE POLARIZATION INTO Ml AND M2 MACROPHAGES

[0203] To determine the effects of FGF-9 on M1/M2 polarization, immunostaining specific for Ml macrophages was performed using iNOS. Specific staining for M2 macrophages was performed with CD206. FIG. 10A shows confocal images of Ml macrophages stained for iNOS antibody and FIG. 1 1A shows CD206 antibody immunostaining specific for M2 macrophages. The quantitative data shows a significant post-MI increase in Ml macrophages in db/db mice when compared with the control and FGF-9 groups (FIG. 10B and FIG. 1 IB). These data indicate that the injured myocardium favored Ml macrophage formation; however, FGF-9 treatment reduced the levels of Ml macrophages.

[0204] Following MI, the M2 macrophage data indciates that the percentage of M2 macrophages in the MI group was lower when compared with control group (FIG. 11B). However, the percentage of M2 macrophages was significantly increased in FGF-9 treatment group, indicating that FGF-9 contributed to the polarization of M2 macrophages (FIG. 1 1B). Specifically, these data indicate that FGF-9 caused monocytes to differentiate into M2 macrophages and caused Ml macrophages to polarize into M2 macrophages. For example, FIG. 9B shows that monocytes were significantly reduced in post-MI animals when treated with FGF-9, and FIG. 10B shows that Ml macrophages were significantly reduced in MI animals when treated with FGF-9.

DETERMINATION OF FGFRl ON MONOCYTES AND CO-LOCALIZATION WITH M2 MACROPHAGES

[0205] Whether infiltrating monocytes in vivo also contained FGFRl was determined. Sections were stained with anti-FGFRl antibody. Sections were also co-stained with monocyte-specific antibody CD 14. FIG. 12A shows FGFRl staining in the MI hearts, which co-localized with staining for the CD 14 antibody specific for monocytes. Merged photomicrographs confirmed co-localized staining for both FGFRl and monocytes. The percentages of monocytes that contained FGFRl was also determined (FIG. 12B).

[0206] Next, whether the presence of FGFRl was significantly up regulated following MI in FGF-9 treated groups was examined. Specifically, the data presented herein demonstrated that M2 macrophages increased following MI and FGF-9 treatment. Whether the increase in M2 macrophages had any relationship with FGFRl, which is present on the infiltrating monocytes, was examined. Double immunostaining for FGFRl and M2 macrophage specific antibody CD206 was performed (FIG. 12C). Confocal data and quantitative analysis (FIG. 12D) indicate that a percentage of M2 macrophages also contained a FGFRl, which was highly up regulated in FGF-9 treated groups. These results indicate that monocytes containing FGFRl had become M2 polarized macrophages that retained FGFRl.

EFFECTS OF FGF-9 ON PRO-INFLAMMATORY AND ANTI-INFLAMMATORY CYTOKINES

[0207] After determination of altered monocytes and macrophage phenotypes following MI with and without FGF-9 treatment, the levels of pro-inflammatory cytokines (TNF-a, MCP-1, and IL-6) were examined (FIG. 13A-FIG. 13C). The data show a significant increase in proinflammatory cytokines in the MI group; however, this increase was reversed with FGF-9 treatment. To confirm the release of anti-inflammatory cytokines by M2 macrophages, the levels of IL-10 and IL-lra were measured (FIG. 13D-FIG. 13E). The data show that these anti-inflammatory cytokines were significantly up regulated following FGF-9 treatment. Collectively, these data indicate that FGF-9 polarized monocytes into M2 macrophages, which were associated with released anti-inflammatory cytokines.

EFFECTS OF FGF-9 ON FGFRl AND RAS AND PI3K-YAP PATHWAY

[0208] To understand the effect of FGF-9 on the signaling pathway, the RAS and PI3K activated- YAP pathway was explored. Homogenates were prepared from hearts that had received and that had not received FGF-9 treatment. Protein content was measured. SDS- PAGE and Western blotting were performed using a standard protocol (Glass et al, 2011a; Yan et al, 2013; Glass et al, 2011b; Singla et al, 2008). FIG. 14 shows that levels of RAS, PI3K, and YAP proteins were significantly increased when compared with sham controls (FIG. 14A-FIG. 14C). Following treatment with FGF-9, a decreased level of RAS, PI3K, and YAP protein was observed. Therefore, the increase in M2 polarized macrophages was associated with decreased levels of RAS and/or PI3K activated YAP pathway, which indicated that the RAS and PI3K pathway was involved in the increased number of M2 polarized macrophages.

ECHOCARDIOGRAPHY MEASUREMENTS OF THE POST-MI HEARTS IN DB/DB MICE

[0209] A Phillips SONOS 5500 Ultrasound system was used to perform M-mode echocardiography to obtain fractional shortening (FS) on day 14 following MI surgery. Heart functions such as FS and ejection fraction were also determined (Glass et al., 2010; Yan et al, 201 1 ; Glass et al, 2011b). The data indicated that FS was significantly reduced when compared with the sham control, indicating that these mice have developed MI. Next, the MI + F-9 treated group showed increased FS in db/db mice indicating that beneficial effects at the functional level had occurred (FIG. 15).

2. EXPERIMENT 1

[0210] Whether FGF-9 and FGF receptors played a role in macrophage polarization and whether increasing levels of FGF-9 correlated with an increased presence of the M2 antiinflammatory macrophage phenotype following MI in db/db hearts was examined. Immunohistochemical and real-time PCR techniques were used to measure the levels of proinflammatory and anti-inflammatory cytokines, specific expression of inflammatory genes, and specific genes for Ml and M2 phenotype macrophages such as arginase 1 and scavenger receptors. The presence and preponderance of specific macrophage phenotypes were identified. [021 1] An established db/db mouse MI model was utilized (Yan et al, 201 1). The MI model is associated with <5% mortality in db/db mice. Animals were divided into three groups: (1) Sham, (2) MI, and (3) MI + FGF-9 (n = 8-10 per group) FGF-9 (100 ng/kg) was administered through intramyocardial injections on the day of the MI surgery. Animals are euthanized on day 14 post-MI. In long-term experiments, animals are euthanized at 4, 12, and 16 weeks. In these long-term experiments, an additional single dose of FGF-9 was given twice a month (intravenously (i.v)). If there was no change, the levels of FGF-9 in the blood circulation were measured every 2-3 weeks. If FGF-9 levels were equal to or below the levels present in control animals, then additional FGF-9 doses were administered. Animals were euthanized, hearts are removed, and subjected to histological (H&E) and immunohistochemistry analysis. DETERMINE TOTAL NUMBER OF MONOCYTES, Ml MACROPHAGES, AND M2 MACROPHAGES FOLLOWING MI

[0212] Serial heart sections were immunostained with (i) a mouse-specific CD 14 antibody to determine monocytes (Abbiotec), (ii) an iNOS antibody (Abeam) to determine Ml macrophages, and (iii) a CD206 antibody (Abeam) to determine M2 macrophages. This immunostaining determined the presence of these cell types following MI. Confocal and fluorescence microscopy were used to acquire images and the NIH image J program is used for analysis.

[0213] To determine the number of infiltrated monocytes that have started polarization into M1/M2 macrophages, BrdU (50 mg/kg body weight, i.p. (Beltrami et al, 1997, Hosoda et al, 2009)) wass administered, which identifies nuclei in S phase to label actively dividing cells. Active cell growth of infiltrated monocytes was identified using double-label immunostaining for monocyte specific marker CD 14 and BrdU (Dako) antibodies. CD 14 staining identified infiltrated cells BrdU staining labels DNA synthesizing cells. The number of cells that have started cell division and cell type polarization at a given time was also determined. Ki-67 is a nuclear antigen associated with cell division and labels proliferating cells (G1-, S-, G2 -phase, and mitosis), but not in quiescent or resting cells (GO-phase). Therefore, double-label immunostaining using a Ki-67 antibody (Dako) identifies proliferating cells and CD 14 antibodies identify monocyte infiltrated cells.

[0214] Monocyte infiltration and polarization was evaluated. Monocytes were identified using the CD14 specific marker at different times post-MI. Cell differentiation (Ml vs. M2 macrophages) was determined using double-label immunostaining to detect monocytes that have been polarized into Ml and M2 cell types. Sections were stained for FGF-9R and combined with an Ml or M2 macrophage cell-specific staining to determine whether monocytes containing FGF-9R have converted into specific phenotypes. If cells were double positive, then then cells were in the process of polarization. Sections were then stained with either FITC or rhodamine-conjugated secondary antibodies and counterstained with DAPI for nuclear visualization. The percentage of cells was calculated. In brief, the total number of positive cells specific for monocytes/Ml or M2 macrophage marker were counted, and then divided by the total number of DAPI nuclei and multiplied by 100. Slides were analyzed using Olympus fluorescence and confocal microscopy.

[0215] Additional experiments were performed using standard Ml and M2 specific markers such as iNOS, MCP-1, and TNF-a to determine Ml macrophages and arginase-1, Fizz, and IL-4 to determine M2 macrophages by using immunostaining/ELISA.

CHARACTERIZE PHENOTYPE SWITCHING

[0216] Phenotype switching was determined by double labeling. iNOS is used to identify Ml macrophages and CD206 and/or arginase 1 for M2 macrophages. If phenotype switching is happening, then some of the Ml marker specific positive cells also stained positively for CD206 or arginase 1 marker. Also, the total number of Ml macrophages decreasds in the area at a particular time point, indicating that Ml macrophages were polarizing into M2 macrophages. Similarly, if there were significantly reduced amounts of M2 positive cells and staining for the Ml specific marker increases, then it indicated that M2 macrophages are polarizing into Ml macrophages under the same conditions at a particular time point.

DETERMINE LEVELS OF PRO-INFLAMMATORY AND ANTI-INFLAMMATORY CYTOKINES

[0217] A cytokine array ELISA method was used to establish the presence of different cytokines and to determine whether the cytokine profile correlated with the presence of one type of macrophage or another type. For example, pro-inflammatory cytokines IL-6, TNF-a, and MCP-1 released by Ml macrophages and anti-inflammatory cytokines IL-10 and IL-1 receptor antagonist were determined in the blood plasma using commercially available kits from Ray Biotech (Rocher et al, 2012).

[0218] Next, real-time PCR methods (Glass et al, 201 la; 201 lb) were used to determine the mRNA levels of these pro-inflammatory and anti-inflammatory cytokines in the macrophage- rich area. Primers specific for these cytokines were designed using Applied Biosystems Primer Express software V2.0.

[0219] Alterations in these cytokines at the protein level following FGF-9 treatment were examined using the standard technique of Western blotting (Glass et al, 201 1a, 2011b; Yan et al, 2013; Singla et al, 2008). MEASURE EXPRESSION OF Ml MACROPHAGE-SPECIFIC AND M2 MACROPHAGE-SPECIFIC GENES FOLLOWING FGF-9 TREATMENT

[0220] Standard protocols were used for the isolation of cytoplasmic RNA (RNAeasy kit) to determine integrity of RNA using Agilent bioanalyzer, which is converted into cDNA (Glass et al, 2011a, 2011b; Yan et al, 2013). Commercially available affymetrix arrays (mouse genome 430 2.0 arrays, Affymetrix, CA) are used for whole genome expression array analysis per manufacturer instructions. Appropriate controls were applied. These data indicate that treatment of db/db mice with FGF-9 induced a specific gene expression pattern that is significantly different compared with respective controls. Highly up-regulated or down-regulated specific genes following FGF-9 data were confirmed with real-time PCR and then with ELISA or western blotting (if needed). Ml macrophage-specific iNOS, IL-Ιβ, and TNF-a gene expression was evaluated using real-time PCR. Arginase-1 gene (specific for M2 macrophages) was also determined. As M-CSF and GM-CSF were involved in Ml and M2 macrophage polarization, specific genes of M-CSF and GM-CSF were identified, confirmed with real time-PCR, and confirmed at the protein level with western blotting.

DETERMINE EFFECTS OF FGF-9 ON INFARCT SIZE, APOPTOSIS, FIBROSIS, AND LEVELS OF GLUCOSE AND INSULIN

[0221] Myocardial infarct size was determined using the tetrazolium method, apoptosis by TUNEL staining combined with caspase 3 staining and activity, and the amount of fibrosis (Yan et al, 2011; Singla et al, 2007, 2011a, 2011b, and Yan et al, 2013). Blood samples were collected to perform plasma cytokine and gene expression. Moreover, levels of blood glucose and insulin were determined (Varma et al, 2012; Yan et al, 2013; Neel et al, 2011) , ASSESS CARDIAC FUNCTION

[0222] Echocardiography is an accurate, noninvasive tool for determining quantitative characterization of heart function (Yan et al, 2011; Singla et al, 2007, 201 la, 201 lb; Neel et al, 2011) Echocardiography was performed on hearts at short-term time points (24 hrs and 14 days) and long-term time points (4, 12, and 16 weeks) following MI.

DETERMINE THE EFFECT OF FGF-9 ON DISRUPTED FGF-9R MONOCYTES

[0223] Monocytes were isolated from two different sources: (1) Bone marrow obtained from femurs, and (2) peripheral blood of 10-12 week old db/db mice (Houthuys et al, 2010; Tamaki et al, 2008). Isolated monocytes were transfected with FGF-9R specific sequences, which are identified using the ABI system. Sequence-specific siRNA was generated and transfected into these cell populations. FGF-9 and FGF-9R sequences are known to those skilled in the art. For example, NCBI Reference Sequence No. NM_002010.2 represents the nucleotide sequence for homo sapiens fibroblast growth factor 9 (FGF-9). Disruption of FGF- 9R was confirmed using RT-PCR and Western blot analysis. Selected cells were isolated and maintained in the cell culture system.

[0224] Cells were maintained in a standard cell culture medium that consists of RPMI supplemented with fetal calf serum and macrophage colony-stimulating factor. The effects of specific FGF-9 at 50 ng/mL were evaluated. Cells were treated with FGF-9 at various times up to 96 hrs to establish the presence of M2 polarized macrophages. The data generated from this set of experiments established the effects of interactions between FGF-9 and its receptor for M2 polarized macrophages. A FGF-9 specific antibody, which forms a complex with FGF-9 protein preventing any binding, was also applied.

[0225] Some of the experiments in non-transfected monocytes were performed to understand the effects of M-CSF. M-CSF was removed from cell culture medium and FGF-9 alone was added. Immunohistochemistry, pro-and anti-inflammatory cytokine levels, protein levels by Western blotting, and by gene expression were performed and/or obtained.

DETERMINE WHETHER FGF-9 ACTS VIA M-CSF AND INDUCES MONOCYTES INTO M2 POLARIZED MACROPHAGES

[0226] Whether FGF-9 independently causes M2 macrophage polarization from monocytes or whether FGF-9 acts in synergy with M-CSF is examined.

DETERMINE THE EFFECTS OF FGF-9 AND M-CSF ON M2 MACROPHAGE POLARIZATION IN VIVO

[0227] Heterozygous mice from op mice (mice specific for M-CSF deletion) and db/db mice were generated using standard protocols (Asakura et al, 1997; Babamusta et al, 2006; Smith et al., 1995). Commercially available op male mice and db/db female mice were obtained (e.g., Jackson Laboratories). These mice were backcrossed 10 times and genotyping is performed using standard PCR methods. (Asakura et al, 1997; Babamusta et al, 2006; Smith et al, 1995). Animals were divided into four groups: (1) Sham, (2) MI, (3) MI + FGF-9, and (4) MI + FGF-9 + M-CSF (n = 8-10 per group). The fourth group enabled an understanding of whether intravenously provided M-CSF restores monocyte function in heterozygous mice and demonstrates enhanced M2 polarization when compared with Group 3 (MI + FGF-9). This established whether FGF-9 and M-CSF were both necessary. FGF-9 (100 ng/kg per injection; intramyocardially) was injected on the day of surgery. For long term experiments, an additional dose using intravenous injection was given. Animals were euthanized at different times as set forth herein. M-CSF (250 μg/kg per injection; i.v., Asakura et. al.) was injected in a similar fashion as used for FGF-9 post-MI. Specifically, M-CSF was given 24 hrs prior to the day of surgery and at any other given times for FGF-9. After euthanization, hearts were removed, and subjected to histological (H&E) and immunohistochemistry analysis. Both FGF-9 and M-CSF plasma concentrations were examined using ELISA (Ray Biotech). Echocardiography wass used to determine heart function.

DETERMINE EFFECTS OF FGF-9 AND M-CSF ON M2 MACROPHAGE POLARIZATION IN CELL CULTURE

[0228] The effect of FGF-9 on THP-1 and primary human monocytes cells was evaluated. To understand the direct effects of FGF-9 and M-CSF in the THP-1 cell culture model, cells were divided into four groups: (1) Control cells, (2) Cells + FGF-9, (3) Cells + FGF-9 + antibody (FGF-9 specific antibody to inhibit its action), and (4) Cells + FGF-9 + soluble form of M-CSF receptor or M-CSF specific antibodies (Kutza et al., 2000). Data were examined at different times up to 96 hrs. The number of M2 polarized macrophages was determined using immunostaining and secreted IL-10 levels were measured. Released levels of M-CSF were obtained using ELISA, gene expression assays, and assays for protein levels.

DETERMINE THE ROLE OF FGF-9 AND MECHANISMS OF M2 POLARIZED MACROPHAGES

[0229] The mechanisms by which monocytes are differentiated into M2 macrophages following MI were examined. The post-MI animals (both db/db mice and heterozygous mouse models) were treated with FGF-9 or specific inhibitors for the PI3K activated-YAP (HIPPO) pathway and RAS, MAP Kinase pathways. MI was induced. Hearts were isolated and homogenatesweare obtained. Cell culture systems were also utilized. (Singla et al, 2008; Assi et al, 2006; Bradley et al, 2008; Dhingra et al, 2007; Su et al, 2010; Razzaque et al, 2013; Mor et al, 2011 ; Serova et al, 2013). Cell lysates were prepared as described herein. M2 macrophage polarization and the levels of pro-inflammatory and anti-inflammatory cytokines were determined.

[0230] SDS-PAGE and Western blot analyses were used to determine the protein expression of PI3K-YAP, RAS, MEK1/2, p38, ERK, and ΓΝΚ (Singla et al, 2008; Assi et al, 2006; Bradley et al, 2008; Dhingra et al, 2007; Su et al, 2010; 2013; Mor et al, 201 1; Serova et al, 2013). Both total and phosphorylated antibodies for each specific protein were utilized (Singla et al, 2008; Assi et al, 2006; Bradley et al, 2008; Dhingra et al, 2007; Su et al, 2010; Razzaque et al, 2013; Mor et al, 201 1 ; Serova et al., 2013). Densitometry was used to measure band density. ELISA was performed with different kits available from Sigma and Ray Biotech Inc. to measure p38, ERK, and ΓΝΚ in the isolated hearts and cell culture models. To determine the role of PI3K-YAP, RAS, MEK1/2, p38, ERK, and ΓΝΚ in M2 polarized macrophages, animals were given selective inhibitors of PI3K (BKM120, 50 mg/kg), YAP in HIPPO pathway, RAS (FTS, 60 mg/kg), MEK1/2 (U0126, 5 mg/kg, i.p.), p38 (SB203580, 50 mg/kg) (Derynck et al, 2003), ERKl/2 (PD98059, 20 μg/kg i.p.), and J K (BI-78D3, 25 mg/kg) pathways (Singla et al, 2008; Assi et al, 2006; Bradley et al, 2008; Dhingra et al, 2007; Mor et al, 2011; Serova et al., 2013). Cell culture doses for each inhibitor were established.

DETERMINE WHETHER M-CSF INDUCED M2 POLARIZED MACROPHAGES ALSO INVOLVE PI3K ACTIVATED-YAP PATHWAY

[0231] Whether FGF-9 and M-CSF have a common pathway for M2 polarized macrophages was examined. Specifically, the PI3K activated-YAP pathway and the Ras/MAP kinase were examined. As discussed herein, FGF-9 acted via the PI3K-YAP pathway in monocytes and M-CSF activated the Ras pathway in osteoclast cells (Bradley et al, 2008). THP-1 cells were used to establish the relevant pathway. Specific inhibitors for the PI3K activator (BKM120) were employed (Serova et al., 2013). Specific inhibitors for the Ras pathway (Ras inhibitor, FTS) were also utilized (Mor et al, 2011). Another associated TEAD activated YAP was examined (Liu-Chittenden et al, 2012). Activation and inhibition of these pathways awere confirmed by ELISA and Western blot methods.

DETERMINE WHETHER INHIBITION OF PI3K-YAP OR RAS PATHWAYS INFLUENCE M2 POLARIZED MACROPHAGES

[0232] Whether inhibition of a FGF-9 specific or a M-CSF specific signaling pathway has an effect on the polarization of M2 macrophages, specific inhibitors for both PI3K-YAP and Ras pathways were utilized. These inhibitors were used in independent set of experiments with respective controls. Groups are divided as follows: (1) THP-1 cells / Primary monocytes, (2) THP-1 cells / Primary monocytes + BKM120 (PI3K inhibitor that blocks YAP pathway), and (3) THP-1 cells / Primary monocytes + FTS (Ras inhibitor). The number of M2 polarized macrophages, the levels of cytokines, and the amount of various pathway-specific proteins were determined using ELISA and Western blot methods.

3. EXPERIMENT 2

[0233] Diabetes is a group of metabolic disorders that results in chronic hyperglycemia. Symptoms include abnormal metabolism of lipids, carbohydrates, and proteins and may also involve impaired insulin secretion (Alberti et al, 1998). Two major classifications of diabetes exist: (1) Type 1 Diabetes Mellitus (T1DM); and (2) Type 2 Diabetes Mellitus (T2DM). T1DM is regarded as an autoimmune condition that involves the destruction of pancreatic β- cells and may lead to total insulin deficiency (Alberti et al, 1998). T2DM accounts for the majority of diabetic patients in the United States (Blonde 2012) and manifests as insulin resistance despite normal pancreatic β-cell function. More recently, the Word Health Organization introduced pre-diabetes (PD) to include patients with impaired fasting glucose (IFG) and impaired glucose tolerance (IGT) (Bergman 2010). Diabetes increases risk for conditions such as cardiovascular disease (D'Souza et al, 2011) and nephropathy (Stolar 2010).

[0234] The exact source of increased immunoregulation and associated oxidative stress in diabetes is complex and not well understood. Monocytes are the first cell subtype to infiltrate the pancreas in rodent models of PD (Cifarelli et al., 2007). Moreover, increased immune cell infiltration and monocytic activity has been reported in diabetic patients (Cifarelli et al, 2007; Bardini et al, 2010).

[0235] Whether monocyte infiltration occurred in the pre-diabetic (PD) hearts and its role in PD cardiomyopathy were examined. The PD heart was significantly populated with monocytes and BMP-7, a mediator of monocyte polarization, activated infiltrated monocytes into anti-inflammatory M2 macrophages, thereby inhibiting apoptosis and fibrosis and improving cardiac function. C57B16 mice were assigned to control, PD, or PD + BMP-7 groups. PD and PD + BMP-7 groups were administered STZ (50 mg/kg) whereas control animals received sodium citrate buffer. Afterwards, the PD + BMP-7 group was administered BMP-7 (200 μg/kg) for three days. The data show significantly increased the number infiltrated monocytes and associated pro-inflammatory cytokines, adverse cardiac remodeling, and heart dysfunction in the PD group (p < 0.05). In the PD + BMP-7 group, M2 macrophage polarization and associated anti-inflammatory cytokines were enhanced, adverse cardiac remodeling was reduced, and cardiac function improved (p < 0.05). These data indicate that PD cardiomyopathy was associated with increased monocyte infiltration and release of pro-inflammatory cytokines, which contributed to adverse cardiac remodeling and cardiac dysfunction. Moreover, BMP-7 possessed therapeutic potential in its ability to polarize monocytes into M2 macrophages and confer cardiac protection in the PD heart.

[0236] Increased monocyte infiltration can be present along with an increased inflammatory immune response, which contributes to adverse cardiac remodeling, in streptozotocin (STZ) induced cardiomyopathy. In addition, the role of BMP-7 in the polarization of proinflammatory monocytes into anti-inflammatory M2 macrophages and associated inhibition of adverse cardiac remodeling in the PD heart was examined. The data show that there were increased numbers of infiltrated monocytes, enhanced pro-inflammatory cytokine expression, and increased adverse cardiac remodeling in the PD group. Treatment with BMP-7 polarized and differentiated monocytes into an enhanced number of M2 macrophages and stimulated the secretion of anti-inflammatory cytokines as well as inhibits oxidative stress in the PD heart. Moreover, the data indicates that there was attenuated cardiomyocyte apoptosis, fibrosis, and improved cardiac function following BMP-7 treatment.

RESEARCH DESIGN AND METHODS

[0237] Streptozotocin-Induced Pre-Diabetes. C57B1/6 mice, 8-10 weeks old, were maintained in a controlled environment and given all appropriate nutritional support ad libitum. The mice were separated into three groups consisting of control, Pre-diabetic (PD), and PD + BMP-7 (n = 7-10 animals/group). Animals were administered 50 mg/kg of STZ via intraperitoneal (i.p.) injections for two days (day 1 (Dl) and D2) in the PD and PD + BMP-7 groups. For the PD + BMP-7 group, animals were additionally treated with an intravenous (i.v.) injection of 200 μg/kg BMP-7. Moreover, control animals were administered sodium citrate buffer injections on Dl and D2. Animals were sacrificed humanly with 4% isoflourane for 10 minutes followed by cervical dislocation.

[0238] Tissue Processing. Hearts from each group were harvested, washed with phosphate buffer saline (PBS), and stored in either RNA Later or formalin solutions for future studies. Formalin-fixed hearts were processed with the Leica TP 1020 system and embedded in paraffin wax with a Tissue Tek TEC machine. 5 μιη sections were cut and placed on plus- plus slides for histochemical studies.

[0239] Immunohistochemistry to Identify Monocytes, Macrophages, and Pro-Inflammatory and Anti Inflammatory Cytokines. Heart sections were deparaffinized and rehydrated as previously described (Singla et al, 2007). Sections were processed for heat-induced epitope retrieval for 20 minutes and then blocked with 10% normal goat serum (Vector Labs) for one hour. Primary antibodies, which included such as CD 14 to identify monocytes, CD206 to identify M2 macrophages, IL-6 and TNF-alpha to identify pro-inflammatory cytokines, and IL-10 to identify anti-inflammatory cytokines, were incubated overnight at 4 °C. Tissues were washed and incubated with appropriate secondary antibodies including goat anti-rabbit Alexa 488, goat anti-rabbit Alexa 568, or goat anti-rat Alexa 594, for one hour at room temperature. Sections were counter stained with mounting medium containing-DAPI (4', 6- diamidino-2-phenylindole) (Vector Labs), and the slides were cover-slipped. Four images per section were taken for data quantification using an Olympic IX-70 fluorescent microscope and representative images were captured using confocal microscopy. Cells positive for CD 14 and CD206 were counted, divided by the total number of nuclei and expressed as a percent of positive cells/section. IL-10, IL-6, and TNF-a intensities were measured and the average corrected integrated density was calculated for each animal using ImageJ software. [0240] Determination of Pro-Inflammatory and Anti-inflammatory Cytokines by ELISA. Whole blood was collected from the jugular vein and serum was separated by centrifugation at 13,000 rpm for 10 minutes. Commercially available ELISA kits were purchased from Raybiotech. Instructions provided in the kits were strictly followed and were used to determine levels of pro-inflammatory cytokines (TNF-a and IL-6) and anti-inflammatory cytokines (IL-10 and IL-1RA).

[0241] Determination of Oxidative Stress by Western Blotting and Dihydroethidium Staining. SDS-PAGE and Western blotting were used to analyze MnSOD expression. For dihydroethidium (DHE) staining, heart sections were stained with 1 μΜ/mL dihydroethidium for 25 minutes as described in Neel et al, 201 1. DHE intensity was then measured in four random images per section and the average corrected integrated density was calculated for each section using ImageJ software.

[0242] Terminal Deoxynucleotidyl Transferase dUTP-Mediated Nick-End Labeling Staining. In brief, heart sections were deparaffinized, rehydrated, and incubated with proteinase-K for antigen retrieval. Terminal deoxynucleotidyl transferase dUTP-mediated nick-end labeling (TUNEL) staining was performed using a cell death detection kit (Roche) following manufacturer's protocol. The sections were then mounted, cover-slipped, and four images per section were quantified utilizing NIH ImageJ software. TUNEL positive (red) cells that merged with nuclei were counted, divided by total nuclei, and multiplied by 100.

[0243] Determination of Apoptotic Proteins by Western Blot. Heart samples were prepared, supernatants collected, and standard sodium dodecyl sulfate polyacrylamide gel electrophoresis was performed. Beta actin was used as a loading control. Proteins were transferred onto PVDF membranes and blocked with 5% milk. Membranes were incubated with primary antibodies, phosphorylated and total AKT and phosphorylated and total PTEN, for one hour at room temperature or overnight at 4 °C. Membranes were then washed and incubated with secondary antibody, goat anti-rabbit HRP, for one hour at room temperature. Membranes were then washed and incubated with an enhanced chemiluminescent substrate (ECL) for 2 minutes and exposed at various times. Blots were scanned and band intensities were measured using ImageJ Software.

[0244] Masson's Trichrome Staining. Sections were stained with Masson's Trichrome staining as previously described (Singla et al, 2007). Using ImageJ software, collagen deposition was measured to quantify fibrotic area (mm²). Interstitial fibrosis was measured by quantifying collagen in three areas/sections while vascular fibrosis was quantified as vessel fibrosis/total vessel area x 100 in six vessels per section. [0245] Determination of Cardiac Function. 3 weeks post-D5, 2D transthoracic echocardiography was performed with a 5500 Ultrasound System. Using a 15-6L Hockey stick transducer, M-mode images of the left ventricle were captured and used to calculate end diastolic volume (EDV), end systolic volume (ESV), ejection fraction (EF%), left ventricular internal dimension-diastole (LVIDd), left ventricular internal dimension-systole (LVIDs), and fractional shortening (FS%). Following heart function assessment, mice were sacrificed with pentobarbital (80 mg/kg) followed by cervical dislocation.

[0246] Statistical Analysis. Statistical analyses of all data were performed utilizing one-way analysis of variance (ANOVA) followed by the Tukey Test. Data are presented as a mean ± SEM with p-value < 0.05 considered statistically significant.

[0247] Results

INCREASED MONOCYTE INFILTRATION AND INFLAMMATORY CYTOKINES IN PD MOUSE HEARTS

[0248] To determine whether inflammatory cells have a direct relationship with the PD heart, infiltrated monocytes were quantified. FIG. 16A shows representative photomicrographs of heart sections stained with CD 14, a marker for monocytes, (a, f, and k), cardiac myocytes (b, g, and 1), nuclei (c, h, and m), merged images (d, i, and n), and enhanced merged images (e, j, and o). Quantitative analyses indicate a significant up-regulation of monocytes in the PD group relative to controls (p < 0.05, FIG. 16B). Conversely, monocytes were significantly reduced upon BMP-7 treatment compared to the PD group (PD + BMP-7: 1.051 + 0.185% vs. PD: 1.759 ± 0.238%, CD14 positive cells/total nuclei, FIG. 16B). No significant difference in the monocytic population was observed between the control and PD + BMP-7 groups (FIG. 16B).

[0249] Next, the consequences of monocyte infiltration and associated pro-inflammatory cytokines were evaluated. FIG. 16C demonstrates representative photomicrographs of heart sections from each group stained with TNF-a (a, f, and k), sarcomeric a-actin (b, g, and 1), and DAPI (c, h, and m). Quantification of stained tissues indicated significantly augmented TNF-a levels in the PD group relative to controls (p < 0.05, FIG. 16D). However, treatment with BMP-7 attenuated levels of TNF-a compared to the PD group (PD + BMP-7: 3.023 + 0.655 A.U. vs. PD: 5.307 ± 0.387 A.U., p < 0.05, FIG. 16D). Although not statistically significant, similar TNF-a expression patterns resulted via TNF-a ELISA analyses using isolated serum (FIG. 16E).

[0250] Levels of the pro-inflammatory cytokine, IL-6, were also measured. FIG. 17A contains representative images of heart sections from each group stained with IL-6, sarcomeric α-actin, and DAPI. Quantitative analysis of PD tissues showed up-regulated IL-6 levels compared to controls (p < 0.05, FIG. 17B). Conversely, significantly decreased IL-6 levels in PD + BMP-7 hearts relative to the PD group were observed (PD + BMP-7: 2.984 + 0.577 A.U. vs. PD: 5.495 ± 0.367 A.U., p < 0.05, FIG. 17B). In addition, an IL-6 ELISA on serum samples from control and experimental groups was performed. The results indicated increased levels of IL-6 in the PD group as compared to controls and that PD + BMP-7 had significantly decreased levels compared to the PD group (p < 0.05, FIG. 17C). Notably, no statistical deviation in IL-6 expression was noted between control and PD + BMP-7 groups (FIG. 17B and FIG. 17C).

BMP-7 AMELIORATED CARDIAC OXIDATIVE STRESS BY UP-REGULATING MNSOD

[0251] To determine the effects of BMP-7 on oxidative stress in the PD heart, superoxide anion production was assessed. FIG. 18A portray representative photomicrographs of heart tissue stained with DHE (a, d, and g), DAPI (b, e, and h), and merged images (c, f, and i). Analysis revealed that PD hearts had significantly increased reactive oxygen species (ROS) relative to controls (p < 0.001, FIG. 18B). Conversely, BMP-7 administration dramatically reduced levels of ROS compared to the PD group (PD + BMP-7: 3.667 + 0.333 A.U. vs. PD: 8.167 ± 0.872 A.U., p < 0.001, FIG. 18B). MnSOD expression was also quantified through western blot analysis with representative blots and beta actin controls illustrated in FIG. 18C. Densitometric analysis indicated decreased expression of MnSOD in the PD group relative to controls (p < 0.05, FIG. 18C). However, treatment with BMP-7 blunted diminished MnSOD expression as compared to the PD group (PD + BMP-7: 0.888 ± 0.0608 A.U. vs. PD: 0.630 ± 0.0550, p < 0.05, FIG. 18C).

BMP-7 INHIBITED APOPTOSIS IN PRE-DlABETIC MICE BY REGULATING PTEN AND AKT

[0252] To determine the effects of BMP-7 treatment on cardiomyocyte apoptosis in vivo, TUNEL staining was performed. FIG. 19A shows representative photomicrographs of heart sections from each group stained with TUNEL (a, d, and g), total nuclei (b, e, and h) and merged images (c, f, and i). Data indicate increased apoptosis in PD hearts relative to controls (p < 0.001, FIG. 19B). Moreover, analysis revealed a significantly decreased percentage of apoptotic nuclei in the PD + BMP-7 group relative to the PD group (PD + BMP-7: 0.286 + 0.0442% vs. PD: 0.505 ± 0.0369%, TUNEL positive nuclei/total nuclei, p < 0.001, FIG. 19B). To demonstrate apoptosis occurred in the cardiomyocytes, sections were stained with TUNEL, sarcomeric a-actin, caspase-3, and DAPI and representative images from the PD group are shown in FIG. 19C. [0253] To elucidate mechanisms of blunted apoptosis noted in the PD + BMP-7 heart, the expression of phosphatase and tensin homo log (PTEN), an inhibitor of PI3K/AKT activation, and AKT, a pro-survival and cell cycle regulator, were evaluated. Densitometric analysis of phosphorylated-PTEN (p-PTEN) western blot bands revealed a significant increase in the expression of activated PTEN in the PD group relative to controls (p < 0.05, FIG. 19D). A significant decrease in p-PTEN expression was observed in the PD + BMP-7 group relative to the PD group (PD + BMP-7: 0.784 + 0.123 A.U. vs. PD: 1.305 ± 0.134 A.U., p < 0.05; FIG. 19D). Inversely proportional to the p-PTEN data, significantly diminished levels of p-AKT in PD hearts relative to controls (p < 0.05) and considerably up-regulated levels of the survival protein in the PD + BMP-7 group compared to the PD group were reported (PD + BMP-7: 0.715 ± 0.0784 A.U. vs. PD: 0.500 ± 0.107 A.U., p < 0.05, FIG. 19E).

BMP-7 AMELIORATED CARDIAC REMODELING IN PRE-DIABETIC MICE

[0254] To assess the influence of BMP-7 on fibrosis formation in the PD heart, heart tissue from each of the study groups were analyzed for collagen deposition using Masson's Trichrome staining. FIG. 20A shows representative photomicrographs from control and experimental groups demonstrating interstitial fibrosis (top panels, a-c) and vascular fibrosis (bottom panels, d-f). Quantitative analyses indicate interstitial fibrosis was dramatically increased in the PD group relative to the control group (p < 0.001, FIG. 20B). However, following BMP-7 treatment, interstitial fibrosis was significantly blunted relative to the PD group (PD + BMP-7: 0.110 ± 0.0222 mm² vs. PD: 0.170 + 0.0159 mm², p < 0.05, FIG. 20B). Fibrosis in the PD group was significantly elevated relative to controls (p < 0.001, FIG. 20C), whereas the addition of BMP-7 significantly abrogated vascular collagen deposition in the PD heart (p < 0.05, FIG. 20C).

BMP-7 AUGMENTED M2 MACROPHAGES AND REDUCES INFLAMMATION IN THE PD HEART

[0255] In order to elucidate mechanisms by which BMP-7 promotes beneficial effects in the PD heart, M2 macrophage and associated anti-inflammatory cytokine expression were evaluated. Representative images of heart sections stained with CD206, an M2 macrophage marker, sarcomeric a-actin, and DAPI are shown in FIG. 21 A. No difference in M2 macrophage concentration was observed between the control and PD groups. However, results showed significantly elevated M2 macrophages in PD + BMP-7 hearts as relative to the PD group, indicating that BMP-7 may play a role in monocyte to M2 macrophage polarization (PD + BMP-7: 2.005 + 0.131% vs. PD: 1.253 + 0.175% CD206 positive cells/total nuclei, p < 0.05, FIG. 2 IB). [0256] The levels of interleukin-1 receptor antagonist (IL-1RA) and interleukin-10 (IL-10), critical mediators of inflammation and immunoregulation, were evaluated. Quantitative analysis of IL-1RA concentration in serum samples indicated a significant decrease in the PD group (p < 0.05, FIG. 21C), which was considerably increased upon BMP-7 treatment (p < 0.05, FIG. 21C). IL-10 analyses performed on heart sections stained with IL-10, sarcomeric a-actin, and DAPI (FIG. 2 ID) indicated an observable decrease in IL-10 levels in the PD group relative to controls, which was significantly increased upon treatment with BMP-7 (FIG. 2 IE). To corroborate immunocytochemistry data, an IL-10 ELISA was performed on serum samples. Significantly reduced levels of IL-10 in the PD group as compared to controls and dramatically elevated IL-10 concentrations upon BMP-7 treatment were noted (p < 0.05, FIG. 2 IF).

EXOGENOUS BMP-7 IMPROVED CARDIAC FUNCTION IN PRE-DIABETIC MICE

[0257] Three weeks post-D5, mice underwent transthoracic echocardiography to assess cardiac function. Raw echocardiogram data is illustrated in FIG. 22A. Data presented indicate impaired left ventricular function in the PD group as demonstrated by deviated LVIDd, LVIDs, EDV, ESV, EF, and FS compared to the control (p < 0.001, FIG. 22B-FIG. 22G). A significant improvement in cardiac function was achieved following BMP-7 administration as indicated by augmented FS and EF relative to the PD alone group (p < 0.05, FIG. 22B-FIG. 22G).

[0258] Monocytes, during pathological circumstances, migrate from circulation and infiltrate tissues where they differentiate into macrophages. Monocyte to macrophage polarization and differentiation yields two distinct phenotypes, including Ml and M2 macrophages. Ml macrophages are pro-inflammatory and heighten the inflammatory response; whereas M2 macrophages are anti-inflammatory and express cytokines that mitigate inflammation (Laskin, 2009). The anti-inflammatory effects of BMP-7 were found in the present study correlate to a report by Gould et al. which showed that treatment with BMP-7 inhibited proinflammatory cytokines in proximal tubular epithelial cells (Gould et al, 2002). Evidences provided indicate the potential cardio-protective effects of BMP-7 against inflammation by directing the secretion of inflammatory cytokines. Moreover, BMP-7 administration significantly inhibited cardiomyocyte apoptosis in the PD heart which correlates to a previous study suggesting bone morphogenetic proteins inhibit apoptosis of cardiomyocytes in vitro (Izumi et al, 2001).

[0259] To elucidate mechanisms of cardiomyocyte apoptosis in the PD heart, expression levels of activated PTEN and AKT were quantified. PTEN is a tumor suppressor that regulates the pro-survival protein, AKT, through inhibition of its upstream activator, PI3K. Densitometric analyses indicated elevated p-PTEN expression in PD mouse hearts relative to controls with a concomitant down-regulation in p-AKT. BMP-7 inhibited the expression of cardiac p-PTEN while significantly up-regulating p-AKT in the PD heart. Collectively, these data indicate the presence of enhanced apoptosis in the monocytic infiltrated PD myocardium consequent to augmentation of normal PI3K/Akt signaling which was ameliorated with BMP-7.

[0260] These data indicate the ratio of Ml to M2 macrophages polarization following monocyte differentiation may determine the severity and progression of inflammation (Galkina et al, 2009; Moore et al, 2011 ; Seimon et al, 2009). M2 macrophages are a class of macrophages that reduce inflammation by secreting IL-10, an anti- inflammatory cytokine (Laskin, 2009). Specifically, IL-10 has been reported to modulate inflammation by regulating the secretion pattern of pro-inflammatory cytokines from inflammatory cells (Moore et al, 1993). The present study provides evidence that infiltrated monocytes in the presence of BMP-7 polarize into beneficial M2 macrophages. The anti-inflammatory effects of BMP-7 have been previously reported (Gould et al, 2002). Monocytes express receptors for BMP-7 and in the presence of BMP-7, polarize into beneficial M2 macrophages in vitro (Rocher et al, 2012). BMP-7 promotes the differentiation of mesenchymal stem cells and white adipocytes into brown adipose tissue (Schulz et al, 2011 ; Tseng et al, 2008). Parallel to increased M2 macrophage concentration, BMP-7 administration significantly enhanced IL-10 secretion. IL-10 can mediate TNF-a-induced cardiomyocyte apoptosis (Dhingra et al, 2011). Down-regulated levels of TNF-a were observed upon BMP-7 treatment; therefore, BMP-7 inhibits TNF-a induced apoptosis in PD cardiomyopathy by up-regulating IL-10. In addition, increased levels of another anti-inflammatory cytokine, IL-IRA, were observed in the BMP-7 treatment group. The present studies demonstrated decreased cardiac function in the PD group, which was significantly improved upon treatment with BMP-7. The decreased apoptosis and fibrosis presented in this study contributed to the improvement in cardiac function in the BMP-7 treatment group.

[0261] In conclusion, the PD myocardium was infiltrated with monocytes and subsequent secreted pro-inflammatory cytokines. PD was associated with myocardial oxidative stress, apoptosis, fibrosis, and cardiac dysfunction. In the presence of BMP-7, monocytes polarized into beneficial M2 macrophages, thereby inhibiting inflammation and protecting the PD heart from cardiomyocyte apoptosis, fibrosis, and cardiac dysfunction. 4. EXPERIMENT 3: BMP-7 ENHANCES M2 MACROPHAGE DIFFERENTIATION INTRODUCTION

[0262] Atherosclerosis (ATH) was once considered a bland lipid storage disease characterized by the deposition of lipids on arterial surfaces leading to restrictive and eventual blockage of normal blood flow with consequential cardiovascular events occurring including myocardial infarction (MI) and stroke. The traditional views of ATH have crumbled with accruing and insurmountable evidence pointing to inflammation as a mediator of all stages of the disease, from its inception and development to the end-stage thrombotic complications. Basic science and growing knowledge, when applied to ATH, has afforded new insight into the mechanisms underlying inflammatory-driven pathophysiological atherosclerotic events.

[0263] In general, during inflammation, monocytes migrate to the injured area and differentiate into one of two antagonistic macrophage subtypes. Ml macrophages, or "classically activated" macrophages, promote and enhance the inflammatory response while upregulating a host of characteristic pro-inflammatory cytokines including monocyte chemoattractant- 1 (MCP-1), tumor necrosis factor-alpha (TNF-a), and interlekin-6 (IL-6). Conversely, M2 macrophages, or "alternatively activated" macrophages, are antiinflammatory and secrete factors such as interleukin- 1 receptor antagonist (IL-IRA), interleukin-10 (IL-10) and Arginase 1. Data have indicated M2 macrophages play an important role in inflammation mediation and resolution with resultant tissue repair and wound healing. In this regard, identification of growth factors/small molecules that mediate monocyte differentiation may offer novel therapeutic options for patients suffering from inflammatory propagated diseases including ATH.

[0264] Bone morphogenetic proteins (BMPs), members of the transforming growth factor beta (TGF-a) superfamily, play a role in variegated processes including bone formation, apoptosis, cell fate and differentiation, and embryogenesis. BMP-7 has the potential to direct cellular plasticity, specifically monocytes into M2 macrophages, in vitro when cultured in "inflammation mimicry" media. However, the role of BMP-7 in inflammation mediation in the context of ATH had never been explored. In this regard, ATH was generated in Apo E-/- mice by partial left carotid artery (PLCA) ligation and following BMP-7 treatment, plaque formation, monocyte infiltration, M1/M2 macrophage differentiation outcomes, pro- and antiinflammatory cytokine expression, BMP-7 receptor (BMP-7R) expression on monocytes and M2 macrophages, and blood flow was evaluated. This data indicates BMP-7 inhibits plaque formation, monocyte infiltration, and pro-inflammatory cytokine secretion as well as enhances M2 macrophage differentiation, anti-inflammatory cytokine secretion, BMP-7R expression on monocytes and M2 macrophages, and systolic velocity in a PLCA ligated mouse model of ATH.

MATERIALS AND METHODS

PARTIAL LEFT CAROTID ARTERY (PLCA) LIGATION

[0265] Male and female Apo E-/- mice between 8 and 10 weeks of age were divided into three groups (n=8 animals/group): Sham, PLCA, and PLCA+BMP-7 (200ug/kg; i.v.). Mice were sedated with inhalatory 4% isoflurane via nose cone on a temperature controlled heating pad. A 4-5 mm ventral midline incision was made in the neck and the visualized muscles were separated using blunt dissection with curved forceps to expose the left carotid artery (LCA). Using a surgical light microscope, three branches (left external carotid, internal carotid, and occipital artery) of the LCA were permanently ligated using a 7-0 silk suture. To provide a source of blood circulation, the superior thyroid artery was left intact. The sham group underwent all aforementioned procedures with the exception that they received a loose suture around the LCA instead of a complete ligation. The incision was then sutured and the mouse remained on the heating pad until consciousness was regained. Following surgery, animals were injected intravenously with BMP-7 (200ug/kg of body weight). Injections were repeated for two following days and mice were left to recover for fourteen days following last injection.

PREPARATION OF PARAFFIN SECTIONS AND HISTOPATHOLOGY

[0266] Two weeks post surgery or injections (PLCA-BMP-7), animals were anesthetized and sacrificed via cervical dislocation. The left and right carotid arteries, trachea, and esophagus were harvested, embedded in paraffin, cut into 5 μιη serial sections, and placed on ColorFrost™ slides as previously described. Following deparaffinization in xylene and rehydration by sequential incubation in 100%, 95%, 70%, and 30% alcohol, sections were stained with Masson's Trichrome to determine plaque area under an Olympus light microscope. % plaque area was obtained by measuring atherosclerotic plaque area using Image J software (NIH), divided by artery area, and multiplied by 100.

IMMUNOHISTOCHEMISTRY

[0267] To determine the presence of infiltrated monocytes and Ml and M2 macrophages in the plaque, carotid artery sections, prepared as aforementioned, were boiled in retrieval buffer for 20 min following deparraffinization in xylene. Once cooled to room temperature, the sections were washed in PBS and blocked in 10% normal goat serum (NGS). Sections were then incubated overnight at 4°C with primary antibodies against either CD 14 (monocytes, Abbiotec, #25156, 1 :50), iNOS (Ml macrophages, Abeam, #abl29372, 1 :50), or CD206 (M2 macrophages, Abeam, #ab64693, 1 :50). To detect BMP-7 receptor (BMP-7R) expression on monocytes and M2 macrophages, previously stained CD 14 and CD206 sections were double stained with BMPRII (Santa Cruz, #sc-5683, 1 :50). Following washings in PBS, slides were incubated with appropriate secondary antibodies for one hr at room temperature. Sections were mounted with Vectashield containing DAPI (4', 6-diamidino-2-phenylindole) (Vector Labs) and cover-slipped. LCA artery images (1-2 sections, n=5 animals/group) were taken for data quantification using an Olympic IX-70 fluorescent microscope and representative images were captured using a confocal microscope. The % Monocytes/Artery was calculated by counting total CD 14 positive cells on the artery/total arterial DAPI x 100. The % Ml Macrophages/Artery was calculated by counting total iNOS positive cells on the artery/total arterial DAPI x 100. The % M2 Macrophages/Artery was calculated by counting total CD206 positive cells on the artery/total arterial DAPI x 100. The % of BMPRII on monocytes was calculated by counting double positive CD14/BMPRII cells on the artery/total CD 14 positive cells x 100. The % of BMPRII on M2 macrophages was calculated by counting double positive CD206/BMPRII cells on the artery/total CD206 positive cells x 100.

PRO- AND ANTI-INFLAMMATORY CYTOKINE ANALYSIS VIA ELISA

[0268] Pro-inflammatory cytokines (RayBiotech, MCP-1 #ELM-MCP-001 and TNF-a #ELM-TNFalpha-001 ), anti-inflammatory cytokines (RayBiotech, IL-10 #ELM-IL 10-001 and IL-1RA #ELM-IL1RA-001), and BMP-7 expression (TSZ Scientific, #M7485) were quantified using commercially available ELISA kits, per manufacture's instructions, on blood serum samples previously collected from each animal. In brief, blood from each animal was collected post-sacrifice in EDTA coated tubes and centrifuged at 13000 rpm for 15 min. Supernatant containing serum was collected into separate tubes and stored at -20°C for future analysis. For each ELISA, serum was loaded into pre-coated 8-well strips for 2.5 hrs at room temperature. After washing, the detection antibody (biotinylated anti-mouse antibodies) was added followed by HRP -conjugated streptavidin and TMB One-Step substrate reagent, as provided in the kit. When the blue color developed, stop solution was added and the wells were measured at 450 nm in a microtiter plate reader (Bio Rad). Results were normalized to protein concentration and data is presented as arbitrary units (A.U.). RT-PCR

[0269] Total RNA was isolated from carotid artery homogenates using TRI® Reagent RNA

Isolation Reagent (Sigma-Aldrich) and retro transcribed into cDNA using iScript™ Select cDNA Synthesis Kit (Bio-Rad). Real-time PCR was carried out with a CI 000™ Thermal

Cycler (Bio-Rad) using SYBR Green (Bio-Rad). TNF-a, IL-6, and Ariginase-1 (Argl) were normalized against GAPDH. Primers used for RT-PCR were as follows:

TNF-a: sense primer, 5'-CACAGAAAGCATGATCCGCGACGT-3'; (SEQ ID NO: 1)

Antisense primer, 5 '-CGGCAGAGAGGAGGTTGACTTTCT-3 '; (SEQ ID NO: 2)

IL-6: sense primer, 5 '-TCCAGTTGCCTTCTTGGGAC-3 '; (SEQ ID NO: 3)

Antisense primer, 5 '-GTACTCCAGAAGACCAGAGG-3 '; (SEQ ID NO: 4)

Argl : sense primer, 5'-CTCCAAGCCAAAGTCCTTAGAG-3';(SEQ ID NO: 5)

Antisense primer, 5 '-AGGAGCTGTCATTAGGGACATC-3 ';(SEQ ID NO: 6)

GAPDH: sense primer, 5'-AACGACCCCTTCATTGAC-3';(SEQ ID NO: 7)

Antisense primer, 5'-TCCACGACATACTCAGCAC-3';(SEQ ID NO: 8)

[0270] Final data are presented as relative fold increase against sham.

SYSTOLIC VELOCITY

[0271] A Phillips Sonos 5500 Ultrasound system was used to assess systolic blood velocity of the left common carotid artery at D14 post ligation. Briefly, mice were anesthetized under 2-4% isoflurane and 2% oxygen via nose cone. Two-dimensional (2D) images were recorded in the long axis projection using a guided M-mode with the ultrasound probe lying on the left side of the neck. Under Doppler vascular settings, three to four 2D images for systolic (SV; cm/s) blood velocity were obtained and averaged.

STATISTICAL ANALYSIS

[0272] Data was analyzed using One-way analysis of variance (ANOVA) followed by the Tukey Test. Data is presented as a mean ± SEM and statistical significance assigned when p<0.05.

RESULTS

BMP-7 INHIBITS ATHEROSCLEROTIC PLAQUE FORMATION IN APO E-/- MICE

[0273] Masson's Trichrome stained sections were examined to determine the extent of plaque formation as a determinant of atherosclerotic lesions in all control and experimental groups (Figure 23A). Two weeks following permanent PLCA ligation, there was a significant increase in plaque formation in the PLCA group relative to sham controls (p<0.05, Figure 23B). Notably, when additionally treated with BMP-7, the extent of plaque formation was significantly abrogated compared with the PLCA alone group (p<0.05, Figure 23B). BMP-7 INHIBITS MONOCYTE INFILTRATION FOLLOWING PLCA LIGATION

[0274] To extrapolate mechanisms by which BMP-7 inhibits plaque formation post-PLCA ligation, inflammatory cells, specifically monocytes, were quantified. Figure 24A contains representative photomicrographs of carotid artery sections stained with CD 14, a marker for monocytes, (top row), DAPI, for nuclei, (second row from top), merged images (third row from top), and enhanced merged images (bottom row). Quantitative analysis indicates a significant upregulation of infiltrated monocytes following PLCA ligation relative to the sham group (p<0.05, Figure 24B). Conversely, monocyte infiltration was significantly reduced upon BMP-7 treatment compared to the PLCA group (p<0.05, Figure 24B).

Ml MACROPHAGE DIFFERENTIATION ENHANCED POST-PLCA LIGATION IN APO E-/- MICE

[0275] To evaluate monocytic reprogramming outcomes post-infiltration into the atherosclerotic lesions, the amount of differentiated pro-inflammatory Ml macrophages was quantified. To demonstrate Ml macrophages, representative photomicrographs are shown in Figure 24C depicting iNOS positive Ml macrophages (top row), total nuclei (second row from top), merged images (third row from top), and enhanced merged images (bottom row) for control and experimental groups. The number of iNOS positive Ml macrophages was significantly enhanced post-PLCA ligation compared to sham controls (p<0.05, Figure 24D). Moreover, treatment with BMP-7 following PLCA ligation had no effect of the number of differentiated Ml macrophages relative to the PLCA group (Figure 24D).

TREATMENT WITH BMP-7 DIMINISHES PRO-INFLAMMATORY CYTOKINE EXPRESSION

[0276] Previous data have suggested that macrophage subtypes have unique cytokine profiles with Ml macrophages contributing to enhanced pro-inflammatory MCP-1, TNF-a and IL-6 secretion. In this regard, via ELISA analysis, pro-inflammatory cytokine expression was evaluated and data presented indicate a significant increase in circulating MCP- 1 and TNF-a levels following PLCA ligation relative to the sham group (p<0.05, Figure 25A and B, respectively). Notably, this increase in MCP-1 and TNF-a expression was significantly abolished following treatment with BMP-7 (p<0.05, Figure 25A and B, respectively). Additionally, mRNA levels of TNF-a and IL-6 were evaluated by RT-PCR and the data demonstrates transcribed levels of TNF-a and IL-6 were significantly elevated in the PLCA group relative to sham control animals (p<0.05, Figure 25C and D, respectively). Importantly, post-PLCA ligation, administration of BMP-7 significantly blunted both TNF-a and IL-6 mRNA levels compared to the PLCA group (p<0.05, Figure 25C and D, respectively). M2 MACROPHAGE DIFFERENTIATION IS ENHANCED IN PLCA LIGATED MICE FOLLOWING BMP-7 TREATMENT

[0277] Representative images of LCA sections shown in Figure 26A-1 depict CD206 positive M2 macrophages (top row), total nuclei (second row from top), merged images (third row from top), and enhanced merged images (bottom row). Although not statistically significant, a decrease in M2 macrophage populations was observed in the PLCA group relative to the sham group (Figure 26). Importantly, results showed significantly elevated M2 macrophage concentrations in the PLCA+BMP-7 group relative to sham and PLCA groups, indicating BMP-7 can play a role in monocyte to M2 macrophage differentiation (p<0.05, Figure 26). ANTI-INFLAMMATORY CYTOKINE EXPRESSION INCREASES FOLLOWING BMP-7 TREATMENT IN PLCA LIGATED APO E-/- MICE

[0278] M2 macrophages secrete proto-typical anti-inflammatory cytokines including IL- lRA, IL-10, and Arginase 1. As such, levels of IL-IRA and IL-10 were assessed via ELISA and data presented indicate a significant decline in circulating pro-inflammatory cytokines following PLCA ligation compared to sham (p<0.05. Figure 27A and B, respectively). Following treatment with BMP-7, a significant increase in IL-IRA and IL-10 were observed as compared to the PLCA group (p<0.05. Figure 27A and B, respectively). Arginase^+ve cells within the LCA arteries as well as Arginase 1 mRNA expression were also evaluated. The number of Arginase+ve cells/artery was significantly decreased following PLCA ligation compared to the sham group (p<0.05, Figure 27C). Conversely, the percentage of Arginase^+ve cells/artery was significantly enhanced following treatment with BMP-7 (p<0.05, Figure 27C). Moreover, albeit transcribed Arginase 1 mRNA levels were unremarkable between PLCA and sham groups, Arginase 1 mRNA was significantly upregulated in the PLCA+BMP-7 group relative to sham and PLCA groups (p<0.05, Figure 27D).

CIRCULATING BMP-7 is DIMINISHED FOLLOWING PLCA LIGATION

[0279] Next, the levels of circulating BMP-7 were evaluated to ascertain a correlation between results obtained and enhanced BMP-7 expression. The BMP-7 expression in the blood was significantly decreased in the PLCA group compared to the sham group indicating potential interference with normal BMP-7 signaling during ATH (p<0.05, Figure 28). Importantly, following BMP-7 treatment, levels of BMP-7 were significantly upregulated in the PLCA-BMP-7 group relative to the PLCA group (p<0.05, Figure 28). BMP-7 RECEPTOR IS UPREGULATED ON MONOCYTES AND M2 MACROPHAGES FOLLOWING BMP-7 TREATMENT POST-PLCA LIGATION

[0280] The effects of exogenous BMP-7 on BMP-7 receptor (BMP-7R) expression on monocytes and M2 macrophages following PLCA ligation were also determined. Representative images of LCAs stained with anti-BMP-7R (top row), anti-CD 14 (monocytes; second row from top) or anti-CD206 (M2 macrophages; second row from top), DAPI (third row from top) and merged images (bottom row) are depicted in Figure 29A 29C. Albeit no difference was observed regarding the percentage of BMP-7R on monocytes between sham and PLCA groups, a significant increase was noted for the PLCA+BMP-7 group relative to both groups (p<0.05, Figure 29B). Additionally, the %BMP-7R on M2 macrophages was also unremarkable between sham and PLCA groups (Figure 29B). However, in the presence of BMP-7, the number of BMP-7 receptors was significantly enhanced on M2 macrophages relative to sham and PLCA ligation operated animals (p<0.05. Figure 29D).

BMP-7 ENHANCES BLOOD FLOW POST-PLCA LIGATION

[0281] Finally, to understand the functional consequences of BMP-7 treatment following PLCA ligation on blood flow, systolic velocity was examined using vascular Doppler ultrasonography. This data demonstrates a significant reduction in systolic blood flow velocity post-PLCA ligation compared with the sham group indicating PLCA ligation interfered with normal blood flow and contributed to the development of ATH (p<0.05, Figure 30). Notably, systolic velocity was significantly improved in the PLCA+BMP-7 group compared to the PLCA group but significantly less than that of the sham group (p<0.05, Figure 30).

DISCUSSION

[0282] Atherosclerosis is a progressive disorder characterized by damaged arterial endothelium with consequential plaque formation comprised of low-density lipoproteins (LDLs) and white blood cells (WBCs) that can lead to arterial blockage and normal blood flow interference. Experimental and clinical evidence now provided have highlighted the role of inflammation in the development and progression of atherogenesis. In general, the nonpathogenic arterial endothelium does not support binding of WBCs. However, in early stages of ATH, areas of arterial endothelial cells initiate expression of variegated adhesion molecules including vascular cell adhesion molecule- 1 (VCAM-1), which bind various types of leukocytes including monocytes and T lymphocytes. Once adhered to the endothelium, the leukocytes are then able to penetrate the intima mediated by various chemoattractants including MCP-1. Once infiltrated, the pro-inflammatory WBCs contribute to and promote a local inflammatory response.

[0283] Upon infiltration, monocytes undergo reprogramming yielding two antagonistic macrophage phenotypes; "classically activated" pro-inflammatory Ml macrophages and "alternatively activated" anti-inflammatory M2 macrophages. Ml macrophages promote the inflammatory response with concomitant upregulation of various cytotoxic effectors including reactive oxygen/nitrogen intermediaries and inflammatory cytokines. Recent reports suggest an association between Ml macrophage differentiation and disease pathophysiology and progression including that of atherosclerosis, cancer, and pre-diabetes. Contrarily, M2 macrophages, which encompass all non-classically differentiated macrophages, are implicated in salubrious mediation of the inflammatory response by bolstering remodeling, repair, and resolution, in part, through secretion of quintessential antiinflammatory cytokines including IL-1RA, IL-10, and Arginase 1. As such, research is now focused on identification of various growth factors and/or small molecules, which can direct and enhance macrophage plasticity towards the M2 phenotype for the development of novel therapeutic treatments for various diseases. BMP-7 enhanced the differentiation of THP-1 monocytes into M2 macrophages with concurrent upregulation of anti-inflammatory cytokines including IL-10 and IL-IRA in vitro. In the present study, the effects of BMP-7 treatment on plaque formation, monocyte infiltration, M1/M2 macrophage differentiation outcomes, pro- and anti-inflammatory cytokine expression, BMP-7R expression on monocytes and M2 macrophages, and blood flow in Apo E-/- mice following PLCA ligation were evaluated.

[0284] Apo E-/- mice, when fed a high fat diet, are regularly used as a model to examine plaque formation and subsequent ATH development, variegated cell infiltration, and vasculature adverse remodeling. An Apo E-/- mouse ATH model in which ATH was attained by PLCA ligation, in absence of a high fat diet, with evidence of plaque formation, lipid deposition, vascular lesions, thickening of the intima, infiltration of pro-inflammatory cells, and upregulation of pro-inflammatory cytokine release has been published. Using this model, ATH was generated and plaque formation was significantly enhanced following PLCA ligation surgery as supported by previous findings using high fat diet models of ATH. Importantly, following treatment with BMP-7, plaque formation was significantly abolished. The data is in accordance with previously published data indicating that BMP-7 can play a palliative, cytoprotective role in vascular proliferative disorders. [0285] Widely accepted, ATH is associated with a significant increase in infiltrated monocytes and Ml macrophage polarization, which was also evident in the PLCA ligated mice. However, following BMP-7 treatment, monocytic infiltration was significantly inhibited. BMP-7 administration has been reported to abrogate inflammatory cell infiltrate in other cell types including renal proximal tubular epithelial cells and alkali-injured corneal cells. Although monocyte infiltration was inhibited following BMP-7 administration, it had no effect on Ml macrophage differentiation.

[0286] Not only is monocyte/Ml macrophage differentiation implicated in inflammatory disease progression, but also the enhanced expression of various pro-inflammatory chemokines/cytokines. Post PLCA ligation, prototypical Ml macrophage cytokine profiles, including MCP-1, TNF-a, and IL-6, were significantly elevated. However, pro-inflammatory cytokine expression was significantly diminished following treatment with BMP-7 in the post-PLCA ligated mouse. These data are supported by studies showing BMP-7 inhibits proinflammatory cytokine expression in vitro and in vivo.

[0287] To elucidate mechanisms by which BMP-7 inhibited plaque formation following PLCA ligation surgery, M2 macrophage differentiation and anti-inflammatory cytokine release were also evaluated. In the ATH model mice, M2 macrophage populations were unremarkable and anti-inflammatory cytokine expression including IL-IRA, IL-10, and Arginase 1 were significantly decreased compared to sham operated mice. Importantly, following treatment with BMP-7 post-PLCA ligation, M2 macrophage differentiation was significantly enhanced compared to sham and PLCA groups and anti-inflammatory cytokine secretion, including IL-IRA, IL-10, and Arginase 1, was significantly upregulated compared to the PLCA ligated mice. The data indicate that BMP-7 has the potential to inhibit the proinflammatory response that is persistent in ATH through enhanced M2 macrophage differentiation and associated anti-inflammatory mediators as has been previously evidenced in in vitro and in vivo models including pre-diabetic cardiomyopathy and inflammatory arthritis.

[0288] BMP signal transduction, in general, is propagated through ligand induced activation and oligomerization of BMP receptors (BMPRs) which in turn induces phosphorylation and activation of Smad 1/5/8 and Smad-independent cascades regulating target gene transcription. Specifically, the BMPR2 (identified as BMP-7R within the current manuscript) is directly involved in BMP-7 ligand induced receptor activation and oligomerization and subsequent signal transduction activities. A correlation between BMP-7 and upregulated BMP-7R during "inflammation mimicry" assault in vitro has been identified, which resulted in decreased monocytic populations and enhanced M2 macrophage differentiation. As such, BMP-7R expression was also assessed and its expression remained unchanged on monocytes and M2 macrophages for both sham and PLCA ligated animals as evidenced by co- localization of BMP-7R/CD14 and BMP-7R/CD206, respectively. However, BMP-7 application resulted in a significant increase in BMP-7R expression on both monocytes and macrophages indicating a correlation between BMP-7R expression and enhanced BMP-7 signal transduction activity.

[0289] Finally, blood flow was assessed for all groups and as expected, systolic velocity was significantly disrupted following PLCA ligation. However, following BMP-7 treatment, blood flow was significantly improved in PLCA ligated Apo E-/- mice. Collectively, presented data indicates that BMP-7 inhibits plaque formation and increases arterial systolic velocity in a PLCA ligation model of ATH through inhibition of monocyte infiltration and associated pro-inflammatory cytokines and induction of M2 macrophage differentiation with consequential enhanced anti-inflammatory cytokine expression. Furthermore, mechanisms promoting monocyte polarization and M2 macrophage differentiation by BMP-7 involve the upregulation and activation of BMP-7R.

5. BMP-7 ATTENUATES ADVERSE CARDIAC REMODELING MEDIATED THROUGH M2

MACROPHAGES IN PREDIABETIC CARDIOMYOPATHY

[0290] Diabetes is a group of metabolic disorders that results in chronic hyperglycemia. Symptoms include abnormal metabolism of lipids, carbohydrates, and protein and may also involve impaired insulin secretion. Two major classifications of diabetes exist: type 1 diabetes mellitus and type 2 diabetes mellitus. Type 1 diabetes mellitus is regarded as an autoimmune condition that involves the destruction of pancreatic β-cells and can lead to total insulin deficiency. Type 2 diabetes mellitus accounts for the majority of diabetic patients in the United States and manifests as insulin resistance despite normal pancreatic β-cell function. More recently, the Word Health Organization introduced prediabetes (PD) to include patients with impaired fasting glucose and impaired glucose tolerance. Diabetes, regardless of the type, increases the risk for conditions such as cardiovascular disease and nephropathy. Recent studies have shown that even subclinical states of hyperglycemia can lead to adverse complications.

[0291] Moreover, the exact source of increased immunoregulation and associated oxidative stress and inflammation in diabetes is complex and not well understood. It has recently been reported that monocytes are the first cell subtype to infiltrate the pancreas in rodent models of PD. Moreover, increased immune cell infiltration and monocytic activity have been reported in diabetic patients.

[0292] Increased monocyte infiltration can be present along with an increased inflammatory immune response, which contributes to the adverse cardiac remodeling, in streptozotocin (STZ)-induced cardiomyopathy. In addition, the present study extrapolated the role of bone morphogenetic protein (BMP)-7 in the polarization of proinflammatory monocytes into antiinflammatory M2 macrophages and the associated inhibition of adverse cardiac remodeling in the PD heart. The data show that there were increased numbers of infiltrated monocytes, enhanced proinflammatory cytokine expression, and increased adverse cardiac remodeling in the PD group. Treatment with BMP-7 differentiated monocytes into an enhanced number of M2 macrophages and stimulated the secretion of anti-inflammatory cytokines as well as inhibited oxidative stress in the PD heart. Moreover, the data indicate attenuated cardiomyocyte apoptosis, fibrosis, and improved cardiac function after BMP-7 treatment. MATERIALS AND METHODS

[0293] STZ-induced PD in mice. C57B1/6 mice (8-10 wk old) were maintained in a controlled environment and given all appropriate nutritional support ad libitum. Mice were separated into three groups consisting of control, PD, and PD + BMP-7 (n 7-10 animals/group). Animals were administered 50 mg/kg STZ via intraperitoneal injections for 2 days (days 1 and 2) in PD and PD + BMP-7 groups. For the PD + BMP-7 group, animals were additionally treated with an intravenous injection of 200 μg/kg BMP-7, as previously reported, on days 3-5. Moreover, control animals were administered sodium citrate buffer injections on days 1 and 2. Twenty-one days following the last STZ injection, animals were euthanized humanely with 4% isoflurane for 10 min followed by cervical dislocation.

[0294] Tissue processing. Hearts from each group were harvested, washed with PBS, and stored in either RNA Later or formalin solutions for future experiments. Formalin-fixed hearts were processed with the Leica TP 1020 system and embedded in paraffin wax with a Tissue Tek TEC machine. Sections (5 μιη) were cut and placed on plus-plus slides for histochemical experiments.

[0295] Glucose levels and tolerance. Blood glucose levels were measured 7 and 21 days after the last STZ injection using an OneTouch Ultra Mini glucose meter. Twenty days after STZ, all groups were subjected to a glucose tolerance test. In brief, mice were fasted for 6 h, and their initial blood glucose was measured. An intraperitoneal injection of glucose (1 g/kg) was administered to each mouse, and blood glucose levels were checked via tail vein puncture every 30 min for a total of 120 min postglucose injection. [0296] Immunohistochemistry to identify monocytes, macrophages, and pro- and antiinflammatory cytokines. Heart sections were deparaffinized and rehydrated as previously described (30). Sections were processed for heat-induced epitope retrieval for 20 min and then blocked with 10% normal goat serum (Vector Labs) for 1 h. Primary antibodies, such as CD 14 to identify monocytes, CD206 to identify M2 macrophages, IL-6 and TNF-a to identify proinflammatory cytokines, and IL-10 to identify anti-inflammatory cytokines, were incubated overnight at 4°C. Tissues were washed and incubated with the appropriate secondary antibodies, including goat anti-rabbit Alexa 488, goat anti-rabbit Alexa 568, or goat anti-rat Alexa 594, for 1 h at room temperature. Sections were counterstained with mounting medium containing 4=,6-diamidino-2-phenylindole (DAPI; Vector Labs), and slides were coverslipped. Four images per section were taken for data quantification using an Olympic ΓΧ-70 fluorescent microscope, and representative images were captured using confocal microscopy. Cells positive for CD 14 and CD206 were counted, divided by the total number of nuclei, and expressed as the percentage of positive cells per section. IL-10, IL-6, and TNF-intensities were measured, and the average corrected integrated density was calculated for each animal using ImageJ software (NIH).

[0297] Determination of pro- and anti-inflammatory cytokines by ELISA. Whole blood was collected from the jugular vein, and serum was separated by centrifugation at 13,000 rpm for 10 min. Commercially available ELISA kits were purchased from Raybiotech. The instructions provided in the kits were strictly followed and used to determine levels of proinflammatory cytokines (TNF-a and IL-6) and anti-inflammatory cytokines [IL-10 and IL-1 receptor antagonist (IL-IRA)].

[0298] Determination of oxidative stress by Western blot analysis and dihydroethidium staining. SDS-PAGE and Western blot analysis were used to analyze MnSOD expression and performed as described elsewhere. For dihydroethidium (DHE) staining, heart sections were stained with 1 μΜ/ml DHE for 25 min. DHE intensity was then measured in 4 random images/section, and the average corrected integrated density was calculated for each section using ImageJ software.

[0299] TUNEL staining. In brief, heart sections were deparaffinized, rehydrated, and incubated with proteinase K for antigen retrieval. TUNEL staining was performed using a cell death detection kit (Roche) following the manufacturer's protocol. Sections were then mounted and coverslipped, and 4 images/section were quantified using ImageJ software. TUNEL-positive (red) cells that merged with nuclei (blue) were counted, divided by total nuclei, and multiplied by 100. [0300] Determination of apoptotic proteins by Western blot analysis. Heart samples were prepared, supernatants were collected, and standard SDS-PAGE was performed. B-actin was used as a loading control. Proteins were transferred onto polyvinylidene difluoride membranes and blocked with 5% milk. Membranes were incubated with primary antibodies [phosphorylated (p-)Akt, total Akt, p-phosphatase and tensin homolog (pPTEN), and total PTEN] for 1 h at room temperature or overnight at 4°C. Membranes were then washed and incubated with secondary antibody (goat anti-rabbit horseradish peroxidase) for 1 h at room temperature. Membranes were then washed, incubated with an enhanced chemiluminescent substrate for 2 min, and exposed at various times. Blots were scanned, and band intensities were measured using ImageJ software.

[0301] Masson's trichrome staining. Sections were stained with Masson's trichrome. Using ImageJ software, collagen deposition (blue) was measured to quantify the fibrotic area (in mm2). Interstitial fibrosis was measured by quantifying collagen in 6 areas/section, whereas vascular fibrosis was quantified as vessel fibrosis/total vessel area x 100 in 6 vessels/section.

[0302] Determination of cardiac function. Three weeks after day 5, twodimensional transthoracic echocardiography was performed with a 5500 Ultrasound System. Using a 15- 6L Hockey stick transducer, M-mode images of the left ventricle (LV) were captured and used to calculate end-diastolic volume, end-systolic volume, ejection fraction (in %), LV internal dimension at diastole, LV internal dimension at systole, and fractional shortening (in %). After the heart function assessment, mice were euthanized with 4% isoflurane followed by cervical dislocation.

[0303] Statistical analysis. Statistical analyses of all data were performed using one-way ANOVA followed by the Tukey test. Data are presented as means ± SE with P values of <0.05 considered statistically significant.

RESULTS

[0304] Increased glucose levels in PD animals. After STZ injections, glucose levels and tolerance were assessed to ascertain that PD mice had been generated. Blood glucose levels were significantly elevated in the PD group on days 7 and 21 after the last STZ injection compared with control mice (P< 0.05; Fig. 31, A and B). Interestingly, the addition of BMP-7 significantly attenuated the hyperglycemia observed in the PD group (P < 0.05; Fig. 31, A and B). During the glucose tolerance test, the ability of PD mice to clear glucose from blood was significantly impaired relative to the control group at all time points assessed after glucose administration (P < 0.05; Fig. 31C). Although not statistically significant, a trend of decreased hyperglycemia was recorded in the PD BMP-7 group at 30 and 60 min postglucose treatment relative to the PD group (Fig. 31C). However, at 90 and 120 min, PD BMP-7 mice had significantly reduced blood glucose compared with PD mice (P < 0.05; Fig. 31C).

[0305] Increased monocyte infiltration and inflammatory cytokines in PD mouse hearts. To determine whether inflammatory cells have a direct relationship with the PD heart, infiltrated monocytes were quantified. Figure 32A shows representative photomicrographs of heart sections stained with CD 14, a marker for monocytes, in red (a, f, and k), cardiac myocytes in green (b, g, and 1), nuclei in blue (c, h, and m), merged images (d, i, and n), and enhanced merged images (e, j, and o). Quantitative analyses suggested a significant upregulation of monocytes in the PD group relative to the control group (P < 0.05; Fig. 32B). Conversely, monocytes were significantly reduced upon BMP-7 treatment compared with the PD group (PD + BMP-7 group: 1.051 ± 0.185% vs. PD group: 1.759 ± 0.238% for CD14-positive cells/total nuclei; Fig. 32B). Additionally, no significant differences in the monocytic population were observed between control and PD + BMP-7 groups (Fig. 32B).

[0306] Next, the consequences of monocyte infiltration and associated proinflammatory cytokines were evaluated. Figure 2C shows representative photomicrographs of heart sections from each group stained with TNF-a (a, f, and k), sarcomeric β-actin (b, g, and 1), and DAPI (c, h, and m). Quantification of stained tissues indicated significantly augmented TNF-a levels in the PD group relative to the control group (P < 0.05; Fig. 32D). However, treatment with BMP-7 attenuated levels of TNF-a compared with the PD group [PD + BMP-7 group: 3.023 ± 0.655 arbitrary units (AU) vs. PD group: 5.307 ± 0.387 AU, P < 0.05; Fig. 32D]. Although not statistically significant, similar TNF-a expression patterns resulted via TNF- ELISA analyses using isolated serum (Fig. 32E).

[0307] Levels of the proinflammatory cytokine IL-6 were also measured. Figure 3A shows representative images of heart sections from each group stained with IL-6, sarcomeric -actin, and DAPI. Quantitative analysis of PD tissues showed upregulated IL-6 levels compared with control tissues (P < 0.05; Fig. 33B). Conversely, significantly decreased IL-6 levels in PD + BMP-7 hearts relative to PD hearts were observed (PD BMP-7 group: 2.984 ± 0.577 AU vs. PD group: 5.495 ± 0.367 AU, P < 0.05; Fig. 33B). In addition, an ELISA for IL-6 on serum samples from control and experimental groups was performed, and the results indicated increased levels of IL-6 in the PD group compared with the control group, whereas the PD + BMP-7 group had significantly decreased levels compared with the PD group (P < 0.05; Fig. 33C). Notably, no statistical deviation in IL-6 expression was noted between control and PDBMP-7 groups (Fig. 33, B and C). [0308] BMP-7 augments M2 macrophages and reduces inflammation in the PD heart. To elucidate the mechanisms by which BMP-7 promotes beneficial effects in the PD heart, M2 macrophage and associated anti-inflammatory cytokine expression were evaluated. Representative images of heart sections stained with CD206, an M2 macrophage marker, sarcomeric β-actin, and DAPI are shown in Fig. 34A. Importantly, no differences in M2 macrophage concentration were observed between control and PD groups. However, the results showed significantly elevated M2 macrophages in PD + BMP-7 hearts relative to PD hearts, suggesting that BMP-7 may play a role in the monocyte to M2 macrophage polarization (PD BMP-7 group: 2.005 ± 0.131% vs. PD group: 1.253 ± 0.175% for CD206- positive cells/total nuclei, P < 0.05; Fig. 34B).

[0309] Previous data have indicated M2 macrophages secrete anti-inflammatory cytokines. To this end, levels of IL-1RA and IL-10, critical mediators of inflammation and immuno- regulation, were evaluated. Quantitative analysis of the IL-1RA concentration in serum samples insinuated a significant decrease in the PD group (P < 0.05; Fig. 34C), which was considerably increased upon BMP-7 treatment (P < 0.05; Fig. 34C). IL-10 analyses performed on heart sections stained with IL-10, sarcomeric -actin, and DAPI (Fig. 34D) indicated an observable decrease in IL-10 levels in the PD group relative to the control group, which was significantly increased upon treatment with BMP-7 (Fig. 34E). To corroborate immunocytochemistry data, an ELISA for IL-10 was performed on serum samples. Coinciding with previous data, significantly reduced levels of IL-10 in the PD group compared with the control group and dramatically elevated IL-10 concentrations upon BMP- 7 treatment were noted (P < 0.05; Fig. 34F).

[0310] BMP-7 ameliorates cardiac oxidative stress by upregulating MnSOD. A previous study has shown that BMP-7 may act as an antioxidant by upregulating levels of MnSOD. To determine the effects of BMP-7 on oxidative stress in the PD heart, superoxide anion production was assessed. Figure 35A shows representative photomicrographs of heart tissue stained with DHE (a, d, and g), DAPI (b, e, and h), and merged images in pink (c, f, and i). Analysis revealed that PD hearts had significantly increased ROS relative to control hearts (P < 0.001 ; Fig. 35B). Conversely, BMP-7 administration dramatically reduced levels of ROS compared with the PD

[031 1] group (PD BMP-7 group: 3.667 ± 0.333 AU vs. PD group: 8.167± 0.872 AU, P < 0.001; Fig. 35B). MnSOD expression was also quantified through Western blot analysis with representative blots and -actin controls shown in Fig. 4C. Densitometric analysis indicated decreased expression of MnSOD in the PD group relative to the control group (P<0.05; Fig. 35C). However, treatment with BMP-7 blunted the diminished Mn-SOD expression compared with the PD group (PD BMP-7 group: 0.888± 0.0608 AU vs. PD group: 0.630 ± 0.0550, P <0.05; Fig. 35C).

[0312] BMP-7 inhibits apoptosis in PD mice by regulating PTEN and Akt. To determine the effects of BMP-7 treatment on cardiomyocyte apoptosis in vivo, TU EL staining was performed. Figure 36A shows representative photomicrographs of heart sections from each group stained with TUNEL (a, d, and g), total nuclei (b, e, and h), and merged images (c, f, and i). The data indicated increased apoptosis in PD hearts relative to control hearts (P < 0.001; Fig. 36B). Moreover, analysis revealed a significantly decreased percentage of apoptotic nuclei in the PD + BMP-7 group relative to the PD group (PD BMP-7 group: 0.286 ± 0.0442% vs. PD group: 0.505 ± 0.0369% for TUNEL-positive nuclei/total nuclei, P < 0.001; Fig. 36B). To demonstrate that apoptosis occurred in the cardiomyocytes, sections were stained with TUNEL, sarcomeric -actin, caspase-3, and DAPI, and representative images from the PD group are shown in Fig. 36C.

[0313] To elucidate the mechanisms of the blunted apoptosis noted in the PD BMP-7 heart, the expression of PTEN, an inhibitor of phosphatidylinositol 3-kinase (PI3K)/Akt activation, and Akt, a prosurvival and cell cycle regulator, were evaluated. Densitometric analysis of p- PTEN Western blot bands revealed a significant increase in the expression of activated PTEN in the PD group relative to the control group (P < 0.05; Fig. 36D). Notably, a significant decrease in p-PTEN expression was observed in the PD + BMP-7 group relative to the PD group (PD + BMP-7 group: 0.784 ± 0.123 AU vs. PD group: 1.305 ± 0.134 AU, P < 0.05; Fig. 36D). Inversely proportional to the p-PTEN data, significantly diminished levels of p- AKT in the PD group relative to the control group (P < 0.05) and considerably upregulated levels of the survival protein in the PD + BMP-7 group compared with the PD group were found (PD + BMP-7 group: 0.715 ± 0.0784 AU vs. PD group: 0.500 ± 0.107 AU, P < 0.05; Fig. 36E).

[0314] BMP-7 ameliorates cardiac remodeling in PD mice. To assess the influence of BMP-7 on fibrosis formation in the PD heart, heart tissue from each of the study groups was analyzed for collagen deposition using Masson's trichrome staining. Figure 37A shows representative photomicrographs from control and experimental groups demonstrating interstitial fibrosis (a- c) and vascular fibrosis (d-f). Quantitative analyses indicated that interstitial fibrosis was dramatically increased in the PD group relative to the control group (P<0.001 ; Fig. 37B). However, after BMP-7 treatment, interstitial fibrosis was significantly blunted relative to the PD group (PD BMP-7 group: 0.110 ± 0.0222 mm² vs. PD group: 0.170 ± 0.0159 mm², P < 0.05; Fig. 37B). Concurrent with the interstitial fibrosis data, vascular fibrosis in the PD group was significantly elevated relative to the control group (P < 0.001 ; Fig. 37C), whereas the addition of BMP-7 significantly abrogated vascular collagen deposition in the PD group (P < 0.05; Fig. 37C).

[0315] Exogenous BMP-7 improves cardiac function in PD mice. Three weeks after day 5, mice underwent transthoracic echocardiography to assess cardiac function. The raw echocardiogram data obtained are shown in Fig. 38A. The data indicated impaired LV function in the PD group as demonstrated by deviated LV internal dimension at diastole, LV internal dimension at systole, end-diastolic volume, end-systolic volume, ejection fraction, and fractional shortening compared with the control group (P < 0.001; Fig. 38B). More importantly, a significant improvement in cardiac function was achieved after BMP-7 administration as indicated by augmented fractional shortening and ejection fraction relative to the PD group (P <0.05; Fig. 38B).

DISCUSSION

[0316] Monocytes, during pathological circumstances, migrate from the circulation and infiltrate tissues, where they differentiate into macrophages. Monocyte to macrophage polarization and differentiation yields two distinct phenotypes, including Ml and M2 macrophages. Ml macrophages are proinflammatory and heighten the inflammatory response, whereas M2 macrophages are anti-inflammatory and express cytokines that mitigate inflammation. Several clinical studies have shown increased levels of monocytes and proinflammatory cytokines, TNF-a and IL-6, in PD patients. Additional evidence further indicates that proinflammatory cytokines promote cardiomyocyte apoptosis, which inevitably leads to cardiac remodeling and depressed cardiac function. However, no animal model has yet been used to examine monocyte infiltration and associated inflammation in the PD heart, nor has any attempt been made to control monocyte polarization. Of particular interest, BMP- 7, a growth factor belonging to the transforming growth factor-β superfamily, has been recently reported to alleviate inflammation after administration. However, whether BMP-7 plays a role in monocyte polarization and differentiation and the subsequent effects on adverse cardiac remodeling in the PD heart remains elusive. To this end, a PD mouse model was generated using multiple low-dose injections of STZ (50 mg/kg) as previously reported, and the impact of BMP-7 on inflammation, apoptosis, fibrosis, and LV output in the diabetic heart was investigated.

[0317] Concurrent with previous studies showing increased monocyte infiltration and monocytic activity in patients with PD, monocyte infiltration was significantly increased in the hearts of PD mice, as evidenced by CD 14 staining. Such findings, indicating increased cardiac monocytic populations, are unique in that no study has previously identified these specific cell types infiltrating the myocardium and residing in the vicinity of cardiomyocytes. Additionally, a correlation between monocyte infiltration and levels of proinflammatory cytokines was confirmed in PD mice by the presence of increased levels of TNF-a and IL-6 in heart tissue as well as in serum samples. These data are in agreement with clinical reports that have shown elevated levels of proinflammatory cytokines in serum samples of PD patients. Upon treatment with BMP-7, the cardiac monocytic population in PD mice was dramatically decreased, which was observed with a concomitant decrease in levels of the proinflammatory cytokines TNF-a and IL-6. The anti-inflammatory effects of BMP-7 found in the present study correlate with a another report which showed that treatment with BMP-7 inhibited proinflammatory cytokines in proximal tubular epithelial cells. The evidence provided indicates potential cardioprotective effects of BMP-7 against inflammation by directing the secretion of anti-inflammatory cytokines.

[0318] The data indicate that the ratio of Ml to M2 macrophage polarization after monocyte differentiation can determine the severity and progression of inflammation. M2 macrophages are a class of macrophages that reduce inflammation by secreting IL-10, an antiinflammatory cytokine. Specifically, IL-10 has been reported to modulate inflammation by regulating the secretion pattern of proinflammatory cytokines from inflammatory cells. In the present study, infiltrated monocytes, in the presence of BMP-7, polarize into beneficial M2 macrophages. Anti-inflammatory effects of BMP-7 have been previously reported. In a previous study, it was shown that monocytes express receptors for BMP-7 and, in the presence of BMP-7, differentiate into beneficial M2 macrophages in vitro. Previous reports have supported that BMP-7 promotes the differentiation of mesenchymal stem cells and white adipocytes into brown adipose tissue. Parallel to increased M2 macrophage concentrations, BMP-7 administration significantly enhanced IL-10 secretion. It has previously been reported that IL-10 can mediate TNF-a-induced cardiomyocyte apoptosis. Downregulated levels of TNF-a upon BMP-7 treatment were observed which indicates that BMP-7 inhibits TNF-a-induced apoptosis in PD cardiomyopathy by upregulating IL-10. In addition, increased levels of another anti-inflammatory cytokine, IL-IRA, were observed in the BMP-7 treatment group. Collectively, the assimilated data indicate that although monocytic infiltration is inevitable in the PD heart, BMP-7 confers cardioprotection by directing monocyte differentiation into M2 macrophages and promoting the secretion of antiinflammatory mediators. [0319] It is widely accepted that diabetes results in cardiac oxidative stress. In fact, a previous study has indicated elevated levels of ROS in the STZ-induced diabetic heart. Similarly, increased levels of ROS and blunted expression of the antioxidant MnSOD were detected in PD hearts relative to control hearts within the present study. Notably, treatment with BMP-7 increased cardiac MnSOD expression and reduced oxidative stress in the PD heart. Whether the diminished oxidative stress noted in the PD + BMP-7 group is consequent with attenuated ROS levels and upregulated antioxidants alone remains obscure. However, the data indicate that BMP-7-conferred PD cardioprotection is a multifactorial chain of events.

[0320] After inflammation and oxidative assault, adverse cardiac remodeling, including cardiomyocyte apoptosis, has been reported in disease-state myocardium. Specifically, clinical and animals studies have reported increased cardiomyocyte apoptosis in diabetic patients as well as in STZ models of diabetes. In corroboration with these previously published reports, this study showed increased cardiomyocyte apoptosis in the PD heart. Moreover, BMP-7 administration significantly inhibited cardiomyocyte apoptosis in the PD heart, which correlates with a previous study BMPs inhibit apoptosis of cardiomyocytes in vitro.

[0321] To elucidate the mechanisms of cardiomyocyte apoptosis in the PD heart, expression levels of activated PTEN and Akt were quantified. PTEN is a tumor suppressor that regulates the prosurvival protein Akt through inhibition of its upstream activator, PI3K. Densitometric analyses indicated elevated p-PTEN expression in PD mouse hearts relative to control mouse hearts with a concomitant downregulation in p-AKT. This data is concurrent with a previous report that showed a downregulation of p-AKT in STZ models of diabetes. Moreover, a previous study reported that overexpression of PTEN encourages cardiomyocyte apoptosis. Importantly, as shown here for the first time, BMP-7 inhibits the expression of cardiac p- PTEN while significantly upregulating p-Akt in the PD heart. Collectively, the data indicates the presence of enhanced apoptosis in the monocytic-infiltrated PD myocardium is consequent to augmentation of normal PI3K/Akt signaling, which was ameliorated with BMP-7.

[0322] Significant cardiac apoptosis promotes collagen deposition and fibrosis formation in an attempt to rescue the indigenous architecture and integrity of the heart. Quantitative analysis revealed significantly elevated interstitial and vascular fibrosis in PD hearts relative to control hearts. The aforementioned data are in conjunction with a previous report that suggested that cardiac remodeling begins as early as the PD stage. Of particular interest, BMP-7 treatment resulted in decreased cardiac remodeling compared with the PD group. Antifibrotic effects of BMP-7 have been previously reported in the diabetic kidney. However, the present study is the first to report the antifibrotic effects of BMP-7 in the PD heart.

[0323] A previous study has implied that cardiac dysfunction can occur during PD. This data correlates with this report and demonstrated decreased cardiac function in the PD group, which was significantly improved upon treatment with BMP-7. Although causes of cardiac dysfunction are complex, it is conceivable that the decreased apoptosis and fibrosis presented in this study contributed to the improvement in cardiac function in the BMP-7 treatment group.

[0324] In conclusion, the PD myocardium is infiltrated with monocytes and subsequent secreted proinflammatory cytokines. Notably, in the presence of BMP-7, monocytes polarize into beneficial M2 macrophages, thereby inhibiting inflammation and protecting the PD heart from cardiomyocyte apoptosis, fibrosis, and cardiac dysfunction.

C. References

[0325] The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference.

[0326] Alberti KG, et al. (1998) Definition, diagnosis and classification of diabetes mellitus and its complications. Part 1 : diagnosis and classification of diabetes mellitus provisional report of a WHO consultation. Diabetic Med. 15:539-53.

[0327] Anzai A, et al. (2012) Regulatory role of dendritic cells in post-infarction healing and left ventricular remodeling. Circulation 125: 1234-1245.

[0328] Asakura E, et al. (1997) Intravenously administered macrophage colony-stimulating factor (M-CSF) specifically acts on the spleen, resulting in the increasing and activating spleen macrophages for cytokine production in mice. Immunopharmacology 37:7-14.

[0329] Assi K, et al. (2006) The specific ΓΝΚ inhibitor SP600125 targets tumour necrosis factor-alpha production and epithelial cell apoptosis in acute murine colitis. Immunology 118: 1 12-121.

[0330] Babamusta F, et al. (2006) Angiotensin II infusion induces site-specific intra-laminar hemorrhage in macrophage colony-stimulating factor-deficient mice. Atheroscl. 186:282-290.

[0331] Bardini G, et al. (2010) Inflammation markers and metabolic characteristics of subjects with 1-h plasma glucose levels. Diabetes Care. 33 :411-3.

[0332] Beltrami CA, et al. (1997) Proliferating cell nuclear antigen (PCNA), DNA synthesis and mitosis in myocytes following cardiac transplantation in man. J. Mol. Cell Cardiol. 29:2789-2802.

[0333] Bergman M. (2010) Inadequacies of absolute threshold levels for diagnosing prediabetes. Diabetes Metab Res Rev. 26:3-6.

[0334] Blonde L. (2012) Benefits and risks for intensive glycemic control in patients with diabetes mellitus. Am J Med Sci. 343: 17-20.

[0335] Boon MR, et al. (201 1) Bone morphogenetic protein 7: a broad-spectrum growth factor with multiple target therapeutic potency. Cytokine Growth Factor Rev. 22:221-9.

[0336] Bradley EW, et al. (2008) Pathway crosstalk between Ras/Rafand PI3K in promotion of M-CSF-induced MEK/ERK-mediated osteoclast survival. J. Cell Biochem. 104: 1439- 1451.

[0337] Chan WL, et al. (2008) BMP-7 protects mesangial cells from injury by polymeric IgA. Kidney Int. 74: 1026-39. [0338] Choi KM, et al. (2010) CD206-positive M2 macrophages that express heme oxygenase-1 protect against diabetic gastroparesis in mice. Gastroenterology 138:2399-409, 2409.

[0339] Chowdhry MF, et al. (2007) Diabetes increases apoptosis and necrosis in both ischemic and nonischemic human myocardium: role of caspases and poly-adenosine diphosphate-ribose polymerase. J Thorac Cardiovasc Surg. 134: 124-31, 131 el-3.

[0340] Cifarelli V, et al. (2007) Increased Expression of Monocyte CDl lb (Mac-1) in

Overweight Recent-Onset Type 1 Diabetic Children. Rev Diabet Stud. 4: 1 12-7.

[0341] Cipolletta C, et al. (2005) Activation of peripheral blood CD 14+ monocytes occurs in diabetes. Diabetes 54:2779-2786 (2005).

[0342] D'Souza A, et al. (201 1) Left ventricle structural remodelling in the prediabetic Goto- Kakizaki rat. Exp Physiol. 96:875-88.

[0343] Davies MR, et al. (2003) BMP-7 is an efficacious treatment of vascular calcification in a murine model of atherosclerosis and chronic renal failure. J Am Soc Nephrol. 14: 1559- 67.

[0344] Derynck R, et al. (2003) Smad-dependent and Smad-independent pathways in TGF- beta family signalling. Nature 425:577-584.

[0345] Dhingra S, et al. (2007) p38 and ERK1/2 MAPKs mediate the interplay of TNF-alpha and IL-10 in regulating oxidative stress and cardiac myocyte apoptosis. Am J. Physiol Heart Circ. Physiol 293 :H3524-H3531.

[0346] Dhingra S, et al. (2011) Akt regulates IL-10 mediated suppression of TNFalpha- induced cardiomyocyte apoptosis by upregulating Stat3 phosphorylation. PloS One. 6:e25009.

[0347] El HK, et al. (2012) Thioredoxin- 1 Promotes Anti-Inflammatory Macrophages of the

M2 Phenotype and Antagonizes Atherosclerosis. Arterioscler. Thromb. Vase. Biol.

[0348] Everett BM, et al. (2013) Rationale and design of the Cardiovascular Inflammation

Reduction Trial: A test of the inflammatory hypothesis of atherothrombosis. Am. Heart J.

166: 199-207.

[0349] Frontini MJ, et al. (201 1) Fibroblast growth factor 9 delivery during angiogenesis produces durable, vasoresponsive microvessels wrapped by smooth muscle cells. Nat. Biotechnol. 29:421-427.

[0350] Galkina E, et al. (2009) Immune and inflammatory mechanisms of atherosclerosis (*). Annu Rev Immunol. 27: 165-97. [0351] Gay D, et al. (2013) Fgf9 from dermal gammadelta T cells induces hair follicle neogenesis after wounding. Nat. Med. 19:916-923.

[0352] Geske MJ, et al. (2008) Fgf9 signaling regulates small intestinal elongation and mesenchymal development. Development 135:2959-2968.

[0353] Glass C, et al (2011) MicroRNA-1 transfected embryonic stem cells enhance cardiac myocyte differentiation and inhibit apoptosis by modulating the PTEN/Akt pathway in the infarcted heart. Am. J. Physiol Heart Circ. Physiol 301 :H2038-H2049.

[0354] Glass C, et al. (2011) ES cells overexpressing microRNA-1 attenuate apoptosis in the injured myocardium. Mol. Cell Biochem. 357: 135-141.

[0355] Glass CE, et al. (2010) Stem cells in the diabetic infarcted heart. Heart Fail.Rev. 15:581-588.

[0356] Gould SE, et al. (2002) BMP-7 regulates chemokine, cytokine, and hemodynamic gene expression in proximal tubule cells. Kidney Int. 61 :51-60.

[0357] Hosoda T, et al. (2009) Clonality of mouse and human cardiomyogenesis in vivo. Proc. Natl. Acad. Sci. U.S.A 106: 17169-17174.

[0358] Houthuys E, et al. (2010) A method for the isolation and purification of mouse peripheral blood monocytes. J. Immunol. Methods 359: 1-10.

[0359] Hung IH, et al. (2007) FGF9 regulates early hypertrophicchondrocyte differentiation and skeletal vascularization in the developing stylopod. Dev. Biol. 307:300-313.

[0360] Izumi M, et al. (2001) Bone morphogenetic protein-2 inhibits serum deprivation- induced apoptosis of neonatal cardiac myocytes through activation of the Smadl pathway. J Biol Chem. 276:31 133-41.

[0361] Jadhav A, et al. (2013) The heme oxygenase system selectively enhances the antiinflammatory macrophage-M2 phenotype, reduces pericardial adiposity, and ameliorated cardiac injury in diabetic cardiomyopathy in Zucker diabetic fatty rats. J. Pharmacol. Exp. Ther. 345:239-249.

[0362] Jain SK, et al. (2007) High glucose and ketosis (acetoacetate) increases, and chromium niacinate decreases, IL-6, IL-8, and MCP-1 secretion and oxidative stress in U937 monocytes. Antioxid. Redox. Signal. 9: 1581-1590.

[0363] Jain SK, et al. (2009) L-cysteine supplementation lowers blood glucose, glycated hemoglobin, CRP, MCP-1, and oxidative stress and inhibits NF-kappaB activation in the livers of Zucker diabetic rats. Free Radic. Biol. Med. 46: 1633-1638. [0364] Jesmin S, et al. (2007) Endothelin antagonism normalizes VEGF signaling and cardiac function in STZ-induced diabetic rat hearts. Am J Physiol Endocrinol Metab. 292:E1030-40.

[0365] Jiang ZS, et al. (2004) Non-angiogenic FGF-2 protects the ischemic heart from injury, in the presence or absence of reperfusion. Cardiovasc. Res. 62: 154-166.

[0366] Kardami E, et al. (2007) Fibroblast growth factor-2 and cardioprotection. Heart Fail. Rev. 12:267-277.

[0367] Kigerl KA, et al. (2009) Identification of two distinct macrophage subsets with divergent effects causing either neurotoxicity or regeneration in the injured mouse spinal cord. J. Neurosci. 29: 13435-13444.

[0368] King GL, et al. (2004) Hyperglycemia-induced oxidative stress in diabetic complications. Histochem. Cell Biol. 122:333-338.

[0369] Korf-Klingebiel M, et al. (2011) Conditional transgenic expression of fibroblast growth factor 9 in the adult mouse heart reduces heart failure mortality after myocardial infarction. Circulation 123 :504-514.

[0370] Kutza J, et al. (2000) Macrophage colony-stimulating factor antagonists inhibit replication of HIV- 1 in human macrophages. J. Immunol. 164:4955-4960.

[0371] Laskin DL. (2009) Macrophages and inflammatory mediators in chemical toxicity: a battle of forces. Chem Res Toxicol. 22: 1376-85.

[0372] Lavine KJ, et al. (2005) Endocardial and epicardial derived FGF signals regulate myocardial proliferation and differentiation in vivo. Dev. Cell 8:85-95.

[0373] Lee TI, et al. (2010) Oxidative stress and inflammation modulate peroxisome proliferator-activated receptors with regional discrepancy in diabetic heart. Eur. J. Clin. Invest 40:692-699.

[0374] Leor J, et al. (2006) Ex vivo activated human macrophages improve healing, remodeling, and function of the infarcted heart. Circulation 1 14:194-100.

[0375] Li M, et al. (2013) Donepezil Markedly Improves Long-Term Survival in Rats With Chronic Heart Failure After Extensive Myocardial Infarction. Circ. J.

[0376] Li Z, et al. (2007) Involvement of endoplasmic reticulum stress in myocardial apoptosis of streptozocin-induced diabetic rats. J Clin Biochem Nutr. 41 :58-67.

[0377] Liu-Chittenden Y, et al (2012) Genetic and pharmacological disruption of the TEAD- YAP complex suppresses the oncogenic activity of YAP. Genes Dev. 26: 1300-1305.

[0378] Lovren F, et al. (2010) Adiponectin primes human monocytes into alternative antiinflammatory M2 macrophages. Am. J. Physiol Heart Circ. Physiol 299:H656-H663. [0379] Mantovani A, et al. (2004) The chemokine system in diverse forms of macrophage activation and polarization. Trends Immun. 25:677-86.

[0380] Martinez FO, et al. (2006) Transcriptional profiling of the human monocyte-to- macrophage differentiation and polarization: new molecules and patterns of gene expression. J. Immunol. 177:7303-7311.

[0381] Mizushige K, et al. (2000) Alteration in left ventricular diastolic filling and accumulation of myocardial collagen at insulin-resistant prediabetic stage of a type II diabetic rat model. Circulation. 101 :899-907.

[0382] Moore KJ, et al. (201 1) Macrophages in the pathogenesis of atherosclerosis. Cell. 145:341-55.

[0383] Moore KW, et al. (1993) Interleukin- 10. Annu Rev Immunol. 11 : 165-90.

[0384] Mor A, et al. (201 1) Ras inhibition induces insulin sensitivity and glucose uptake.

PLoS. One. 6:e21712.

[0385] Muller S, et al. (2002) Impaired glucose tolerance is associated with increased serum concentrations of interleukin 6 and co-regulated acute-phase proteins but not TNF-alpha or its receptors. Diabetologia. 45:805-12.

[0386] Neel S, et al. (2011) Induced pluripotent stem (iPS) cells inhibit apoptosis and fibrosis in streptozotocin-induced diabetic rats. Mol. Pharm. 8:2350-2357.

[0387] Oh J, et al. (2012) Endoplasmic reticulum stress controls M2 macrophage differentiation and foam cell formation. J. Biol. Chem. 287: 1 1629-1 1641.

[0388] Parthasarathy S, et al. (1988) Is oxidized low density lipoprotein involved in the recruitment and retention of monocyte/macrophages in the artery wall during the initiation of atherosclerosis? Basic Life Sci. 49:375-380.

[0389] Pickup JC, et al. (2000) Plasma interleukin-6, tumour necrosis factor alpha and blood cytokine production in type 2 diabetes. Life Sci. 67:291-300.

[0390] Pirvola U, et al. (2004) Fgf9 signaling regulates inner ear morphogenesis through epithelial-mesenchymal interactions. Dev. Biol. 273 :350-360.

[0391] Poucher SM, et al. (2012) Effects of saxagliptin and sitagliptin on glycaemic control and pancreatic beta-cell mass in a streptozotocin-induced mouse model of type 2 diabetes. Diabetes Obes Metab. 14:918-26.

[0392] Quan W, et al. (2013) Magnesium lithospermate B reduces myocardial ischemia/reperfusion injury in rats via regulating the inflammation response. Pharm. Biol.

[0393] Razzaque MA, et al. (2013) An endogenously produced fragment of cardiac Myosin- binding protein C is pathogenic and can lead to heart failure. Circ. Res. 1 13 :553-561. [0394] Rocher C, et al. (2012) Bone morphogenetic protein 7 polarizes THP-1 cells into M2 macrophages. Can. J. Physiol Pharmacol. 90:947-951.

[0395] Sawatis K, et al. (2010) Kinins in cardiac inflammation and regeneration: insights from ischemic and diabetic cardiomyopathy. Neuropeptides 44: 119-125.

[0396] Schulz TJ, et al. (201 1) Identification of inducible brown adipocyte progenitors residing in skeletal muscle and white fat. PNAS. 108: 143-8.

[0397] Schwartzbauer G, et al. (2001) The tumor suppressor gene PTEN can regulate cardiac hypertrophy and survival. J Biol Chem. 276:35786-93.

[0398] Seimon T, et al. (2009) Mechanisms and consequences of macrophage apoptosis in atherosclerosis. J Lipid Res. 50 Suppl:S382-7.

[0399] Serova M, et al. (2013) Benchmarking effects of mTOR, PI3K, and dual PI3K/mTOR inhibitors in hepatocellular and renal cell carcinoma models developing resistance to sunitinib and sorafenib. Cancer Chemother. Pharmacol. 71 : 1297-1307.

[0400] Sierra-Filardi E, et al. (2010) Heme Oxygenase- 1 expression in M-CSF-polarized M2 macrophages contributes to LPS-induced IL-10 release. Immunobiology 215:788-795.

[0401] Singla DK, et al. (2007) Transplanted embryonic stem cells following mouse myocardial infarction inhibit apoptosis and cardiac remodeling. Am. J. Physiol Heart Circ. Physiol 293:H1308-H1314.

[0402] Singla DK, et al. (2008) Factors released from embryonic stem cells inhibit apoptosis in H9c2 cells through PI3K/Akt but not ERK pathway. Am. J. Physiol Heart Circ. Physiol 295:H907-H913.

[0403] Singla DK, et al. (201 1) iPS Cells Repair and Regenerate Infarcted Myocardium. Mol. Pharm.

[0404] Singla DK, et al. (2011) TGF-beta2 treatment enhances cytoprotective factors released from embryonic stem cells and inhibits apoptosis in infarcted myocardium. Am. J. Physiol Heart Circ. Physiol 300:H1442-H1450.

[0405] Smith JD, et al. (1995) Decreased atherosclerosis in mice deficient in both macrophage colony-stimulating factor (op) and apolipoprotein E. Proc. Natl. Acad. Sci. U.S. A 92:8264-8268.

[0406] Stolar M. (2010) Glycemic control and complications in type 2 diabetes mellitus. Am J Med. 123 :S3-1 1.

[0407] Su J, et al. (2010) SB203580, a p38 inhibitor, improved cardiac function but worsened lung injury and survival during Escherichia coli pneumonia in mice. J. Trauma 68: 1317- 1327. [0408] Tamaki Y, et al. (2008) Enhanced osteolytic potential of monocytes/macrophages derived from bone marrow after particle stimulation. J. Biomed. Mater. Res. B Appl. Biomater. 84: 191-204.

[0409] Tomanek RJ, et al. (2010) Embryonic coronary vasculogenesis and angiogenesis are regulated by interactions between multiple FGFs and VEGF and are influenced by mesenchymal stem cells. Dev. Dyn. 239:3182-3191.

[0410] Trinanes J, et al. (2012) Type 1 diabetes increases the expression of proinflammatory cytokines and adhesion molecules in the artery wall of candidate patients for kidney transplantation. Diabetes Care 35:427-433.

[041 1] Tseng YH, et al. al. (2008) New role of bone morphogenetic protein 7 in brown adipogenesis and energy expenditure. Nature. 454: 1000-4.

[0412] Van der Mieren G, et al. (2012) Angiotensin-converting enzyme inhibition and food restriction in diabetic mice do not correct the increased sensitivity for ischemia-reperfusion injury. Cardiovasc. Diabetol. 11 :89.

[0413] Van LS, et al. (2007) Anti-inflammatory effects of atorvastatin improve left ventricular function in experimental diabetic cardiomyopathy. Diabetologia 50: 1977-1986.

[0414] van Tits LJ, et al. (2011) Oxidized LDL enhances pro-inflammatory responses of alternatively activated M2 macrophages: a crucial role for Kruppel-like factor 2. Atherosclerosis 214:345-349.

[0415] Varma A, et al. (2012) Anti-inflammatory and cardioprotective effects of tadalafil in diabetic mice. PLoS. One. 7:e45243.

[0416] Wang J, et al. (2013) FGF-2 protects cardiomyocytes from doxorubicin damage via protein kinase C-dependent effects on efflux transporters. Cardiovasc. Res. 98:56-63.

[0417] Westermann D, et al. (2007) Cardioprotective and anti-inflammatory effects of interleukin converting enzyme inhibition in experimental diabetic cardiomyopathy. Diabetes 56: 1834-1841.

[0418] Westermann D, et al. (2007) Tumor necrosis factor-alpha antagonism protects from myocardial inflammation and fibrosis in experimental diabetic cardiomyopathy. Basic Res. Cardiol. 102:500-507.

[0419] White AC, et al. (2007) FGF9 and SHH regulate mesenchymal Vegfa expression and development of the pulmonary capillary network. Development 134:3743-3752.

[0420] Xu JH, et al. (2010) Effects of mouse recombinant bone morphogenetic protein-7 transfection on cell apoptosis, NF-kappaB, and downstream genes in cultured primary cardiomyocytes after simulated ischemia and reperfusion injury. J Cardiovasc Pharmacol. 56:69-77.

[0421] Yamamoto S, et al. (2011) Macrophage polarization by angiotensin II-type 1 receptor aggravates renal injury-acceleration of atherosclerosis. Arterioscler. Thromb. Vase. Biol. 31 :2856-2864.

[0422] Yan B, et al. (2011) Transplanted induced pluripotent stem cells improve cardiac function and induce neovascularization in the infarcted hearts of db/db mice. Mol. Pharm. 8: 1602-1610.

[0423] Yan X, et al. (2013) Temporal dynamics of cardiac immune cell accumulation following acute myocardial infarction. J. Mol. Cell Cardiol. 62:24-35.

[0424] Yu LY, et al. (2007) Effect of fibroblast growth factor 9 on Runx2 gene promoter activity in MC3T3-E1 and C2C12 cells. Chin Med. J. (Engl.) 120:491-495.

[0425] Zeng XY, et al. (2012) Oleanolic acid reduces hyperglycemia beyond treatment period with Akt/FoxOl -induced suppression of hepatic gluconeogenesis in type-2 diabetic mice. PloS One. 7:e42115.

[0426] Zhang X, et al. (2006) Reciprocal epithelial-mesenchymal FGF signaling is required for cecal development. Development 133 : 173-180.

[0427] Bentzon JF, Otsuka F, Virmani R, and Falk E. Mechanisms of plaque formation and rupture. Circulation research 114: 1852-1866, 2014.

[0428] Boon MR, van der Horst G, van der Pluijm G, Tamsma JT, Smit JW, and Rensen PC.

Bone morphogenetic protein 7: a broad-spectrum growth factor with multiple target therapeutic potency. Cytokine & growth factor reviews 22: 221-229, 2011.

[0429] Brown BN, et al. Macrophage polarization: an opportunity for improved outcomes in biomaterials and regenerative medicine. Biomaterials 33 : 3792-3802, 2012.

[0430] Cheng C, et al. Atherosclerotic lesion size and vulnerability are determined by patterns of fluid shear stress. Circulation 1 13 : 2744-2753, 2006.

[0431] Dorai H, Vukicevic S, and Sampath TK. Bone morphogenetic protein-7 (osteogenic protein- 1) inhibits smooth muscle cell proliferation and stimulates the expression of markers that are characteristic of SMC phenotype in vitro. Journal of cellular physiology 184: 37-45, 2000.

[0432] Fadini GP, et al. Monocyte-macrophage polarization balance in pre-diabetic individuals. Acta diabetologica 50: 977-982, 2013. [0433] Guedj K, et al. Ml macrophages act as LTbetaR- independent lymphoid tissue inducer cells during atherosclerosis-related lymphoid neogenesis. Cardiovascular research 101 : 434- 443, 2014.

[0434] Hartman J, and Frishman WH. Inflammation and atherosclerosis: a review of the role of interleukin-6 in the development of atherosclerosis and the potential for targeted drug therapy. Cardiology in review 22: 147-151, 2014.

[0435] Italiani P, and Boraschi D. From Monocytes to M1/M2 Macrophages: Phenotypical vs. Functional Differentiation. Frontiers in immunology 5: 514, 2014.

[0436] Korshunov VA, and Berk BC. Flow- induced vascular remodeling in the mouse: a model for carotid intima-media thickening. Arteriosclerosis, thrombosis, and vascular biology 23 : 2185-2191, 2003.

[0437] Laskin DL. Macrophages and inflammatory mediators in chemical toxicity: a battle of forces. Chemical research in toxicology 22: 1376-1385, 2009.

[0438] Lee MJ, et al. Bone morphogenetic protein-7 inhibits constitutive and interleukin- 1 beta-induced monocyte chemoattractant protein- 1 expression in human mesangial cells: role for J K/AP-1 pathway. Journal of immunology 170: 2557-2563, 2003.

[0439] Lessner SM, Prado HL, Waller EK, and Galis ZS. Atherosclerotic lesions grow through recruitment and proliferation of circulating monocytes in a murine model. The American journal of pathology 160: 2145-2155, 2002.

[0440] Mahmood DF, et al. Truncated thioredoxin (Trx-80) promotes pro-inflammatory macrophages of the Ml phenotype and enhances atherosclerosis. Journal of cellular physiology 228: 1577-1583, 2013.

[0441] Mantovani A, et al. The chemokine system in diverse forms of macrophage activation and polarization. Trends in immunology 25: 677-686, 2004.

[0442] Marie I, et al. Bone morphogenetic protein-7 reduces the severity of colon tissue damage and accelerates the healing of inflammatory bowel disease in rats. Journal of cellular physiology 196: 258-264, 2003.

[0443] Merino H, Parthasarathy S, and Singla DK. Partial ligation-induced carotid artery occlusion induces leukocyte recruitment and lipid accumulation-a shear stress model of atherosclerosis. Molecular and cellular biochemistry 372: 267-273, 2013.

[0444] Meyrelles SS, et al. Endothelial dysfunction in the apolipoprotein E-deficient mouse: insights into the influence of diet, gender and aging. Lipids in health and disease 10: 211,

2011. [0445] Moore KJ, and Tabas I. Macrophages in the pathogenesis of atherosclerosis. Cell 145: 341-355, 201 1.

[0446] Mosser DM, and Edwards JP. Exploring the full spectrum of macrophage activation. Nature reviews Immunology 8: 958-969, 2008.

[0447] Ni CW, et al. Discovery of novel mechanosensitive genes in vivo using mouse carotid artery endothelium exposed to disturbed flow. Blood 1 16: e66-73, 2010.

[0448] Papadopoulou C, et al. The role of the chemokines MCP-1, GRO-alpha, IL-8 and their receptors in the adhesion of monocytic cells to human atherosclerotic plaques. Cytokine 43 : 181-186, 2008.

[0449] Pendse AA, et al. Apolipoprotein E knock-out and knock-in mice: atherosclerosis, metabolic syndrome, and beyond. Journal of lipid research 50 Suppl: S 178-182, 2009.

[0450] Perron JC, and Dodd J. ActRIIA and BMPRII Type II BMP receptor subunits selectively required for Smad4-independent BMP7-evoked chemotaxis. PloS one 4: e8198, 2009.

[0451] Reddick RL, Zhang SH, and Maeda N. Atherosclerosis in mice lacking apo E.

Evaluation of lesional development and progression. Arteriosclerosis and thrombosis : a journal of vascular biology / American Heart Association 14: 141-147, 1994.

[0452] Rocher C, and Singla DK. SMAD-PI3K-Akt-mTOR pathway mediates BMP-7 polarization of monocytes into M2 macrophages. PloS one 8: e84009, 2013.

[0453] Rocher C, et al. Bone morphogenetic protein 7 polarizes THP-1 cells into M2 macrophages. Canadian journal of physiology and pharmacology 90: 947-951, 2012.

[0454] Saika S, et al. Therapeutic effects of adenoviral gene transfer of bone morphogenic protein-7 on a corneal alkali injury model in mice. Laboratory investigation; a journal of technical methods and pathology 85: 474-486, 2005.

[0455] Shimizu T, Kayamori T, Murayama C, and Miyamoto A. Bone morphogenetic protein (BMP)-4 and BMP-7 suppress granulosa cell apoptosis via different pathways: BMP -4 via PI3K/PDK-1/Akt and BMP-7 via PI3K/PDK-1/PKC. Biochemical and biophysical research communications 417: 869-873, 2012.

[0456] Takahashi T, Muneta T, Tsuji K, and Sekiya I. BMP-7 inhibits cartilage degeneration through suppression of inflammation in rat zymosan-induced arthritis. Cell and tissue research 344: 321-332, 2011.

[0457] Torroella-Kouri M, Rodriguez D, and Caso R. Alterations in macrophages and monocytes from tumor-bearing mice: evidence of local and systemic immune impairment. Immunologic research 57: 86-98, 2013. [0458] Urbina P, and Singla DK. BMP-7 attenuates adverse cardiac remodeling mediated through M2 macrophages in prediabetic cardiomyopathy. American journal of physiology Heart and circulatory physiology 307: H762-772, 2014.

[0459] Zhang XL, et al. Bone morphogenic protein-7 inhibits monocyte-stimulated TGF- betal generation in renal proximal tubular epithelial cells. Journal of the American Society of Nephrology: JASN 16: 79-89, 2005.

Claims

CLAIMS What is claimed is:

1. A method of modulating polarization of a population of monocytes in a subject, comprising:

administering to the subject a composition comprising an effective amount of one or more of FGF-9, FGF-8, BMP-7, or a combination thereof, thereby modulating the polarization of a population of monocytes.

2. The method of claim 1, wherein the composition comprises an effective amount of FGF-9.

3. The method of Claim lor 2, wherein the subject has experienced a myocardial infarction.

4. The method of Claim 1 or 2, wherein the subject has diabetes.

5. The method of Claim 1 or 2, wherein the subject has pre-diabetes.

6. The method of Claim 2, wherein the composition comprising FGF-9 is intravenously administered to the subject.

7. The method of Claim 1 or 2, wherein the composition is administered one or more times.

8. The method of Claim 1 or 2, further comprising administering to the subject a second composition comprising an effective amount of M-CSF, an agent that modulates the polarization of monocytes, or a combination thereof.

9. The method of Claim 2, further comprising administering to the subject a second composition comprising an effective amount of FGF-8, BMP-7, or a combination thereof.

10. The method of Claim 8 or Claim 9, wherein the second composition is intravenously administered to the subject.

1 1. The method of Claim 8 or Claim 9, wherein the second composition is administered one or more times.

12. The method of Claim 8 or Claim 9, wherein the second composition polarizes monocytes independently of the composition comprising FGF-9.

13. The method of Claim 8 or Claim 9, wherein the composition comprising FGF-9 and the second composition are co-administered.

14. The method of Claim 8 or Claim 9, wherein the composition comprising FGF-9 and the second composition are separately administered.

15. The method of Claim 8 or Claim 9, wherein the composition comprising FGF-9 and the second composition are administered one or more times.

16. The method of Claim 2, wherein the composition comprising FGF-9 further comprises an effective amount of M-CSF, an agent that modulates the polarization of monocytes, or a combination thereof.

17. The method of Claim 2, wherein the composition comprising FGF-9 further comprises an effective amount of FGF-8, BMP-7, or a combination thereof.

18. The method of Claim 2, wherein the population of monocytes express a FGF receptor.

19. The method of Claim 18, wherein the FGF receptor is FGFR1.

20. The method of Claim 1 or 2, wherein modulating the polarization of the population of monocytes comprises increasing the polarization of the monocytes into M2 macrophages.

21. The method of Claim 1 or 2, wherein modulating the polarization of the population of monocytes comprises decreasing the polarization of monocytes into Ml macrophages.

22. The method of any one of Claims 1-21, further comprising altering one or more administering steps.

23. The method of any of Claims 1-22, wherein the composition comprising FGF-9 changes the phenotype of Ml macrophages into M2 macrophages.

24. A method of modulating polarization of a population of monocytes in a subject, comprising:

administering to the subject a composition comprising an effective amount of BMP-7, thereby modulating the polarization of a population of monocytes.

25. A method of modulating polarization of a population of monocytes in a subject having a skin disease or skin disorder, comprising:

administering to the subject a composition comprising an effective amount of FGF-9, thereby modulating the polarization of a population of monocytes.

26. The method of Claim 25, wherein the skin disease or skin disorder comprises an inflammatory reaction.

27. The method of Claim 25, wherein the subject has experienced a myocardial infarction.

28. The method of Claim 25, wherein the subject has diabetes.

29. The method of Claim 25, wherein the subject has pre-diabetes.

30. The method of Claim 25, wherein the composition comprising FGF-9 is intravenously administered to the subject.

31. The method of Claim 25, wherein the composition comprising FGF-9 is topically administered to the subject.

32. The method of Claim 31, wherein topical administration comprises applying the composition to the subject's skin.

33. The method of Claim 25, wherein the composition is administered one or more times.

34. The method of Claim 25, further comprising administering to the subject a second composition comprising an effective amount of M-CSF, an agent that modulates the polarization of monocytes, or a combination thereof.

35. The method of Claim 25, further comprising administering to the subject a second composition comprising an effective amount of FGF-8, BMP-7, or a combination thereof.

36. The method of Claim 34 or Claim 35, wherein the second composition is intravenously administered to the subject.

37. The method of Claim 34 or Claim 35, wherein the second composition is topically administered to the subject.

38. The method of Claim 34 or Claim 35, wherein topical administration comprises applying the composition to the subject's skin.

39. The method of Claim 34 or Claim 35, wherein the second composition is administered one or more times.

40. The method of Claim 34 or Claim 35, wherein the second composition polarizes monocytes independently of the composition comprising FGF-9.

41. The method of Claim 34 or Claim 35, wherein the composition comprising FGF-9 and the second composition are co-administered.

42. The method of Claim 34 or Claim 35, wherein the composition comprising FGF-9 and the second composition are separately administered.

43. The method of Claim 34 or Claim 35, wherein the composition comprising FGF-9 and the second composition are administered one or more times.

44. The method of Claim 25, wherein the composition comprising FGF-9 further comprises M-CSF, an agent that modulates the polarization of monocytes, or a combination thereof.

45. The method of Claim 25, wherein the composition comprising FGF-9 further comprises FGF-8, BMP-7, or a combination thereof.

46. The method of Claim 25, wherein the population of monocytes express a FGF receptor.

47. The method of Claim 46, wherein the FGF receptor is FGFR1.

48. The method of Claim 25, wherein modulating the polarization of the population of monocytes comprises increasing the polarization of the monocytes into M2 macrophages.

49. The method of Claim 25, wherein modulating the polarization of the population of monocytes comprises decreasing the polarization of monocytes into Ml macrophages.

50. The method of any one of Claims 25-49, further comprising altering one or more administering steps.

51. The method of any of Claims 25-50, wherein the composition comprising FGF-9 changes the phenotype of Ml macrophages into M2 macrophages.

52. A method of ameliorating cellular injury in a subject, comprising:

administering to the subject a composition comprising an effective amount of FGF-9, thereby modulating the polarization of infiltrating monocytes in the subject; and

reducing the severity of the cellular injury in the subject.

53. The method of Claim 52, wherein the subject has experienced a myocardial infarction.

54. The method of Claim 52, reducing the severity of the cellular injury comprises reducing the size of the myocardial infarction, reducing the amount of fibrosis, reducing the amount of apoptosis, or a combination thereof.

55. The method of Claim 52, wherein the subject has diabetes.

56. The method of Claim 52, wherein the subject has pre-diabetes.

57. The method of Claim 52, wherein the composition comprising FGF-9 is intravenously administered to the subject.

58. The method of Claim 52, wherein the composition comprising FGF-9 is administered one or more times.

59. The method of Claim 52, further comprising administering to the subject a second composition comprising an effective amount of M-CSF, an agent that modulates the polarization of monocytes, or a combination thereof.

60. The method of Claim 52, further comprising administering to the subject a second composition comprising an effective amount of FGF-8, BMP-7, or a combination thereof.

61. The method of Claim 59 or Claim 60, wherein the second composition is intravenously administered to the subject.

62. The method of Claim 59 or Claim 60, wherein the second composition is administered one or more times.

63. The method of Claim 59 or Claim 60, wherein the second composition polarizes monocytes independently of the composition comprising FGF-9.

64. The method of Claim 59 or Claim 60, wherein the composition comprising FGF-9 and the second composition are co-administered.

65. The method of Claim 59 or Claim 60, wherein the composition comprising FGF-9 and the second composition are separately administered.

66. The method of Claim 59 or Claim 60, wherein the composition comprising FGF-9 and the second composition are administered one or more times.

67. The method of Claim 52, wherein the composition comprising FGF-9 further comprises M-CSF, an agent that modulates the polarization of monocytes, or a combination thereof.

68. The method of Claim 52, wherein the composition comprising FGF-9 further comprises FGF-8, BMP-7, or a combination thereof.

69. The method of Claim 52, wherein modulating the polarization of the infiltrating monocytes comprises increasing the polarization of the monocytes into M2 macrophages.

70. The method of Claim 52, wherein modulating the polarization of the infiltrating monocytes comprises decreasing the polarization of monocytes into Ml macrophages.

71. The method of Claim 52, wherein, following the administration of the composition comprising FGF-9, blood velocity is improved, fibrosis is reduced, or a combination thereof.

72. The method of Claim 52, wherein, following the administration of the composition comprising FGF-9, cardiac function is enhanced.

73. The method of Claim 52, wherein enhanced cardiac function comprises (i) improving left ventricular function, (ii) improving fractional shortening, (iii) improving ejection fraction, (iv) reducing end-diastolic volume, (v) decreasing left ventricular mass, and (v) normalizing of heart geometry, or (vi) a combination thereof.

74. The method of any one of Claims 52-73, further comprising altering one or more administering steps.

75. A method of ameliorating cellular injury in a subject, comprising:

administering to the subject a composition comprising an effective amount of one or more of FGF-9, FGF-8, BMP-7, or a combination thereof, thereby modulating the polarization of infiltrating monocytes in the subject; and

reducing the severity of the cellular injury in the subject.

76. A method of ameliorating cellular injury in a subject having a skin disease or skin disorder, comprising:

reducing the severity of the skin disease or skin disorder in the subject.

77. The method of Claim 76, wherein the skin disease or skin disorder comprises an inflammatory reaction.

78. The method of Claim 76, wherein the subject has experienced a myocardial infarction.

79. The method of Claim 76, wherein the subject has diabetes.

80. The method of Claim 76, wherein the subject has pre-diabetes.

81. The method of Claim 76, wherein the composition comprising FGF-9 is intravenously administered to the subject.

82. The method of Claim 76, wherein the composition comprising FGF-9 is topically administered to the subject.

83. The method of Claim 82, wherein topical administration comprises applying the composition to the subject's.

84. The method of Claim 76, wherein the composition comprising FGF-9 is administered one or more times.

85. The method of Claim 76, further comprising administering to the subject a second composition comprising an effective amount of M-CSF, an agent that modulates the polarization of monocytes, or a combination thereof.

86. The method of Claim 76, further comprising administering to the subject a second composition comprising an effective amount of FGF-8, BMP-7, or a combination thereof.

87. The method of Claim 85 or Claim 86, wherein the second composition is intravenously administered to the subject.

88. The method of Claim 85 or Claim 86, wherein the second composition is administered one or more times.

89. The method of Claim 85 or Claim 86, wherein the second composition polarizes monocytes independently of the composition comprising FGF-9.

90. The method of Claim 85 or Claim 86, wherein the composition comprising FGF-9 and the second composition are co-administered.

91. The method of Claim 85 or Claim 86, wherein the composition comprising FGF-9 and the second composition are separately administered.

92. The method of Claim 85 or Claim 86, wherein the composition comprising FGF-9 and the second composition are administered one or more times.

93. The method of Claim 76, wherein modulating the polarization of the infiltrating monocytes comprises increasing the polarization of the monocytes into M2 macrophages.

94. The method of Claim 76, wherein modulating the polarization of the infiltrating monocytes comprises decreasing the polarization of monocytes into Ml macrophages.

95. The method of Claim 76, wherein, following the administration of the composition comprising FGF-9, blood velocity is improved, fibrosis is reduced, or a combination thereof.

96. The method of Claim 76, wherein, following the administration of the composition comprising FGF-9, cardiac function is enhanced.

97. The method of Claim 96, wherein enhanced cardiac function comprises (i) improving left ventricular function, (ii) improving fractional shortening, (iii) improving ejection fraction, (iv) reducing end-diastolic volume, (v) decreasing left ventricular mass, and (v) normalizing of heart geometry, or (vi) a combination thereof.

98. The method of any one of Claims 76-97, further comprising altering one or more administering steps.

99. A method of modulating polarization of a population of monocytes, comprising:

(i) administering FGF-9 to a population of monocytes; and

(ii) modulating the polarization of the population of monocytes.

100. A method of modulating polarization of a population of monocytes, comprising:

(i) administering to a population of monocytes one or more of FGF-9, FGF-8, BMP-7, or a combination thereof; and

(ii) modulating the polarization of the population of monocytes.

101. A method of modulating cellular phenotype of macrophages, comprising:

(i) administering FGF-9 to a population of macrophages; and

(ii) modulating the cellular phenotype of the population of macrophages such that the macrophages exhibit characteristics of M2 macrophages.

102. A method of modulating cellular phenotype of macrophages, comprising:

(i) administering to a population of macrophages one or more of FGF-9, FGF-8, BMP-7, or a combination thereof; and

103. A method of ameliorating cellular injury in a subject in need thereof, comprising:

(i) administering FGF-9 to the subject;

(ii) modulating the polarization of infiltrating monocytes in the subject; and

(iii) increasing the expression of anti-inflammatory cytokines in the subject.

104. A method of ameliorating cellular injury in a subject in need thereof, comprising:

(i) administering to the subject one or more of FGF-9, FGF-8, BMP-7, or a combination thereof;

(ii) modulating the polarization of infiltrating monocytes in the subject; and

(iii) increasing the expression of anti-inflammatory cytokines in the subject.

105. A method of attenuating fibrosis in a subject in need thereof, comprising: (i) administering FGF-9 to the subject;

(ii) modulating the polarization of infiltrating monocytes in the subject; and

(iii) increasing the expression or activity of one or more anti-inflammatory cytokines in the subject.

106. A method of attenuating fibrosis in a subject in need thereof, comprising:

(ii) modulating the polarization of infiltrating monocytes in the subject; and

107. A method of modulating FGF receptor expression in monocytes, comprising:

(i) administering FGF-9 to a population of monocytes; and

(ii) increasing the expression of a FGF receptor in the monocytes.

108. A method of modulating FGF receptor expression in monocytes, comprising:

(ii) increasing the expression of a FGF receptor in the monocytes.

109. A pharmaceutical composition, comprising:

FGF-9;

M-CSF, an agent that modulates the polarization of monocytes, or a combination thereof; and

a pharmaceutically acceptable carrier.

1 10. The composition of Claim 109, wherein the composition acts at a FGF receptor.

1 11. The composition of Claim 109, wherein the FGF receptor is FGFR1.

1 12. The composition of Claim 109, wherein the composition modulates the polarization of a population of monocytes.

1 13. The composition of Claim 109, wherein the population of monocytes are infiltrating monocytes.

1 14. The composition of Claim 109, wherein modulating the polarization of a population of monocytes comprises increasing the polarization of the monocytes into M2 macrophages.

1 15. The composition of Claim 109, wherein modulating the polarization of a population of monocytes comprises decreasing the polarization of the monocytes into Ml macrophages.

1 16. The composition of Claim 109, wherein the composition changes the phenotype of Ml macrophages into M2 macrophages.

1 17. The composition of any one of Claims 109-1 15, wherein the population of monocytes is in a subject.

1 18. The composition of Claim 109, wherein the subject has experienced a myocardial infarction.

1 19. The composition of Claim 109, wherein the subject has diabetes.

120. The composition of Claim 109, wherein the subject has pre-diabetes.

121. The composition of Claim 109, wherein the composition increases the expression of one or more anti-inflammatory cytokines.

122. The composition of Claim 109, wherein anti-inflammatory cytokines comprise IL-10 and IL-lra.

123. The composition of Claim 109, wherein the composition decreases the expression of one or more pro-inflammatory cytokines.

124. The composition of Claim 109, wherein pro-inflammatory cytokines comprise interleukin-6, monocyte chemotactic protein- 1, and tumor necrosis factor alpha.

125. The composition of Claim 109, wherein the composition ameliorates cellular injury.

126. The composition of Claim 125, wherein ameliorating cellular injury comprises reducing the size of the myocardial infarction, reducing the amount of fibrosis in the infarcted tissue, reducing the amount of apoptosis in the infarcted tissue, or a combination thereof.

127. The composition of Claim 109, wherein the composition enhances cardiac function is enhanced.

128. The method of Claim 127, wherein enhancing cardiac function comprises (i) improving left ventricular function, (ii) improving fractional shortening, (iii) improving ejection fraction, (iv) reducing end-diastolic volume, (v) decreasing left ventricular mass, and (v) normalizing of heart geometry, or (vi) a combination thereof.

129. A pharmaceutical composition, comprising:

FGF-9, FGF-8, BMP-7, or a combination thereof; and

a pharmaceutically acceptable carrier.

130. A pharmaceutical composition, comprising:

FGF-9;

FGF-8, BMP-7, or a combination thereof; and

a pharmaceutically acceptable carrier.

131. The composition of Claim 130, wherein the composition acts at a FGF receptor.

132. The composition of Claim 131, wherein the FGF receptor is FGFR1.

133. The composition of Claim 130, wherein the composition modulates the polarization of a population of monocytes.

134. The composition of Claim 130, wherein the population of monocytes are infiltrating monocytes.

135. The composition of Claim 133, wherein modulating the polarization of a population of monocytes comprises increasing the polarization of the monocytes into M2 macrophages.

136. The composition of Claim 133, wherein modulating the polarization of a population of monocytes comprises decreasing the polarization of the monocytes into Ml macrophages.

137. The composition of Claim 130, wherein the composition changes the phenotype of Ml macrophages into M2 macrophages.

138. The composition of any one of Claims 130-136, wherein the population of monocytes is in a subject.

139. The composition of Claim 130, wherein the subject has experienced a myocardial infarction.

140. The composition of Claim 130, wherein the subject has diabetes.

141. The composition of Claim 130, wherein the subject has prediabetes.

142. The composition of Claim 130, wherein the subject has a skin disease or skin disorder.

143. The composition of Claim 130, wherein the skin disease or skin disorder comprises an inflammatory reaction.

144. The composition of Claim 130, wherein the composition increases the expression of one or more anti-inflammatory cytokines.

145. The composition of Claim 144, wherein anti-inflammatory cytokines comprise IL-10 and IL-lra.

146. The composition of Claim 130, wherein the composition decreases the expression of one or more pro-inflammatory cytokines.

147. The composition of Claim 146, wherein pro-inflammatory cytokines comprise interleukin-6, monocyte chemotactic protein- 1, and tumor necrosis factor alpha.

148. The composition of Claim 130, wherein the composition ameliorates cellular injury.

149. The composition of Claim 148, wherein ameliorating cellular injury comprises reducing the size of the myocardial infarction, reducing the amount of fibrosis in the infarcted tissue, reducing the amount of apoptosis in the infarcted tissue, or a combination thereof.

150. The composition of Claim 130, wherein the composition enhances cardiac function is enhanced.

151. The method of Claim 150, wherein enhancing cardiac function comprises (i) improving left ventricular function, (ii) improving fractional shortening, (iii) improving ejection fraction, (iv) reducing end-diastolic volume, (v) decreasing left ventricular mass, and (v) normalizing of heart geometry, or (vi) a combination thereof.

152. The method of claim 24, wherein the subject is pre-diabetic.

153. The method of claim 24, wherein the subject has diabetes.

154. The method of claim 24, wherein the subject has atherosclerosis.

155. The method of claim 24, wherein modulating the polarization of the population of monocytes comprises increasing the polarization of the monocytes into M2 macrophages.

156. The method of claim 152, wherein modulating the polarization of the population of monocytes comprises decreasing the polarization of monocytes into Ml macrophages.

157. The method of claim 152, wherein the composition comprising BMP-7 changes the phenotype of Ml macrophages into M2 macrophages.

158. A method of treating a subject with atherosclerosis comprising administering a composition comprising an effective amount of BMP-7.

159. The method of claim 159, wherein plaque formation is reduced.

160. The method of claim 159, wherein monocyte infiltration into arteries is reduced.

161. The method of claim 159, wherein one or more pro-inflammatory cytokines are reduced.

162. The method of claim 162, wherein the pro-inflammatory cytokine is TNF-a, MCP-1, IL-6, or a combination thereof.

163. The method of claim 159, wherein one or more anti-inflammatory cytokines are reduced.

164. The method of claim 164, wherein the anti-inflammatory cytokine is IL-10, IL-1RA, Arginase 1, or a combination thereof.

165. The method of claim 159, wherein the BMP-7 causes an increase in M2 macrophages.

166. The method of claim 166, wherein the increase in M2 macrophages is due to an increase in monocyte to M2 macrophage polarization.

167. A method of increasing BMP-7 receptor (BMP-7R) on M2 macrophages comprising administering a composition comprising an effective amount of BMP-7.