WO2015164800A2 - Compositions et procédés permettant de moduler la polarisation des monocytes - Google Patents

Compositions et procédés permettant de moduler la polarisation des monocytes Download PDF

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WO2015164800A2
WO2015164800A2 PCT/US2015/027614 US2015027614W WO2015164800A2 WO 2015164800 A2 WO2015164800 A2 WO 2015164800A2 US 2015027614 W US2015027614 W US 2015027614W WO 2015164800 A2 WO2015164800 A2 WO 2015164800A2
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composition
fgf
monocytes
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polarization
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WO2015164800A3 (fr
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Dinender Singla
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University Of Central Florida Research Foundation, Inc.
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1875—Bone morphogenic factor; Osteogenins; Osteogenic factor; Bone-inducing factor
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1825—Fibroblast growth factor [FGF]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/193—Colony stimulating factors [CSF]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0014—Skin, i.e. galenical aspects of topical compositions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner

Definitions

  • MI Myocardial infarction
  • 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.
  • monocyte 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.
  • Ml major classical
  • M2 alternative
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • composition comprising FGF-9, FGF-8, BMP-
  • composition comprising (i) FGF-9; (ii) FGF-8,
  • BMP-7 BMP-7, or a combination thereof; and (iii) a pharmaceutically acceptable carrier.
  • composition comprising FGF-9, FGF-8, BMP-7,
  • M-CSF or a combination thereof, and a pharmaceutically acceptable carrier.
  • 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. 1A shows percentage of Arginase-1 (M2 macrophages) cells expressing p-FGFRl.
  • 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).
  • 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).
  • 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).
  • 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) ⁇
  • 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.
  • 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.
  • IL-10 and IL-1 receptor antagonist IL-lra
  • 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).
  • 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).
  • 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).
  • 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).
  • 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).
  • 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).
  • 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).
  • 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 ⁇ -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).
  • 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).
  • 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).
  • FIG. 16B shows quantitative analysis of percent monocytes.
  • 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
  • FIG. 16D shows quantitative analysis of the TNF-a immunostaining.
  • 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. 17B shows quantitative analysis of the IL-6 immunostaining.
  • 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. 18B shows quantitative analysis of DHE immunostaining.
  • 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. 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. 20A-FIG. 20C are graphs (20B and 20C) and photomicrographs (20A) showing that BMP-7 blunted cardiac fibrosis in the pre-diabetic heart.
  • 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).
  • 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).
  • 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),
  • FIG. 20B shows quantitative analysis of percent M2 macrophages.
  • FIG. 2 IE shows quantitative analysis of IL-10 expression per group.
  • 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).
  • Figures 23A and 23B are 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.
  • 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).
  • 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).
  • FIGS 25A, 25B, 25C, and 25D are graphs showing pro-inflammatory cytokine secretion is significantly diminished following BMP-7 treatment.
  • D Transcribed IL-6 is significantly enhanced in the PLCA group whereas IL-6 mRNA is significantly diminished following BMP-7 treatment.
  • U arbitrary units. *p ⁇ 0.05 vs. sham and #p ⁇ 0.05 vs. PLCA.
  • Figure 26A and 26B are photomicrographs (26A) and a graph (26B) showing post- PLCA ligation, BMP-7 enhances M2 macrophage differentiation.
  • FIGS 27A, 27B, 27C, and 27D are graphs showing that BMP-7 enhances circulating anti-inflammatory cytokine secretion.
  • U arbitrary units. *p ⁇ 0.05 vs. sham and #p ⁇ 0.05 vs. PLCA.
  • 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.
  • 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 ⁇ and 25 ⁇ , respectively.
  • BMP-7 treatment significantly enhances BMP-7R on monocytes (left histogram).
  • 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.
  • 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.
  • FIGS 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.
  • 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.
  • White boxes are enlarged in merged images in e, j, and o. Scale bar 100 ⁇ . 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 ⁇ -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 ⁇ . 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.
  • 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 ⁇ - actin (b, g, and 1), total DAPI (c, h, and m), and merged images (d, i, and n).
  • 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.
  • 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 ⁇ -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 ⁇ . n 4 animals/group.
  • 34B quantitative analysis of percent M2 macrophages.
  • 34E quantitative analysis of IL-10 expression per group.
  • FIGS. 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.
  • 35B quantitative analysis of DHE immunostaining.
  • 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.
  • Figures 37A, 37B, and 37C are graphs (37B and 37C) and photomicrographs (37A) showing that BMP-7 blunts cardiac fibrosis in the PD heart.
  • 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.
  • 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.
  • compositions comprising active molecules, which may be used in methods disclosed herein.
  • 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.
  • 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.
  • a composition can act at a FGF receptor.
  • a FGF receptor can be FGFR1.
  • a disclosed composition can modulate the polarization of a population of monocytes.
  • modulating the polarization of a population of monocytes can comprise increasing the polarization of the monocytes into M2 macrophages.
  • modulating the polarization of a population of monocytes can comprise decreasing the polarization of monocytes into Ml macrophages.
  • 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.
  • a disclosed composition can change the phenotype of Ml macrophages into M2 macrophages.
  • a population of monocytes can be infiltrating monocytes.
  • a population of monocytes can be in a subject.
  • 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.
  • a subject can have a skin disease or disorder.
  • 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.
  • a disclosed composition can increase the expression of one or more antiinflammatory cytokines.
  • anti-inflammatory cytokines can comprise IL-10 and IL-lra.
  • a composition can decrease the expression of one or more proinflammatory cytokines.
  • pro-inflammatory cytokines can comprise interleukin- 6, monocyte chemotactic protein- 1, and tumor necrosis factor alpha.
  • a disclosed composition can ameliorate cellular injury.
  • 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.
  • a disclosed composition can enhance cardiac function.
  • 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.
  • a pharmaceutical composition comprising (i) FGF-9; (ii) FGF-8, BMP-7, or a combination thereof; and optionally, (iii) a pharmaceutically acceptable carrier.
  • a composition can comprise FGF-9, FGF-8, and a pharmaceutically acceptable carrier.
  • a pharmaceutical composition can comprise FGF-9, BMP- 7, and a pharmaceutically acceptable carrier.
  • a pharmaceutical composition can comprise FGF-9, FGF-8, BMP-7, and a pharmaceutically acceptable carrier.
  • a disclosed composition can act at a FGF receptor.
  • a FGF receptor can be FGFRl .
  • a composition can modulate the polarization of a population of monocytes.
  • modulating the polarization of a population of monocytes can comprise increasing the polarization of the monocytes into M2 macrophages.
  • modulating the polarization of a population of monocytes can comprise decreasing the polarization of monocytes into Ml macrophages.
  • 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.
  • a composition can change the phenotype of Ml macrophages into M2 macrophages.
  • a population of monocytes can be infiltrating monocytes.
  • a population of monocytes can be in a subject.
  • 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.
  • 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.
  • a subject can have a skin disease or skin disorder.
  • a skin disease or skin disorder can comprise an inflammatory reaction.
  • a disclosed composition can increase the expression of one or more antiinflammatory cytokines.
  • anti-inflammatory cytokines can comprise IL-10 and IL-lra.
  • a composition can decrease the expression of one or more proinflammatory cytokines.
  • pro-inflammatory cytokines can comprise interleukin- 6, monocyte chemotactic protein- 1, and tumor necrosis factor alpha.
  • a disclosed composition can ameliorate cellular injury.
  • 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.
  • a disclosed composition can ameliorate one or more signs or symptoms associated with a skin disease or disorder.
  • a disclosed composition can enhance cardiac function.
  • 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.
  • composition comprising FGF-9, FGF-8, BMP-7, or a combination thereof; and and for a pharmaceutical composition, further comprise a pharmaceutically acceptable carrier.
  • 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.
  • 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.
  • 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.
  • 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.
  • a composition disclosed herein for example, a composition comprising FGF-9 can be intravenously administered to a subject.
  • a composition can be administered one or more times.
  • 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.
  • a composition disclosed herein, for example, a composition comprising FGF-9 can be administered continuously.
  • 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.
  • 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.
  • a second composition can be intravenously administered to a subject.
  • a second composition can be administered one or more times.
  • a second composition can polarize monocytes independently of a composition comprising FGF-9.
  • 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.
  • a second composition can be administered continuously.
  • 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.
  • a disclosed method can comprise separately administering a composition comprising FGF-9 and a second composition.
  • a disclosed method can comprise administering a composition comprising FGF-9 and a second composition one or more times.
  • 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.
  • 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.
  • 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.
  • 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.
  • a disclosed method of modulating polarization of a population of monocytes a population of monocytes can express a FGF receptor.
  • a FGF receptor can be FGFR1.
  • a disclosed method of modulating polarization of a population of monocytes can comprise altering one or more administering steps.
  • 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.
  • 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.
  • a skin disease or skin disorder can comprise an inflammatory reaction.
  • a disclosed composition can ameliorate one or more signs or symptoms associated with a skin disease or disorder.
  • 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.
  • 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.
  • 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.
  • a composition disclosed herein for example, a composition comprising FGF-9 can be intravenously administered to a subject.
  • a composition disclosed herein, for example, a composition comprising FGF-9 can be topically administered to a subject.
  • topical administration can comprise applying a composition to a subject's skin.
  • a composition can be administered one or more times.
  • 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.
  • a composition comprising FGF-9 can be administered continuously.
  • 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.
  • 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.
  • a second composition can be intravenously administered to a subject.
  • a second composition can be topically administered to a subject.
  • topical administration can comprise applying a composition to a subject's skin.
  • a composition can be administered one or more times.
  • 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.
  • a second composition can be administered continuously.
  • a second composition can polarize monocytes independently of a composition comprising FGF-9.
  • 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.
  • a disclosed method can comprise separately administering a composition disclosed herein, for example, a composition comprising FGF-9 and a second composition.
  • a disclosed method can comprise administering a composition comprising FGF-9 and a second composition one or more times.
  • 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.
  • 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.
  • 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.
  • a population of monocytes can express a FGF receptor.
  • a FGF receptor can be FGFR1.
  • a disclosed method of modulating polarization of a population of monocytes can comprise altering one or more administering steps.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • a disclosed composition disclosed herein for example, a composition comprising FGF-9 can be intravenously administered to a subject.
  • a composition can be administered one or more times.
  • 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.
  • a composition comprising FGF-9 can be administered continuously.
  • 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.
  • 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.
  • a second composition can be intravenously administered to a subject.
  • a second composition can be administered one or more times.
  • 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.
  • a second composition can be administered continuously.
  • a second composition can polarize monocytes independently of a composition disclosed herein, for example, a composition comprising FGF-9.
  • 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.
  • a disclosed method can comprise separately administering a composition disclosed herein, for example, a composition comprising FGF-9 and a second composition.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • a composition comprising FGF-9 blood velocity can be improved, fibrosis can be reduced, or a combination thereof.
  • cardiac function can be enhanced.
  • 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.
  • a disclosed method ameliorating cellular injury in a subject can comprise altering one or more administering steps.
  • 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.
  • 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.
  • 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.
  • 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.
  • a skin disease or skin disorder can comprise an inflammatory reaction.
  • 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.
  • 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.
  • a composition disclosed herein for example, a composition comprising FGF-9 can be intravenously administered to a subject.
  • a composition disclosed herein, for example, a composition comprising FGF-9 can be topically administered to a subject.
  • topical administration can comprise applying the composition to a subject's skin.
  • a composition can be administered one or more times.
  • composition disclosed herein for example, a composition comprising FGF-9
  • 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.
  • a composition disclosed herein, for example, a composition comprising FGF-9 can be administered continuously.
  • 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.
  • 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.
  • a second composition can be intravenously administered to a subject.
  • a second composition can be topically administered to a subject.
  • topical administration can comprise applying a composition to a subject's skin.
  • a composition can be administered one or more times.
  • 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.
  • a second composition can be administered continuously.
  • a second composition can polarize monocytes independently of a composition comprising FGF-9.
  • 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.
  • a disclosed method can comprise separately administering a composition disclosed herein, for example, a composition comprising FGF-9 and a second composition.
  • 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.
  • 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.
  • cardiac function can be enhanced.
  • 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.
  • a disclosed composition can ameliorate one or more signs or symptoms associated with a skin disease or disorder.
  • a disclosed method of ameliorating cellular injury in a subject can comprise altering one or more administering steps.
  • 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.
  • 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.
  • 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.
  • 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.
  • a composition disclosed herein for example, a composition comrpising FGF-9
  • 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
  • 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.
  • 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.
  • 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.
  • 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.
  • a composition disclosed herein for example, a composition comprising FGF-9 can be intravenously administered to a subject.
  • a composition can be administered one or more times.
  • 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.
  • a composition comprising FGF-9 can be administered continuously.
  • 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.
  • a second composition can be administered continuously.
  • an effective amount of a composition disclosed herein, for example, a composition comprising FGF-9 can be intravenously administered to a subject.
  • a composition disclosed herein, for example, a composition comprising FGF-9 can be topically administered to a subject.
  • topical administration can comprise applying the composition to a subject's skin.
  • a composition disclosed herein, for example, a composition comprising FGF-9 can be administered one or more times.
  • 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.
  • FGF-9 can be administered continuously.
  • a disclosed method of ameliorating cellular injury in a subject can comprise altering one or more administering steps.
  • 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.
  • 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.
  • 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.
  • a composition disclosed herein for example, a composition comprising one or more of FGF-9, FGF-8, BMP-7, or a combination thereof.
  • 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.
  • 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.
  • 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.
  • a subject can have a skin disease or disorder or the subject can have an inflammatory disease or disorder.
  • a disclosed composition can ameliorate one or more signs or symptoms associated with a skin disease or disorder.
  • a composition disclosed herein for example, a composition comprising FGF-9 can be intravenously administered to a subject.
  • a composition can be administered one or more times.
  • 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.
  • a composition disclosed herein, for example, a composition comprising FGF-9 can be administered continuously.
  • a composition comprising FGF-9 blood velocity can be improved, fibrosis can be reduced, or a combination thereof.
  • cardiac function can be enhanced.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • BMP-7R BMP-7 receptor
  • 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.
  • 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.
  • 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.
  • a disclosed method can optionally comprise one or more additional steps, such as, for example, repeating an administering step or altering an administering step.
  • 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.).
  • the subject of the herein disclosed methods can be a vertebrate, such as a mammal, a fish, a bird, a reptile, or an amphibian.
  • 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.
  • a subject can be a human patient.
  • 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.
  • a patient can have an inflammatory disorder.
  • a patient can have one or more diseases or disorder and can have experienced a myocardial infarction.
  • 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).
  • 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).
  • active treatment that is, treatment directed specifically toward the improvement of a disease, pathological condition, or disorder
  • causal treatment that is, treatment directed toward removal of the cause of the associated disease, pathological condition, or disorder.
  • 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.
  • 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.
  • a mammal e.g., a human
  • 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.
  • preventing monocyte infiltration is intended.
  • preventing monocyte polarization into Ml macrophages is intended.
  • diagnosisd 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.
  • diagnosis 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.
  • diagnosisd 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.
  • 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.
  • 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.
  • 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
  • the identification can, in one aspect, be performed by a person different from the person making the diagnosis.
  • the administration can be performed by one who performed the diagnosis.
  • administering refers 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.
  • a preparation can be administered therapeutically; that is, administered to treat an existing disease or condition.
  • a preparation can be administered prophylactically; that is, administered for prevention of a disease or condition.
  • 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.
  • 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.
  • altering one or more administering steps can comprise altering the administration of one or more disclosed compositions or disclosed complexes.
  • 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.
  • 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.
  • altering one or more administering steps can comprise altering the administering of a disclosed composition.
  • contacting 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).
  • an intended target such as, e.g., a cell or population of cells, a receptor, an antigen, or other biological entity
  • 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.
  • a cell or population of cells such as, for example, a population of monocytes, such as infiltrating monocytes.
  • 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.
  • 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.
  • aqueous and nonaqueous carriers, diluents, solvents or vehicles examples 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.
  • ethanol polyols
  • carboxymethylcellulose such as glycerol, propylene glycol, polyethylene glycol and the like
  • vegetable oils such as olive oil
  • 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.
  • 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.
  • 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.
  • Peptide refers to any peptide, oligopeptide, polypeptide, gene product, expression product, or protein.
  • 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.
  • 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).
  • enzyme refers to any peptide that catalyzes a chemical reaction of other substances without itself being destroyed or altered upon completion of the reaction.
  • 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.
  • 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.
  • ATCC American Type Culture Collection
  • eukaryotic 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.
  • 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.
  • "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.
  • 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.
  • 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 ⁇ , 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.
  • 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.
  • IL interleukin
  • IL-lra interleukin-l receptor antagonist
  • 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.
  • 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).
  • modulate is meant to alter, i.e., by an increase or a decrease.
  • 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.
  • 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.
  • 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.
  • modulation of the prevalence of a specific cell type does not have to be complete.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • a preparation can be administered in a "prophylactically effective amount"; that is, an amount effective for prevention of a disease or condition.
  • 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.
  • 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.
  • Thl and Th2 cells see Nagai et al, 2001.
  • 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.
  • compositions of the invention 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.
  • FGF-9 fibroblast growth factor
  • FGF-2 fibroblast growth factor
  • 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).
  • MI myocardial infarction
  • fibrosis fibrosis
  • 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).
  • 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).
  • 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).
  • 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.
  • FGFR1 FGF receptor 1
  • 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).
  • M2 macrophages were confirmed with specific marker arginase 1 (FIG. 1C, FIG. ID).
  • FIG. 1C The number of M2 macrophages expressing FGFRl increased following FGF-9 treatment (FIG. 1C, FIG. ID).
  • 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.
  • 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.
  • HM cells 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.
  • mice The effect of MI on db/db mice was examined.
  • MI was performed as described herein (Yan et al, 201 1; Singla et al, 2007, 2011a, and 201 1b).
  • animals were injected intramyocardially with either mouse recombinant FGF-9 (100 ng/kg) or saline.
  • FGF-9 100 ng/kg
  • 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).
  • 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.
  • FIG. 10A shows confocal images of Ml macrophages stained for iNOS antibody
  • 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).
  • 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.
  • 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).
  • 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.
  • M2 polarized macrophages were 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.
  • 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.
  • 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).
  • the MI model is associated with ⁇ 5% mortality in db/db mice.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • Ml macrophage-specific iNOS, IL- ⁇ , and TNF-a gene expression was evaluated using real-time PCR.
  • Arginase-1 gene (specific for M2 macrophages) was also determined.
  • 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.
  • 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
  • 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.
  • 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.
  • FGF-9R homo sapiens fibroblast growth factor 9
  • 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.
  • 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.
  • FGF-9 independently causes M2 macrophage polarization from monocytes or whether FGF-9 acts in synergy with M-CSF is examined.
  • mice 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).
  • M-CSF 250 ⁇ g/kg per injection; i.v., Asakura et. al.
  • M-CSF was given 24 hrs prior to the day of surgery and at any other given times for FGF-9.
  • hearts were removed, and subjected to histological (H&E) and immunohistochemistry analysis.
  • H&E histological
  • M-CSF plasma concentrations were examined using ELISA (Ray Biotech). Echocardiography wass used to determine heart function.
  • mice 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 ⁇ g/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.
  • FGF-9 and M-CSF have a common pathway for M2 polarized macrophages. 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.
  • 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 ⁇ - 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 ⁇ -cell function.
  • T1DM Type 1 Diabetes Mellitus
  • T2DM Type 2 Diabetes Mellitus
  • PD pre-diabetes
  • IGF impaired fasting glucose
  • ITT impaired glucose tolerance
  • cardiovascular disease D'Souza et al, 2011
  • nephropathy Stolar 2010
  • 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).
  • PD pre-diabetic
  • 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 ⁇ g/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).
  • M2 macrophage polarization and associated anti-inflammatory cytokines were enhanced, adverse cardiac remodeling was reduced, and cardiac function improved (p ⁇ 0.05).
  • PD cardiomyopathy was associated with increased monocyte infiltration and release of pro-inflammatory cytokines, which contributed to adverse cardiac remodeling and cardiac dysfunction.
  • BMP-7 possessed therapeutic potential in its ability to polarize monocytes into M2 macrophages and confer cardiac protection in the PD heart.
  • Increased monocyte infiltration can be present along with an increased inflammatory immune response, which contributes to adverse cardiac remodeling, in streptozotocin (STZ) induced cardiomyopathy.
  • STZ streptozotocin
  • BMP-7 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.
  • the data indicates that there was attenuated cardiomyocyte apoptosis, fibrosis, and improved cardiac function following BMP-7 treatment.
  • 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.
  • ELISA 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).
  • 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.
  • TUNEL Terminal deoxynucleotidyl Transferase dUTP-Mediated Nick-End Labeling
  • EDV end diastolic volume
  • ESV end systolic volume
  • EF ejection fraction
  • LVIDd left ventricular internal dimension-diastole
  • LVIDs left ventricular internal dimension-systole
  • FS fractional shortening
  • 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).
  • 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).
  • 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).
  • FIG. 17A contains representative images of heart sections from each group stained with IL-6, sarcomeric ⁇ -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.
  • 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).
  • ROS reactive oxygen species
  • 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).
  • 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).
  • TUNEL sarcomeric a-actin
  • caspase-3 sarcomeric a-actin
  • DAPI DAPI and representative images from the PD group are shown in FIG. 19C.
  • PTEN phosphatase and tensin homo log
  • AKT a pro-survival and cell cycle regulator
  • 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 mm 2 vs.
  • PD 0.170 + 0.0159 mm 2 , 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).
  • 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).
  • IL-1RA interleukin-1 receptor antagonist
  • IL-10 interleukin-10
  • IL-10 analyses performed on heart sections stained with IL-10, sarcomeric a-actin, and DAPI (FIG.
  • FIG. 22A 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).
  • 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).
  • PTEN is a tumor suppressor that regulates the pro-survival protein, AKT, through inhibition of its upstream activator, PI3K.
  • BMP-7 inhibited the expression of cardiac p-PTEN while significantly up-regulating p-AKT in the PD heart.
  • M2 macrophages are a class of macrophages that reduce inflammation by secreting IL-10, an anti- inflammatory cytokine (Laskin, 2009).
  • 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.
  • BMP-7 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).
  • BMP-7 inhibits TNF-a induced apoptosis in PD cardiomyopathy by up-regulating IL-10.
  • BMP-7 inhibits TNF-a induced apoptosis in PD cardiomyopathy by up-regulating IL-10.
  • 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.
  • Atherosclerosis 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.
  • MI myocardial infarction
  • 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.
  • M2 macrophages 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).
  • 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.
  • IL-IRA interleukin- 1 receptor antagonist
  • IL-10 interleukin-10
  • Arginase 1 Arginase 1.
  • Bone morphogenetic proteins 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.
  • inflammation mimicry media.
  • 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.
  • 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.
  • LCA left carotid artery
  • 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.
  • 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.
  • PLCA-BMP-7 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 ⁇ serial sections, and placed on ColorFrostTM 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.
  • NASH Image J software
  • 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).
  • CD 14 monoocytes, Abbiotec, #25156, 1 :50
  • iNOS Ml macrophages, Abeam, #abl29372, 1 :50
  • CD206 M2 macrophages, Abeam, #ab64693, 1 :50.
  • BMP-7R BMP-7 receptor
  • 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-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.
  • 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)
  • 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.
  • Doppler vascular settings three to four 2D images for systolic (SV; cm/s) blood velocity were obtained and averaged.
  • FIG. 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).
  • 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).
  • 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
  • FIG. 26A-1 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).
  • a decrease in M2 macrophage populations was observed in the PLCA group relative to the sham group ( Figure 26).
  • 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).
  • M2 macrophages secrete proto-typical anti-inflammatory cytokines including IL- lRA, IL-10, and Arginase 1.
  • 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).
  • BMP-7 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
  • BMP-7R BMP-7 receptor
  • 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.
  • LDLs low-density lipoproteins
  • WBCs white blood cells
  • 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.
  • monocytes 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.
  • 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.
  • cytokines including IL-1RA, IL-10, and Arginase 1.
  • 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.
  • 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.
  • 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.
  • M2 macrophage differentiation and anti-inflammatory cytokine release were also evaluated.
  • 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.
  • 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.
  • 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.
  • 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.
  • BMPRs BMP receptors
  • the BMPR2 identified as BMP-7R within the current manuscript
  • BMP-7R BMP-7 receptors
  • 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.
  • 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.
  • 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.
  • 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 ⁇ -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 ⁇ -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.
  • PD prediabetes
  • 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.
  • STZ streptozotocin
  • 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.
  • BMP-7 bone morphogenetic protein-7
  • 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.
  • mice [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.
  • animals were additionally treated with an intravenous injection of 200 ⁇ g/kg BMP-7, as previously reported, on days 3-5.
  • 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.
  • 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.
  • DAPI diamidino-2-phenylindole
  • 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.
  • FIG. 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).
  • 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).
  • FIG. 2C shows representative photomicrographs of heart sections from each group stained with TNF-a (a, f, and k), sarcomeric ⁇ -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).
  • FIG. 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).
  • FIG. 34A Representative images of heart sections stained with CD206, an M2 macrophage marker, sarcomeric ⁇ -actin, and DAPI are shown in Fig. 34A.
  • M2 macrophage concentration were observed between control and PD groups.
  • 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).
  • 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.
  • FIG 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).
  • FIG. 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).
  • PD group 1.305 ⁇ 0.134 AU, P ⁇ 0.05; Fig. 36D).
  • PD + BMP-7 group 0.715 ⁇ 0.0784 AU vs. PD group: 0.500 ⁇ 0.107 AU, P ⁇ 0.05; Fig. 36E).
  • BMP-7 ameliorates cardiac remodeling in PD mice.
  • 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 mm 2 vs.
  • PD group 0.170 ⁇ 0.0159 mm 2 , P ⁇ 0.05; Fig. 37B).
  • 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).
  • Exogenous BMP-7 improves cardiac function in PD mice.
  • the raw echocardiogram data obtained are shown in Fig. 38A.
  • 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).
  • 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.
  • Ml macrophages are proinflammatory and heighten the inflammatory response
  • M2 macrophages are anti-inflammatory and express cytokines that mitigate inflammation.
  • M2 macrophages are a class of macrophages that reduce inflammation by secreting IL-10, an antiinflammatory cytokine.
  • IL-10 has been reported to modulate inflammation by regulating the secretion pattern of proinflammatory cytokines from inflammatory cells.
  • infiltrated monocytes, in the presence of BMP-7 polarize into beneficial M2 macrophages.
  • Anti-inflammatory effects of BMP-7 have been previously reported.
  • monocytes express receptors for BMP-7 and, in the presence of BMP-7, differentiate into beneficial M2 macrophages in vitro.
  • BMP-7 promotes the differentiation of mesenchymal stem cells and white adipocytes into brown adipose tissue.
  • BMP-7 administration significantly enhanced IL-10 secretion.
  • 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.
  • IL-IRA another anti-inflammatory cytokine
  • BMP-7 confers cardioprotection by directing monocyte differentiation into M2 macrophages and promoting the secretion of antiinflammatory mediators.
  • diabetes results in cardiac oxidative stress.
  • a previous study has indicated elevated levels of ROS in the STZ-induced diabetic heart.
  • increased levels of ROS and blunted expression of the antioxidant MnSOD were detected in PD hearts relative to control hearts within the present study.
  • treatment with BMP-7 increased cardiac MnSOD expression and reduced oxidative stress in the PD heart.
  • PTEN is a tumor suppressor that regulates the prosurvival protein Akt through inhibition of its upstream activator, PI3K.
  • BMP-7 inhibits the expression of cardiac p- PTEN while significantly upregulating p-Akt in the PD heart.
  • 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.
  • the PD myocardium is infiltrated with monocytes and subsequent secreted proinflammatory cytokines.
  • monocytes polarize into beneficial M2 macrophages, thereby inhibiting inflammation and protecting the PD heart from cardiomyocyte apoptosis, fibrosis, and cardiac dysfunction.
  • 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.
  • Cifarelli V et al. (2007) Increased Expression of Monocyte CDl lb (Mac-1) in
  • 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.
  • Galkina E et al. (2009) Immune and inflammatory mechanisms of atherosclerosis (*). Annu Rev Immunol. 27: 165-97.
  • Gay D et al. (2013) Fgf9 from dermal gammadelta T cells induces hair follicle neogenesis after wounding. Nat. Med. 19:916-923.
  • 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.
  • 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.
  • 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.
  • Mor A, et al. (201 1) Ras inhibition induces insulin sensitivity and glucose uptake.
  • 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.
  • Singla DK, et al. (201 1) iPS Cells Repair and Regenerate Infarcted Myocardium. Mol. Pharm.
  • 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.
  • 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.
  • 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.
  • Fadini GP et al. Monocyte-macrophage polarization balance in pre-diabetic individuals. Acta diabetologica 50: 977-982, 2013.
  • 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.
  • 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.
  • 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.
  • 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,
  • 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.
  • 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.

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Abstract

Selon un aspect, la présente divulgation concerne des compositions contenant le facteur FGF-9 et des procédés d'utilisation des compositions décrites. Selon un autre aspect, la présente divulgation concerne des compositions contenant un ou plusieurs facteurs FGF-9, FGF-8, et/ou protéines BMP-7 et des procédés d'utilisation des compositions décrites. Les compositions et les procédés ci-décrits permettent de moduler la polarisation des monocytes. Le présent abrégé est destiné à servir d'outil d'exploration à des fins de recherche dans la technique particulière et n'est pas destiné à limiter la présente divulgation.
PCT/US2015/027614 2014-04-24 2015-04-24 Compositions et procédés permettant de moduler la polarisation des monocytes WO2015164800A2 (fr)

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