US20120014879A1

US20120014879A1 - USE OF TUMOR NECROSIS FACTOR-alpha RECEPTOR p75 FOR TREATMENT OF ISCHEMIA-INDUCED NEOVASCULARIZATION

Info

Publication number: US20120014879A1
Application number: US12/091,405
Authority: US
Inventors: Douglas W. Losordo; David A. Goukassian
Original assignee: St Elizabeths Medical Center of Boston Inc
Current assignee: STEWARD RESEARCH AND SPECIALTY PROJECTS Corp
Priority date: 2005-10-24
Filing date: 2006-10-24
Publication date: 2012-01-19
Also published as: US9360526B2; EP1948816A4; US20160376645A1; DK1948816T3; US20130040300A9; US20110059435A1; US10837050B2; EP1948816B1; US10150991B2; CY1112525T1; HK1121193A1; EP2428579A1; WO2007050465A2; EP2428579B1; PL1948816T3; WO2007050465A3; ES2381204T3; US20200123600A1; ATE536420T1; EP1948816A2

Abstract

Improvements on the basic method used for BEAMing increase sensitivity and increase the signal-to-noise ratio. The improvements have permitted the determination of intrinsic error rates of various DNA polymerases and have permitted the detection of rare and subtle mutations in DNA isolated from plasma of cancer patients.

Description

BACKGROUND OF THE INVENTION

Aging is associated with an increased risk of atherosclerotic disease of the coronary and peripheral arteries. In either vascular system, the extent of ischemic damage and degree of subsequent functional recovery after arterial obliteration largely depends on the development of new collateral blood vessels. Aging is associated with impaired angiogenesis in murine and rabbit limb ischemia models. Aging is also accompanied by a steady decline in immune functions, such as defects in signaling pathways and altered expression of cytokines, such as interferon-gamma (IFNγ) and vascular endothelial growth factor (VEGF). Angiogenesis is associated with perivascular inflammation and monocyte/macrophage accumulation.
Tumor necrosis factor alpha (TNF-α), a macrophage/monocyte-derived pluripotent mediator, can function as an angiogenic factor in one system and as an anti-angiogenic factor in another. These mutually exclusive effects have been attributed to TNF-α concentration and duration of exposure; that is, low concentrations and short exposure is angiogenic, whereas high concentrations and prolonged exposure is anti-angiogenic. TNF-α has been reported to induce the expression of many important immune- and angiogenesis-related genes through two different TNF-α receptors: TNF-αR1 (p55) and TNF-αR2 (p75). In various vascular endothelial cells, TNF-α increased the expression of the well-known angiogenic factors VEGF, basic fibroblast growth factor (bFGF), and interleukin-6 (IL-6). The role of the two distinct TNF-α receptors in mediating these responses are still unclear.
Given that angiogenesis is impaired in elderly individuals, this dysfunction may be related to alterations in TNF-α receptor expression. Methods of enhancing blood flow in the elderly are required to treat or prevent ischemia.

SUMMARY OF THE INVENTION

The invention generally provides methods and compositions for modulating p75 receptor/TNFR2 expression for the treatment or prevention of ischemia.
In one aspect, the invention generally features a method of treating, reducing the severity of, or preventing ischemia in a subject having or at risk of developing ischemia. The method involves contacting a cell of the subject (e.g., human or veterinary patient) with a nucleic acid molecule encoding a p75/TNFR2 polypeptide or a fragment thereof; and expressing the p75/TNFR2 polypeptide in the cell, where the method treats or prevents ischemia in the subject.
In another aspect, the invention generally features a method of enhancing angiogenesis in a tissue before, during, or after an ischemic event. The method involves contacting a cell with a nucleic acid molecule encoding a p75/TNFR2 polypeptide or a fragment thereof; and expressing the p75/TNFR2 polypeptide in the cell, where the method enhances angiogenesis in the tissue.
In yet another aspect, the invention generally features a method of enhancing angiogenesis in a subject. The method involves contacting a cell isolated from the subject with a nucleic acid molecule encoding a p75/TNFR2 polypeptide or a fragment thereof; and administering the cell to the subject, where the method enhances angiogenesis.
In yet another aspect, the invention generally features method of reducing apoptosis in a subject having or at risk of developing ischemia. The method involves contacting a cell of the subject with a nucleic acid molecule encoding a p75 TNFR2 receptor polypeptide or a fragment thereof; and expressing the p75 TNFR2 receptor polypeptide in the cell, where the method reduces apoptosis in the subject.
In one embodiment of the above aspects, the method enhances the local release of angiogenic growth factors and cytokines in the tissue. In another embodiment of the above aspects, the method further includes the steps of administering to the subject an angiogenic factor selected from the group consisting of: vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), basic fibroblast growth factor (bFGF), angiopoietin 1, angiopoietin 2 and monocyte chemotactic protein-1 (MCP-1). In yet another embodiment of the above aspects, the method further includes the steps of administering to the subject an endothelial cell mitogen selected from the group consisting of acidic and basic fibroblast growth factors, vascular endothelial growth factor, epidermal growth factor, transforming growth factor α and β, platelet-derived endothelial growth factor, platelet-derived growth factor, tumor necrosis factor α, hepatocyte growth factor, insulin like growth factor, erythropoietin, colony stimulating factor, macrophage-CSF, granulocyte/macrophage CSF and nitric oxide synthase. In yet another embodiment of the above aspects, the cell is in vivo or in vitro. In yet another embodiment of the above aspects, the method further comprises the step of delivering the cell to a subject (e.g., the cell is delivered directly to an ischemic tissue or the cell is delivered systemically). In still another embodiment of the above aspects, the nucleic acid molecule is present in a vector (e.g., a viral vector). In another embodiment of the above aspects, the nucleic acid molecule is positioned for expression.
In another aspect, the invention generally features a method of enhancing p75/TNFR2 expression in a cell. The method involves contacting a cell with a nucleic acid molecule encoding a p75/TNFR2 polypeptide or a fragment thereof; and expressing the p75/TNFR2 polypeptide in the cell (e.g., a cell in vitro or in vivo).
In a related aspect, the invention generally features an expression vector comprising a nucleic acid molecule encoding a mammalian p75/TNFR2 polypeptide or a fragment thereof operably linked to a promoter sufficient to direct expression of the p75 TNFR2 receptor polypeptide in a cell. In one embodiment, the expression vector is a mammalian expression vector, such as an expression vector suitable for expression in a human cell. In another embodiment, the expression vector is a viral expression vector. In yet another embodiment, the viral vector is selected from the group consisting of adenoviral vectors, adeno-associated viral vectors, retroviral vectors, lentiviral vectors, alphaviral vectors, and herpes virus vectors. In another embodiment, the mammalian p75/TNFR2 polypeptide is a murine polypeptide or a human polypeptide. In another embodiment, the promoter drives expression in an endothelial progenitor cell or in a bone marrow derived cell.
In another related aspect, the invention generally features a host cell (e.g., a mammalian cell, such as a human or murine cell) comprising the vector of any previous aspect. In one embodiment, the cell is an endothelial progenitor cell or is a bone marrow-derived cell. In another embodiment, the cell is in vitro or in vivo.
In another aspect, the invention generally features a pharmaceutical composition comprising an effective amount of an expression vector encoding a human p75/TNFR2 polypeptide or a fragment thereof in a pharmaceutically acceptable excipient, where the p75/TNFR2 polypeptide is operably linked to a promoter sufficient to drive expression of the p75/TNFR2 polypeptide in a mammalian cell. In one embodiment, the vector is a viral vector. In another embodiment, the promoter is sufficient to drive expression in an endothelial progenitor cell or a bone marrow-derived cell.
In yet another aspect, the invention generally features kit for the treatment or prevention of ischemia, the kit comprising an effective amount of an expression vector encoding a human p75/TNFR2 polypeptide or a fragment thereof in a pharmaceutically acceptable excipient, where the p75/TNFR2 polypeptide is operably linked to a promoter sufficient to drive expression of the p75/TNFR2 polypeptide in a mammalian cell.
In yet another aspect, the invention generally features a method of monitoring a subject being treated for ischemia. The method involves administering a treatment that enhances the expression of a p75/TNFR2 polypeptide in a cell of the subject; and measuring angiogenesis in a tissue of the subject relative to a reference, where an increase in angiogenesis indicates a reduced severity of ischemia in the subject.
In a related aspect, the invention generally features a method of monitoring a subject being treated for ischemia. The method involves administering a treatment that enhances the expression of a p75/TNFR2 polypeptide in a cell of the subject; and measuring apoptosis in a tissue of the subject relative to a reference, where a decrease in apoptosis indicates a reduced severity of ischemia in the subject. In one embodiment, the reference is the level of angiogenesis or apoptosis previously present in the subject or in a biological sample derived from the subject at an earlier time point. In another embodiment, the reference is a baseline level of apoptosis or angiogenesis present prior to therapy. In yet another embodiment, the reference is the level of angiogenesis or apoptosis present in a normal subject (e.g., a human subject) sample.
In another aspect, the invention generally features a method for identifying a candidate compound useful for the treatment of ischemia. The method involves contacting a cell expressing p75/TNFR2 nucleic acid molecule with a candidate compound; and detecting an increase in p75/TNFR2 expression in the cell relative to a reference, where an increase in p75/TNFR2 expression identifies the candidate compound as a compound useful for the treatment of ischemia. In one embodiment, the method identifies a compound that increases transcription or translation of a p75/TNFR2 nucleic acid molecule.
In a related aspect, the invention generally features an method for identifying a candidate compound a candidate compound useful for the treatment of ischemia. The method involves contacting a cell expressing p75/TNFR2 polypeptide with a candidate compound; and detecting an increase in the level of p75/TNFR2 polypeptide in the cell relative to a reference level, where an increase in the level of p75/TNFR2 polypeptide identifies a candidate compound useful for the treatment of ischemia.
In another related aspect, the invention generally features a method for identifying a candidate compound useful for the treatment of ischemia. The method involves the steps of: contacting a cell expressing a p75/TNFR2 polypeptide with a candidate compound; and detecting an increase in the biological activity of the p75/TNFR2 polypeptide in the cell contacted with the candidate compound with a reference level of biological activity where the candidate compound as a candidate compound that useful for the treatment of ischemia.
In various embodiments of any of the above aspects, the method further includes the step of delivering the cell to a subject (e.g., a human or veterinary patient) having or at risk of developing ischemia. In still other embodiments of the above aspects, the cell is a mammalian cell (e.g., a human or murine cell), such as an endothelial progenitor cell or bone marrow derived cell. In yet other embodiments of the above aspects, the method includes administering an angiogenic factor or an endothelial cell mitogen. Factors that can be administered in combination with a method of the invention include, for example, TNF, TGF-α, TGF-β, hemoglobin, interleukin-1, interleukin-2, interleukin-3, interleukin-4, interleukin-5, interleukin-6, interleukin-7, interleukin-8, interleukin-9, interleukin-10, interleukin-11, interleukin-12 etc., GM-CSF, G-CSF, M-CSF, human growth factor, co-stimulatory factor B7, insulin, factor VIII, factor IX, PDGF, EGF, NGF, IL-ira, EPO, β-globin. Other factors that may be administered in combination with any of the above methods include endothelial cell mitogens, acidic and basic fibroblast growth factors, vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), platelet-derived endothelial cell growth factor (PD-ECGF), hepatocyte growth factor (HGF), insulin like growth factor (IGF), erythropoietin, colony stimulating factor (CSF), macrophage-CSF (M-CSF), granulocyte/macrophage CSF (GM-CSF), monocyte chemotactic protein-1, and nitric oxide synthase (NOS).

Definitions

By “p75/TNFR2 polypeptide” is meant a protein or fragment thereof having substantial identity to the amino acid sequence of p75/TNFR2 provided at GenBank Accession No. NP_—001057 that promotes angiogenesis or has TNF binding activity.
By “p75/TNFR2 nucleic acid molecule” is meant a polynucleotide that encodes a p75/TNFR2 polypeptide.
By “p75/TNFR2 biological activity” is meant TNF binding activity or angiogenesis enhancing activity.
By “angiogenesis” is meant any alteration that benefits tissue perfusion. Angiogenesis includes the growth by sprouting of endothelial cells from existing blood vessels or the remodeling of existing vessels to alter size, maturity, direction or flow properties to improve blood perfusion of tissues. In one embodiment, angiogenesis increases the density of an existing vascular network. Angiogenesis is measured by any method known in the art, including by determining the number of capillaries per muscle fiber.
By “apoptosis” is meant the process of cell death wherein a dying cell displays a set of well-characterized biochemical hallmarks that include cell membrane blebbing, cell soma shrinkage, chromatin condensation, and DNA laddering. Cells that die by apoptosis include neurons (e.g., during the course of a stroke or ischemic injury) and myocytes, such as cardiomyocytes (e.g., after myocardial infarction or over the course of congestive heart failure).
By “effective amount” is meant the amount of a compound required to prevent, treat, or ameliorate the symptoms of a disease.
By “enhance” is meant increase. For example, an increase of at least 5%, 10%, 25%, 50%, 75% or 100% relative to a reference.
By “ischemia” is meant reduced blood flow to a tissue or organ relative to the level required for the maintenance of normal cell metabolism. Exemplary ischemic events include primary myocardial infarction, secondary myocardial infarction, angina pectoris (including both stable and unstable angina), congestive heart failure, sudden cardiac death, cerebral infarction, restenosis, syncope, ischemia, reperfusion injury, vascular occlusion, carotid obstructive disease, transient ischemic attack, and the like.
By “angiogenic factor” is meant any polypeptide or fragment thereof that enhances angiogenesis.
By “endothelial cell mitogen” is meant any polypeptide or fragment thereof that supports the proliferation of an endothelial cell.
By “ameliorate” is meant decrease, suppress, attenuate, diminish, arrest, or stabilize the development or progression of a disease.
By “disease” is meant any condition or disorder that damages or interferes with the normal function of a cell, tissue, or organ. Examples of diseases include bacterial invasion or colonization of a host cell.
An “expression vector” is a nucleic acid construct, generated recombinantly or synthetically, bearing a series of specified nucleic acid elements that enable transcription of a particular gene in a host cell. Typically, gene expression is placed under the control of certain regulatory elements, including constitutive or inducible promoters, tissue-preferred regulatory elements, and enhancers.
By “fragment” is meant a portion of a protein or nucleic acid that is substantially identical to a reference protein or nucleic acid. In some embodiments the portion retains at least 50%, 75%, or 80%, or more preferably 90%, 95%, or even 99% of the biological activity of the reference protein or nucleic acid described herein.
By “immunological assay” is meant an assay that relies on an immunological reaction, for example, antibody binding to an antigen. Examples of immunological assays include ELISAs, Western blots, immunoprecipitations, and other assays known to the skilled artisan.
By “isolated nucleic acid molecule” is meant a nucleic acid (e.g., a DNA) that is free of the genes which, in the naturally-occurring genome of the organism from which the nucleic acid molecule of the invention is derived, flank the gene.
By “operably linked” is meant that the polynucleotide of the invention (e.g., a DNA molecule) is positioned adjacent to a DNA sequence that directs transcription and translation of the sequence (i.e., facilitates the production of, for example, a recombinant polypeptide of the invention, or an RNA molecule).
By “promoter” is meant a polynucleotide sufficient to direct transcription.
By “reduces” or “increases” is meant a negative or positive alteration, respectively, of at least 10%, 25%, 50%, 75%, or 100%.
By “reference” is meant a standard or control condition.
By “subject” is meant a mammal, including, but not limited to, a human or non-human mammal, such as a bovine, equine, canine, ovine, or feline.
“Therapeutic compound” means a substance that has the potential of affecting the function of an organism. A therapeutic compound may decrease, suppress, attenuate, diminish, arrest, or stabilize the development or progression of disease or disorder an organism.
By “vector” is meant a DNA molecule, usually derived from a plasmid or bacteriophage, into which fragments of DNA may be inserted or cloned. A recombinant vector will contain one or more unique restriction sites, and may be capable of autonomous replication in a defined host or vehicle organism such that the cloned sequence is reproducible. A vector contains a promoter operably linked to a gene or coding region such that, upon transfection into a recipient cell, an RNA is expressed.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1G show that ischemia-induced angiogenesis is impaired in old TNFR2 KO mice. FIG. 1A is a graph showing blood flow recovery presented as the laser Doppler perfusion ratio up to 28 days after hind limb surgery in wild-type (WT) young mice (FIG. 1A) (black bars, n=15); wild-type old mice (gray bars, n=12), and young mice lacking (i.e., having a p75 knock-out (p75KO)) (clear bars, n=15). FIG. 1B shows blood flow recovery in old mice lacking p75 (p75KO) (black bars, n=12) and (inset) a representative image of 5 old p75KO mice showing autoamputation of operated limb. FIG. 1C (upper panel) shows representative images of hind limb muscles immunostained with isolectin B4 to identify capillaries. FIG. 1C (lower panel) is a graph showing relative capillary density at post-operative day twenty-eight in young wild-type (WT), in young mice lacking p75 (p75KO) and old wild-type mice. Capillary density is expressed as a number of capillaries per muscle fiber. Results represent the mean±SEM of 12 randomly chosen areas (0.06 mm²) of muscle fiber from 3 mice per genotype. FIG. 1D is a graph showing the kinetics of mobilization of bone marrow-derived endothelial progenitor cells (EPCs) into peripheral blood (PB) after hind limb surgery in wild-type (WT) and p75KO mice, measured by staining the mononuclear (MN) fraction of PB with antibodies against VEGF receptors Flk1 and Sca1. FIG. 1E shows five (41.6%) p55KO mice that underwent hindlimb surgery and has intact limbs at the time post-ischemic recovery was complete. Limbs were evaluated 28 days post-surgery. FIG. 1F shows two animals (16.8%) that manifested distal toe necrosis (shown with white arrows). FIG. 1G shows that only five (41.6%) old p55KO mice lost their limbs 2-3 weeks after hind limb surgery.

FIGS. 2A and B show that ischemia-induced VEGF expression is lower in the limbs of p75KO mice. FIG. 2A is a series of eight representative confocal images of ischemic muscles from young wild-type (FIG. 2A, upper panel) and p75KO mice (FIG. 2A, lower panel) after hind limb surgery stained with anti-VEGF antibodies. Fluorescent cells are TUNEL-positive cells that are presumably apoptotic. Blue fluorescent cells represent TopRo3-positive cells used to visualize nuclei. FIG. 2B shows densitometric analysis of VEGF, bFGF, and angiopoeitin-1 mRNA expression, using multiprobe RPA, in post-ischemic HIND LIMB muscle tissue homogenates of wild-type (clear bars) and p75KO (black bars) mice three and ten days post-surgery.

FIGS. 3A and 3B show VEGF mRNA expression, determined by RT-PCR, in post-ischemic hind limb muscle tissue homogenates. FIG. 3A is a graph showing mRNA expression in wild-type (WT) (clear bars) and p75KO (black bars)

mice

3 and 10 days post-surgery. FIG. 3B shows representative confocal images of ischemic muscles from young (8-10 weeks old) WT and p75KO mice after hindlimb surgery stained with anti-VEGF antibodies (fluorescence). Post-ischemic muscles of at least 3 animals per genotype/time point were examined and similar VEGF expression pattern, as shown in these representative images, was observed in the tissues of WT vs. p75KO mice.

FIGS. 4A-4C shows that ischemia-induced apoptosis is greater in the limbs of p75KO mice. FIG. 4A is a series of twelve representative merged images of triple stained hind limb muscle of wild-type (upper panel) and p75KO (lower panel) mice up to 10 days after hind limb ischemia (HU) surgery showing a significant increase in the number of TUNEL/EC (+) cells in

p75KO mice

1 and 10 days post-hind limb surgery. FIG. 4B is a graph that provides a quantitative analysis of immunofluorescence (number of double TUNEL/EC positive cells) in at least 8-10 randomly selected areas of approximately 10,600 μm2 (measured by computer assisted software of the confocal microscope) of hind limb muscle from at least 3 animals/treatment group. All samples were coded, and then evaluated by a single blinded observer to eliminate the bias and inter-observer variability. Compared to wild-type, by

days

1 and 10 there was a statistically significant increase in the number of TUNEL/EC (+) cells in p75KO mice after HL surgery (day 1—26±5 vs. 7±5; and day 10—37±8 vs. 3±0.5, p<0.0001, p75KO vs. WT mice), representing a 67% and 93% increase in EC apoptosis in p75KO mice on

days

1 and 10, respectively. FIG. 4C is a series of four representative confocal images of triple stained hindlimb muscles of p75KO mice. TUNEL positive/endothelial cells (EC) are visualized 1 day after hind limb surgery. Arrows show a few examples of immunostaining with Isolectin B4, which identified endothelial lineage cells, TUNEL-identified apoptotic cells, TopRo3-visualized nuclei, and merged image to identify single, double, and triple-stained cells.

FIGS. 5A-5D show that loss of p75 impairs the function of cultured endothelial progenitor cells (EPCs). FIG. 5A is a graph showing that no difference was found in the migration of wild-type (WT) and P75KO bone marrow-derived EPCs toward specific chemotactic agents. Graphs represent data pooled from independent experiments completed in triplicate. FIG. 5B is a series of eight photomicrographs showing that baseline and TNF-induced tubulogenesis was inhibited in bone marrow-derived p75KO or wild-type WT) EPCs, as assessed by the ability to form tube-like structures on VEGF-enriched matrigel. FIG. 5C shows a representative radiogram of multiprobe RPA after TNF stimulation using 5 μg total RNA/lane. FIG. 5D is a series of three graphs, which provide a graphic representation of bFGF, VEGF and angiopoetin-1 mRNA expression after densitometric analysis. All values are adjusted relative to actin mRNA expression.

FIGS. 6A-6E show that signaling through TNFR2 p75 is required for NFκB VEGF gene expression. FIG. 6A is a pair of representative confocal images of NFκB nuclear translocation in wild-type and p75KO EPCs 30 minutes after treatment with TNF. NFκB nuclear translocation was evaluated by immunostaining with NFκB (p65) antibodies. FIG. 6B shows NFκB DNA binding activity in TNF-treated wild-type and p75KO EPCs up to two hours after TNF treatment. Specificity of NFκB bands was confirmed by (25× mutant NF_B/p65 competition—

lane

5 and 25× cold probe competition—lane 6). FIG. 6C shows VEGF promoter activity in TNF-treated wild-type (WT) and p75KO EPCs transfected with VEGF/luciferase reporter plasmid deletion constructs. Results are expressed relative to control pGL2 plasmid activity and represent data pooled from independent experiments completed in triplicate. FIG. 6D is a schematic diagram showing VEGF promoter deletion constructs (kind gift of Dr. Debabrata Mukhopadhyay, Beth Israel Deaconess Medical Center, Boston, Mass.). A 2.6 kb promoter fragment represents complete promoter region of VEGF gene and contains several consensus transcriptional response elements: AP-1, AP-2, GATA, IL-6RE, hypoxia-inducible enhancer sequences, several SP1 sites and clusters and several estrogen response elements (EREs) and NFkB putative but not classical response elements. A 0.35 kb promoter contains only two putative NFkB sites (bp −220 and −125, relative to the transcription origination site) and one Sp1 cluster and a 0.07 kb promoter contains an incomplete Sp1 cluster and has no remaining promoter activity. FIG. 6E is a graph that shows a concentration-dependent increase in VEGF promoter activity in wild-type cells treated with 1 and 20 ng/ml TNF, whereas 40 ng/ml TNF treatment inhibited the activity of VEGF promoter, consistent with the previous reports of low TNF doses being angiogenic and high doses being antiangiogenic. To determine an angiogenic TNF concentration in EPCs from WT and p75KO mice these cells were transfected with 2.6 kb VEGF/Luciferase promoter constructs and treated with different concentrations of TNF (1 ng, 20 ng and 40 ng/ml) for 16 hours. No increase in VEFG promoter activity was observed in p75KO EPCs after treatment with any of the TNF concentrations. Hence, in subsequent studies 1 ng/ml TNF concentration was used. Moreover, 0.35 kb VEGF promoter construct was used to elucidate the TNF-mediated NFkB-dependent regulation of VEGF gene expression in EPCs from WT and p75KO mice.

FIG. 7 provides a diagram of murine bone marrow transplantation models.

FIGS. 8A and 8B are graphs showing the evaluation of bone barrow engraftment and peripheral blood mononuclear cells in wild-type and p75KO mice. FIG. 8A is a graph showing that by day 28 recipients bone marrow (BM) was almost completely (88-93%) reconstituted with donor marrow and no difference between wild-type or p75KO engraftment was observed. FIG. 8B is a graph showing that although, percent of GFP (+) mononuclear cells in wild-type and p75KO bone marrow-transplanted mice was 26-34% lower than in wild-type GFP control mice the number of bone marrow -derived peripheral blood mononuclear cells (PB MNCs) were similar in recipient-mice of both genotypes, as evaluated by FACS analysis.

FIGS. 9A-9D show that p75/TNFR2 is required for efficient contribution of bone marrow-derived EPCs in post-ischemic recovery. FIG. 9A is a graph showing limb autoamputation after hind limb surgery in old p75KO mice transplanted 28 days prior to hind limb surgery with bone marrow-derived mononuclear cells from young wild-type (WT) and p75KO mice (n=4 per transplanted group). FIG. 9B is a graph showing loss of muscle tissue after hind limb surgery in old wild-type (WT) mice transplanted twenty-eight days prior to hind limb surgery with bone marrow-derived mononuclear cells from young wild-type and p75KO mice (n=7-8 per transplanted group). FIG. 9C shows representative images of old (10-12 months) p75KO mice transplanted with bone marrow from young (3-4 weeks) p75KO and WT mice (n=4/BMT group). 50% of old p75KO mice transplanted with bone marrow from p75KO mice (upper panel) have lost limbs (indicated by arrows) by day 14 post-HL surgery, whereas 100% of mice transplanted with WT BM (lower panel) preserved limbs. FIG. 9D shows representative images of old (8-10 months) WT mice transplanted with bone marrow from young (3-4 weeks) WT and p75KO mice (n=8/ bone marrow transplant (BMT) group). Although, no limb loss was observed in mice of either bone marrow transplant group, compared to the WT bone marrow transplant group there was more than 50% statistically significant muscle loss in p75KO bone marrow transplant group (indicated by arrow).

FIG. 10A shows representative confocal images of non-ischemic (right) mouse limb 56 days after transplantation with bone marrow-derived mononuclear cells from WT/GFP mice and 28 days after hind limb surgery on contralateral (left) limb. Immunofluorescence in the far left panels is TopRo3 that was used to visualize nuclei. No GFP-positive cells were detected in non-ischemic limbs of operated mice. Intact muscle fibers emit negligible autofluorescence. FIG. 10B shows representative confocal images of operated ischemic limb in the border zone (indicated by dotted line). Intact and ischemic areas of operated limb muscle fibers were first delineated after hematoxylin and eosin (H&E) staining in adjacent sections. GFP-positive cells (green fluorescence) horned only to the ischemic areas of operated limbs. FIG. 10C shows a representative confocal images of operated limbs showing abundance of GFP-positive bone marrow-derived cells in ischemic areas.

FIGS. 11A and 11B show representative confocal images of ischemic and ischemic border zone muscles of operated limbs of mice transplanted with WT/GFP bone marrow-derived mononuclear cells 28 days prior to hind limb surgery. Immunostaining with isolectin B4 (middle panels) identifies endothelial lineage cells and GFP-positive cells (left panels) identifies bone marrow-derived cells. In merged images, double-positive positive (right panels) cells represent bone marrow-derived EPCs.

DETAILED DESCRIPTION OF THE INVENTION

In general, the invention provides compositions and methods for modulating p75 receptor/TNFR2 expression or activity for the prevention or treatment of ischemia.
As reported in more detail below, the invention is based in part on the discovery that signaling through the p75 receptor/TNFR2 was required for collateral vessel development in ischemia-induced neovascularization. Accordingly, the invention provides methods and compositions for treating or preventing ischemia by enhancing p75 receptor/TNFR2 signaling to increase the growth of collateral blood vessels.
p75/TNFR2
The p75 receptor/TNFR2 is a 415-amino acid polypeptide with a single membrane-spanning domain and has an extracellular domain with sequence similarity to nerve growth factor receptor (Schall et al., “Molecular cloning and expression of a receptor for human tumor necrosis factor,” Cell 61: 361-370, 1990). Human p75/TNFR2 gene spans nearly 43 kb and consists of 10 exons and 9 introns (Beltinger et al., Physical mapping and genomic structure of the human TNFR2 gene. Genomics 35: 94-100, 1996). The amino acid sequence of p75/TNFR2 is provided at GenBank Accession No. NP_—001057. A nucleic acid sequence encoding the p75/TNFR2 polypeptide is provided at NM_—001066.

P75/TNFR2 Polypeptides and Analogs

Overexpression of a P75/TNFR2 polypeptide or fragment thereof promotes angiogenesis and is useful for the treatment of ischemia. Included in the invention are P75/TNFR2 polypeptides, analogs, or fragments thereof, that are modified in ways that enhance their ability to promote angiogenesis. In one embodiment, the invention provides methods for optimizing a P75/TNFR2 amino acid sequence or nucleic acid sequence by producing an alteration in the sequence. Such alterations may include certain mutations, deletions, insertions, or post-translational modifications. The invention further includes analogs of any naturally-occurring polypeptide of the invention. Analogs can differ from a naturally-occurring polypeptide of the invention by amino acid sequence differences, by post-translational modifications, or by both. Analogs of the invention will generally exhibit at least 85%, more preferably 90%, and most preferably 95% or even 99% identity with all or part of a naturally-occurring amino, acid sequence of the invention. The length of sequence comparison is at least 5, 10, 15 or 20 amino acid residues, preferably at least 25, 50, or 75 amino acid residues, and more preferably more than 100 amino acid residues. Again, in an exemplary approach to determining the degree of identity, a BLAST program may be used, with a probability score between e⁻³and e⁻¹⁰⁰indicating a closely related sequence. Modifications include in vivo and in vitro chemical derivatization of polypeptides, e.g., acetylation, carboxylation, phosphorylation, or glycosylation; such modifications may occur during polypeptide synthesis or processing or following treatment with isolated modifying enzymes. Analogs can also differ from the naturally-occurring polypeptides of the invention by alterations in primary sequence. These include genetic variants, both natural and induced (for example, resulting from random mutagenesis by irradiation or exposure to ethanemethylsulfate or by site-specific mutagenesis as described in Sambrook, Fritsch and Maniatis, Molecular Cloning: A Laboratory Manual (2d ed.), CSH Press, 1989, or Ausubel et al., supra). Also included are cyclized peptides, molecules, and analogs which contain residues other than L-amino acids, e.g., D-amino acids or non-naturally occurring or synthetic amino acids, e.g., .beta. or .gamma. amino acids.
In addition to full-length polypeptides, the invention also includes fragments of any one of the polypeptides of the invention. As used herein, the term “a fragment” means at least 10, 25, 50, 75, 100, 150, or 200 amino acids. In other embodiments a fragment is at least 20 contiguous amino acids, at least 30 contiguous amino acids, or at least 50 contiguous amino acids, and in other embodiments at least 60 to 80 or more contiguous amino acids. Fragments of the invention can be generated by methods known to those skilled in the art or may result from normal protein processing (e.g., removal of amino acids from the nascent polypeptide that are not required for biological activity or removal of amino acids by alternative mRNA splicing or alternative protein processing events).
Non-protein p75/TNFR2 analogs having a chemical structure designed to mimic p75/TNFR2 functional activity can be administered according to methods of the invention. p75/TNFR2 analogs may exceed the physiological activity of the original polypeptide. Methods of analog design are well known in the art, and synthesis of analogs can be carried out according to such methods by modifying the chemical structures such that the resultant analogs exhibit the angiogenesis promoting activity of a reference p75/TNFR2 polypeptide. These chemical modifications include, but are not limited to, substituting alternative R groups and varying the degree of saturation at specific carbon atoms of a reference p75/TNFR2 polypeptide. Preferably, the p75/TNFR2 analogs are relatively resistant to in vivo degradation, resulting in a more prolonged therapeutic effect upon administration. Assays for measuring functional activity include, but are not limited to, those described in the Examples below.

Treatment of an Ischemic Disease

The increased expression of p75/TNFR2 in a cell prevents or treats ischemia. Ischemia results when blood flow to a cell, tissue, or organ is interrupted. Tissue damage related to apoptotic cell death often results. Ischemic diseases are characterized by cell or tissue damage related to hypoxia. Exemplary ischemic diseases include, but are not limited to, ischemic injuries caused by a myocardial infarction, a stroke, a transient ischemic episode, a reperfusion injury, physical injury, renal failure, a secondary exsanguination, or blood flow interruption resulting from any other primary diseases. The effects of ischemia are particularly devastating in the brain, when stroke, traumatic brain injury, myocardial infarction, or a transient ischemic attack limits blood flow to the tissues of the CNS. Accordingly, the invention provides therapeutic and prophylactic compositions useful for the treatment of ischemia that affects a variety of cells, tissues, or organs. Such therapies include polynucleotide therapies, polypeptide therapies, as well as the delivery of endothelial progenitor cells (EPCs) expressing a heterologous p75/TNFR2. If desired, such cells may be removed from the patient, transfected, and then returned to the patient for the treatment of ischemia.
Delivery of therapeutic agents to ischemic tissues (e.g., muscle tissue, including cardiac tissue, and neural tissue, including the CNS) can be achieved by several methods. One method relies on neurosurgical techniques. In the case of gravely ill patients, surgical intervention is warranted despite its attendant risks. For instance, therapeutic agents can be delivered by direct physical introduction into the CNS, such as intraventricular, intralesional, or intrathecal injection. Intraventricular injection can be facilitated by an intraventricular catheter, for example, attached to a reservoir, such as an Ommaya reservoir. Methods of introduction are also provided by rechargeable or biodegradable devices.
In addition, the invention provides methods of screening for compounds that increase the biological activity or expression of p75/TNFR2 or that inhibit the biological activity or expression of p75/TNFR2. Such compounds are useful for enhancing angiogenesis, limiting the tissue damage associated with ischemia (e.g., by enhancing the survival of cells at risk of cell death associated with ischemia). Compounds that enhance p75/TNFR2 biological activity (e.g., angiogenesis enhancing activity) or expression may be used to treat or prevent ischemia in cells, tissues, or organs. Individuals at increased risk of an ischemic disease due to a hereditary condition are also candidates for such treatment.
p75/TNFR2 Polynucleotide Therapy
Therapy featuring a nucleic acid molecule encoding a p75 receptor/TNFR2 polypeptide, variant, or fragment thereof is one therapeutic approach for treating ischemia. Such nucleic acid molecules can be delivered to cells (e.g., endothelial progenitor cells, or bone marrow derived cells) of a subject before, during, or after an ischemic episode. Such delivery may take place in vivo or ex vivo. In one embodiment, a human EPC is removed from a donor, tranfected with a polynucleotide ex vivo, and then injected into a recipient patient in need thereof. Polynucleotide therapy has been successfully used to enhance angiogenesis. For example, the promotion of angiogenesis in the treatment of ischemia was demonstrated in a rabbit model and in human clinical trials with VEGF using a Hydrogel-coated angioplasty balloon as the gene delivery system (Takeshita, et al., Laboratory Investigation, 75:487-502 (1996); Isner, et al., Lancet, 348:370 (1996)). Successful transfer and sustained expression of the VEGF gene in the vessel wall subsequently augmented neovascularization in the ischemic limb (Takeshita, et al., Laboratory Investigation, 75:487-502 (1996); Isner, et al., Lancet, 348:370 (1996)). In addition, it has been demonstrated that direct intramuscular injection of DNA encoding VEGF into ischemic tissue induces angiogenesis, providing the ischemic tissue with increased blood vessels (U.S. Ser. No. 08/545,998; Tsurumi et al., Circulation 94(12):3281-90, 1996). The nucleic acid molecules must be delivered to the cells of a subject in a form in which they can be taken up so that therapeutically effective levels of an p75/TNFR2 polypeptide or fragment thereof can be produced.
Transducing viral (e.g., retroviral, adenoviral, and adeno-associated viral) vectors can be used for somatic cell gene therapy, especially because of their high efficiency of infection and stable integration and expression (see, e.g., Cayouette et al., Human Gene Therapy 8:423-430, 1997; Kido et al., Current Eye Research 15:833-844, 1996; Bloomer et al., Journal of Virology 71:6641-6649, 1997; Naldini et al., Science 272:263-267, 1996; and Miyoshi et al., Proc. Natl. Acad. Sci. U.S.A. 94:10319, 1997). For example, a polynucleotide encoding a p75/TNFR2 receptor polypeptide variant, or a fragment thereof, can be cloned into a retroviral vector and expression can be driven from its endogenous promoter, from the retroviral long terminal repeat, or from a promoter specific for a target cell type of interest. Other viral vectors that can be used include, for example, a vaccinia virus, a bovine papilloma virus, or a herpes virus, such as Epstein-Barr Virus (also see, for example, the vectors of Miller, Human Gene Therapy 15-14, 1990; Friedman, Science 244:1275-1281, 1989; Eglitis et al., BioTechniques 6:608-614, 1988; Tolstoshev et al., Current Opinion in Biotechnology 1:55-61, 1990; Sharp, The Lancet 337:1277-1278, 1991; Cornetta et al., Nucleic Acid Research and Molecular Biology 36:311-322, 1987; Anderson, Science 226:401-409, 1984; Moen, Blood Cells 17:407-416, 1991; Miller et al., Biotechnology 7:980-990, 1989; Le Gal La Salle et al., Science 259:988-990, 1993; and Johnson, Chest 107:77S-83S, 1995). Retroviral vectors are particularly well developed and have been used in clinical settings (Rosenberg et al., N. Engl. J. Med 323:370, 1990; Anderson et al., U.S. Pat. No. 5,399,346). In one embodiment, a viral vector is used to administer a p75/TNFR2 receptor nucleic acid molecule systemically.
Non-viral approaches can also be employed for the introduction of a therapeutic nucleic acid molecule to a cell of a subject requiring modulation of angiogenesis or vascularization. For example, a nucleic acid molecule can be introduced into a cell by administering the nucleic acid in the presence of lipofection (Feigner et al., Proc. Natl. Acad. Sci. U.S.A. 84:7413, 1987; Ono et al., Neuroscience Letters 17:259, 1990; Brigham et al., Am. J. Med. Sci. 298:278, 1989; Staubinger et al., Methods in Enzymology 101:512, 1983), asialoorosomucoid-polylysine conjugation (Wu et al., Journal of Biological Chemistry 263:14621, 1988; Wu et al., Journal of Biological Chemistry 264:16985, 1989), or by micro-injection under surgical conditions (Wolff et al., Science 247:1465, 1990). Preferably the nucleic acids are administered in combination with a liposome and protamine.
Gene transfer can also be achieved using non-viral means involving transfection in vitro. Such methods include the use of calcium phosphate, DEAE dextran, electroporation, and protoplast fusion. Liposomes can also be potentially beneficial for delivery of DNA into a cell. Transplantation of genes into the affected tissues of a subject (e.g., tissues subject to ischemia or requiring enhanced vascularization) can also be accomplished by transferring a normal nucleic acid into a cultivatable cell type ex vivo (e.g., an autologous or heterologous primary cell or progeny thereof), after which the cell (e.g., a bone marrow-derived cell or EPC) (or its descendants) are injected into a targeted tissue (e.g., an ischemic tissue).
cDNA expression for use in polynucleotide therapy methods can be directed from any suitable promoter (e.g., the human cytomegalovirus (CMV), simian virus 40 (SV40), or metallothionein promoters), and regulated by any appropriate mammalian regulatory element. For example, if desired, enhancers known to preferentially direct gene expression in specific cell types can be used to direct the expression of a nucleic acid. The enhancers used can include, without limitation, those that are characterized as tissue- or cell-specific enhancers. Alternatively, if a genomic clone is used as a therapeutic construct, regulation can be mediated by the cognate regulatory sequences or, if desired, by regulatory sequences derived from a heterologous source, including any of the promoters or regulatory elements described above.
Another therapeutic approach included in the invention involves administration of a recombinant therapeutic, such as a recombinant p75 receptor/TNFR2 polypeptide, variant, or fragment thereof, either directly to the site of a potential or actual disease-affected tissue or systemically (for example, by any conventional recombinant protein administration technique). The dosage of the administered polypeptide depends on a number of factors, including the size and health of the individual subject. For any particular subject, the specific dosage regimes should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions.

Endothelial Progenitor Cell Therapy

Transplantation of hematopoietic stem cells derived from peripheral blood can provide sustained hematopoietic recovery. (See, for example, Messinger et al., Blood 77, 211 (1991); Sheridan et al., Lancet 339, 640 (1992); Shpall et al., J. Clin. Oncol. 12, 28 (1994). This observation is now being exploited clinically as an alternative to bone marrow transplantation. By using techniques similar to those employed for hematopoietic stem cells, endothelial progenitor cells can be isolated from circulating blood. Such cells, once isolated, can be expanded in vitro and engineered to express one or more heterologous nucleic acid molecules. The cells are then delivered back to the donor, or to another subject, to achieve a therapeutic result.
To obtain the endothelial progenitor cell from peripheral blood about 5 ml to about 500 ml of blood is taken from a donor. Preferably, about 50 ml to about 200 ml of blood is taken. Endothelial progenitor cells are expanded in vivo by administration of recruitment growth factors, e.g., GM-CSF and IL-3, to the donor prior to removing the progenitor cells. Methods for obtaining and using hematopoietic progenitor cells in autologous transplantation are disclosed in U.S. Pat. No. 5,199,942, the disclosure of which is incorporated by reference. Alternatively, the cells are expanded ex vivo using, for example, the method disclosed by U.S. Pat. No. 5,541,103. Endothelial progenitor cells may be obtained from human mononuclear cells obtained from peripheral blood or bone marrow of the subject before treatment. Such cells may also be obtained from heterologous or autologous umbilical cord blood. In particular, endothelial progenitor cells may be obtained from the leukocyte fraction of peripheral blood. Endothelial progenitor cells may be isolated using antibodies that recognize endothelial progenitor cell specific antigens on immature human hematopoietic progenitor cells. For example, CD34 is commonly shared by endothelial progenitor cells and hematopoietic stem cells. CD34 is expressed by all hematopoietic stem cells but is lost by hematopoietic cells as they differentiate. Flk-1, a receptor for vascular endothelial growth factor (VEGF) is also expressed by both early hematopoietic stem cells and endothelial cells, but ceases to be expressed in the course of hematopoietic differentiation.
In vitro, endothelial progenitor cells differentiate into endothelial cells. Indeed, one can use a multipotentiate undifferentiated cell as long as it is still capable of becoming an endothelial cell in the presence of agents that promote its differentiation. In vivo, heterologous, homologous, and autologous endothelial cell progenitor grafts incorporate into sites of active angiogenesis or blood vessel injury by selectively migrating to such locations. Angiogenesis can be promoted in a subject by administering a potent angiogenesis factor, such as VEGF, alone or in combination with endothelial progenitor cells. Once the progenitor cells are obtained by a particular separation technique, they may be administered to a selected subject to treat a number of conditions including, for example, unregulated angiogenesis or blood vessel injury. The cells may also be stored in cryogenic conditions.
The progenitor cells are administered to a subject by any suitable means, including, for example, intravenous infusion, bolus injection, and site directed delivery via a catheter. Preferably, the progenitor cells obtained from the subject are readministered. Generally, from about 10⁶to about 10¹⁸progenitor cells are administered to a subject for transplantation. Depending on the use of the progenitor cells, various genetic material may be delivered to the cell (e.g., a polynucleotide encoding p75/TNFR2). Such genetic material includes nucleic acid sequences both exogenous and endogenous to cells into which a virus vector, for example, a pox virus such as swine pox containing the human TNF gene may be introduced. Additionally, it is of interest to use genes encoding polypeptides for secretion from the endothelial progenitor cells so as to provide for a systemic effect by the protein encoded by the gene. Specific genes of interest include those encoding TNF, TGF-α, TGF-β, hemoglobin, interleukin-1, interleukin-2, interleukin-3, interleukin-4, interleukin-5, interleukin-6, interleukin-7, interleukin-8, interleukin-9, interleukin-10, interleukin-11, interleukin-12 etc., GM-CSF, G-CSF, M-CSF, human growth factor, co-stimulatory factor B7, insulin, factor VIII, factor IX, PDGF, EGF, NGF, IL-ira, EPO, β-globin, endothelial cell mitogens, as well as biologically active variants of these proteins. The gene may further encode a product that regulates expression of another gene product or blocks one or more steps in a biological pathway. Similarly, the gene may encode a therapeutic protein fused to a targeting polypeptide, to deliver a therapeutic effect to a diseased tissue or organ. To further enhance angiogenesis, endothelial cell mitogens may also be administered to the subject in conjunction with, or subsequent to, the administration of the endothelial progenitor cells. Endothelial cell mitogens can be administered directly, e.g., intra-arterially, intramuscularly, or intravenously, or a nucleic acid molecule encoding the mitogen may be used. See, Baffour, et al., J Vase Surg. 16(2):181-91 (1992). (bFGF); Pu, et al, Circulation, 88:208-215 (1993) (aFGF); Yanagisawa-Miwa, et al., Science. 257(5075):1401-3 (1992). (bFGF); Ferrara, et al., Biochem. Biophys. Res. Commun., 161:851-855 (1989) (VEGF); (Takeshita, et al., Circulation, 90:228-234 (1994)).
The nucleic acid encoding the endothelial cell mitogen can be administered to a blood vessel by perfusing the ischemic tissue or to a site of vascular injury via a catheter, for example, a hydrogel catheter, as described by U.S. Ser. No. 08/675,523, the disclosure of which is herein incorporated by reference. The nucleic acid also can be delivered by injection directly into the ischemic tissue using the method described in U.S. Ser. No. 08/545,998.
As used herein the term “endothelial cell mitogen” means any protein, polypeptide, variant or portion thereof that is capable of, directly or indirectly, inducing endothelial cell growth. Such proteins include, for example, acidic and basic fibroblast growth factors (aFGF) (GenBank Accession No. NP_—149127) and bFGF (GenBank Accession No. AAA52448), vascular endothelial growth factor (VEGF) (GenBank Accession No. AAA35789 or NP_—001020539), epidermal growth factor (EGF) (GenBank Accession No. NP_—001954), transforming growth factor α (TGF-α) (GenBank Accession No. NP_—003227) and transforming growth factor β (TFG-β) (GenBank Accession No. 1109243A), platelet-derived endothelial cell growth factor (PD-ECGF) (GenBank Accession No. NP_—001944), platelet-derived growth factor (PDGF) (GenBank Accession No. 1109245A), tumor necrosis factor α (TNF-α) (GenBank Accession No. CAA26669), hepatocyte growth factor (HGF) (GenBank Accession No. BAA14348), insulin like growth factor (IGF) (GenBank Accession No. P08833), erythropoietin (GenBank Accession No. P01588), colony stimulating factor (CSF), macrophage-CSF (M-CSF) (GenBank Accession No. AAB59527), granulocyte/macrophage CSF (GM-CSF) (GenBank Accession No. NP_—000749), monocyte chemotactic protein-1 (GenBank Accession No. P13500) and nitric oxide synthase (NOS) (GenBank Accession No. AAA36365). See, Klagsbrun, et al., Annu. Rev. Physiol., 53:217-239 (1991); Folkman, et al., J. Biol. Chem., 267:10931-10934 (1992) and Symes, et al., Current Opinion in Lipidology, 5:305-312 (1994). Variants or fragments of a mitogen may be used as long as they induce or promote endothelial cell or endothelial progenitor cell growth. Preferably, the endothelial cell mitogen contains a secretory signal sequence that facilitates secretion of the protein. Proteins having native signal sequences, e.g., VEGF, are preferred. Proteins that do not have native signal sequences, e.g., bFGF, can be modified to contain such sequences using routine genetic manipulation techniques. See, Nabel et al., Nature, 362:844 (1993).
The nucleotide sequence of numerous endothelial cell mitogens, are readily available through a number of computer data bases, for example, GenBank, EMBL and Swiss-Prot. Using this information, a DNA segment encoding the desired may be chemically synthesized or, alternatively, such a DNA segment may be obtained using routine procedures in the art, e.g, PCR amplification. A DNA encoding VEGF is disclosed in U.S. Pat. No. 5,332,671, the disclosure of which is herein incorporated by reference.
In certain situations, it may be desirable to use nucleic acids encoding two or more different proteins in order optimize the therapeutic outcome. For example, DNA encoding two proteins, e.g., VEGF and bFGF, can be used, and provides an improvement over the use of bFGF alone, or an angiogenic factor (e.g., fibroblast growth factor (bFGF), acidic FGF (aFGF), FGF-5, vascular endothelial growth factor isoforms (VEGF), angiopoietin-1 (Ang-1), angiopoietin-2 (Ang-2), platelet-derived endothelial cell growth factor (PD-ECGF), hepatocyte growth factor (HGF) (GenBank Accession No. BAA14348), interleukin-8 (IL-8) (GenBank Accession No. NP_—000575), granulocyte-colony stimulating factor (G-CSF), placental growth factor (GenBank Accession No. NP_—002623.), proliferin (GenBank Accession No. S48671), angiogenin (NP_—001136), TNFα, Transforming growth factor-β (GenBank Accession No. 1109243A)) can be combined with other genes or their encoded gene products to enhance the activity of targeted cells, while simultaneously inducing angiogenesis, including, for example, nitric oxide synthase, L-arginine, fibronectin, urokinase, plasminogen activator and heparin.
The effective dose of the nucleic acid will be a function of the particular expressed protein, the target tissue, the subject and his or her clinical condition. Effective amount of DNA are between about 1 and 4000 μg, more preferably about 1000 and 2000, most preferably between about 2000 and 4000.

Kits

The invention provides kits for the treatment or prevention of ischemia or related tissue damage. In one embodiment, the kit includes a therapeutic or prophylactic composition containing an effective amount of a p75 receptor/TNFR2 polypeptide or an expression vector encoding the p75 receptor/TNFR2 polypeptide in unit dosage form. In some embodiments, the kit comprises a sterile container which contains a therapeutic or prophylactic composition; such containers can be boxes, ampules, bottles, vials, tubes, bags, pouches, blister-packs, or other suitable container forms known in the art. Such containers can be made of plastic, glass, laminated paper, metal foil, or other materials suitable for holding medicaments.
If desired an expression vector of the invention is provided together with instructions for administering it to a subject having or at risk of developing ischemia. The instructions will generally include information about the use of the composition for the treatment or prevention of ischemia or for enhancing angiogenesis to a tissue in need thereof. In other embodiments, the instructions include at least one of the following: description of the expression vector; dosage schedule and administration for treatment or prevention of ischemia or symptoms thereof; precautions; warnings; indications; counter-indications; overdosage information; adverse reactions; animal pharmacology; clinical studies; and/or references. The instructions may be printed directly on the container (when present), or as a label applied to the container, or as a separate sheet, pamphlet, card, or folder supplied in or with the container.

Screening Assays

Signaling through the p75/TNFR2 polypeptide was required for collateral vessel development in ischemia-induced neovascularization. Based in part on this discovery, compositions of the invention are useful for the high-throughput low-cost screening of candidate compounds that enhance p75/TNFR2 expression or activity. Such compounds are useful for the treatment or prevention of ischemia. Tissues or cells treated with a candidate compound are compared to untreated control samples to identify therapeutic agents that enhance the p75 receptor/TNFR2 expression or activity. If desired, such compounds are further tested in vitro or in vivo for their effects on angiogenesis using any method known in the art. Any number of methods are available for carrying out screening assays to identify new candidate compounds that bind a p75 receptor/TNFR2 polypeptide and enhance the angiogenesis promoting activity of the receptor.
In one working example, candidate compounds are added at varying concentrations to the culture medium of cultured cells. p75 receptor/TNFR2 expression (e.g., polypeptide or mRNA expression) is then measured using standard methods. The expression of a p75 receptor/TNFR2 in the presence of the candidate compound is compared to the level measured in a control culture medium lacking the candidate molecule. A compound that increases the expression of a p75 receptor/TNFR2 is useful for promoting an increase in angiogenesis. Such compounds are considered useful in the invention; such a compound may be used, for example, as a therapeutic to prevent, delay, ameliorate, stabilize, reduce the severity of, or treat ischemia. In other embodiments, the candidate compound prevents, delays, ameliorates, stabilizes, or treats a disease or disorder related to ischemia or reduces tissue damage or apoptosis associated with ischemia. Such therapeutic compounds are useful in vivo as well as ex vivo.
In some embodiments, a compound that promotes an increase in the biological activity of a p75 receptor/TNFR2 of the invention is considered useful. Such compounds are added to a culture containing p75 receptor/TNFR2 expressing cells. The effect of the compound on p75 receptor/TNFR2 biological activity is measured and compared to p75 receptor/TNFR2 biological activity in the absence of the candidate compound. Again, a candidate compound that enhances the biological activity of a p75 receptor/TNFR2 may be used, for example, as a therapeutic to treat or prevent ischemia.
One skilled in the art appreciates that the effects of a candidate compound on the p75 receptor/TNFR2 expression or biological activity are typically compared to the expression or activity of the p75 receptor/TNFR2 in the absence of the candidate compound. Thus, the screening methods include comparing the value of a cell modulated by a candidate compound to a reference value of an untreated control cell.
Expression levels can be compared by procedures well known in the art such as RT-PCR, Northern blotting, Western blotting, flow cytometry, immunocytochemistry, binding to magnetic and/or antibody-coated beads, in situ hybridization, fluorescence in situ hybridization (FISH), flow chamber adhesion assay, and ELISA, microarray analysis, or colorimetric assays, such as the Bradford Assay and Lowry Assay. Changes in angiogenesis can be assayed by methods described herein or by any method known in the art, including Angiogram, Computed Tomography Angiography (CTA), Duplex Ultrasound, magenetic resonance angiography, vascular ultrasound, or angiogram.
Molecules that increase the p75 receptor/TNFR2 expression or activity include organic molecules, peptides, peptide mimetics, polypeptides, nucleic acids, and antibodies that bind to a the p75 receptor/TNFR2 nucleic acid sequence or polypeptide and increase its expression or biological activity are preferred.
In yet another example, candidate compounds are screened for those that specifically bind to a p75 receptor/TNFR2. The efficacy of such a candidate compound is dependent upon its ability to interact with the p75 receptor/TNFR2, or with functional equivalents thereof. Such an interaction can be readily assayed using any number of standard binding techniques and functional assays (e.g., those described in Ausubel et al., supra). In one embodiment, the compound is assayed in vitro for receptor binding.
In one particular working example, a candidate compound that binds to a p75 receptor/TNFR2 is identified using a chromatography-based technique. For example, a recombinant polypeptide of the invention may be purified by standard techniques from cells engineered to express the polypeptide (e.g., those described above) and may be immobilized on a column. A solution of candidate compounds is then passed through the column, and a compound specific for the p75 receptor/TNFR2 is identified on the basis of its ability to bind to the polypeptide and be immobilized on the column. To isolate the compound, the column is washed to remove non-specifically bound molecules, and the compound of interest is then released from the column and collected. Similar methods may be used to isolate a compound bound to a polypeptide microarray. Compounds identified using such methods are then assayed for their effect on angiogenesis as described herein.
In another example, the compound, e.g., the substrate, is coupled to a radioisotope or enzymatic label such that binding of the compound to the p75 receptor/TNFR2 can be determined by detecting the labeled compound, e.g., substrate, in a complex. For example, compounds can be labeled with ¹²⁵I, ³⁵S, ¹⁴C, or ³H, either directly or indirectly, and the radioisotope detected by direct counting of radioemmission or by scintillation counting. Alternatively, compounds can be enzymatically labeled with, for example, horseradish peroxidase, alkaline phosphatase, or luciferase, and the enzymatic label detected by determination of conversion of an appropriate substrate to product.
In yet another embodiment, a cell-free assay is provided in which the p75 receptor/TNFR2 or a biologically active portion thereof is contacted with a test compound and the ability of the test compound to bind to the polypeptide thereof is evaluated.
The interaction between two molecules can also be detected, e.g., using fluorescence energy transfer (FET) (see, for example, Lakowicz et al., U.S. Pat. No. 5,631,169; Stavrianopoulos et al., U.S. Pat. No. 4,868,103). A fluorophore label on the first, ‘donor’ molecule is selected such that its emitted fluorescent energy will be absorbed by a fluorescent label on a second, ‘acceptor’ molecule, which in turn is able to fluoresce due to the absorbed energy. Alternately, the ‘donor’ protein molecule may simply utilize the natural fluorescent energy of tryptophan residues. Labels are chosen that emit different wavelengths of light, such that the ‘acceptor’ molecule label may be differentiated from that of the ‘donor’. Since the efficiency of energy transfer between the labels is related to the distance separating the molecules, the spatial relationship between the molecules can be assessed. In a situation in which binding occurs between the molecules, the fluorescent emission of the ‘acceptor’ molecule label in the assay should be maximal. An FET binding event can be conveniently measured through standard fluorometric detection means well known in the art (e.g., using a fluorimeter).
In another embodiment, determining the ability of a test compound to bind to the p75 receptor/TNFR2 can be accomplished using real-time Biomolecular Interaction Analysis (BIA) (see, e.g., Sjolander, S. and Urbaniczky, C., Anal. Chem. 63:2338-2345, 1991; and Szabo et al., Curr. Opin. Struct. Biol. 5:699-705, 1995). “Surface plasmon resonance” or “BIA” detects biospecific interactions in real time, without labeling any of the interactants (e.g., BIAcore). Changes in the mass at the binding surface (indicative of a binding event) result in alterations of the refractive index of light near the surface (the optical phenomenon of surface plasmon resonance (SPR)), resulting in a detectable signal that can be used as an indication of real-time reactions between biological molecules.
It may be desirable to immobilize either the candidate compound or its p75 receptor/TNFR2 target to facilitate separation of complexed from uncomplexed forms of one or both of the proteins, as well as to accommodate automation of the assay. Binding of a candidate compound to the p75 receptor/TNFR2, or interaction of a test compound with a target molecule in the presence and absence of a candidate compound, can be accomplished in any vessel suitable for containing the reactants. Examples of such vessels include microtiter plates, test tubes, and micro-centrifuge tubes. In one embodiment, a fusion protein can be provided which adds a domain that allows one or both of the proteins to be bound to a matrix. For example, glutathione-S-transferase/p75 receptor/TNFR2 fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) or glutathione derivatized microtiter plates, which are then combined with the test compound or the test compound and a sample comprising the GST-tagged p75 receptor/TNFR2 polypeptide, and the mixture incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH). Following incubation, the beads or microtiter plate wells are washed to remove any unbound components, the matrix immobilized in the case of beads, complex determined either directly or indirectly, for example, as described above.
Other techniques for immobilizing a complex of a compound and the p75 receptor/TNFR2 polypeptide on matrices include using conjugation of biotin and streptavidin. For example, biotinylated proteins can be prepared from biotin-NHS (N-hydroxy-succinimide) using techniques known in the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, Ill.), and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemical).
In order to conduct the assay, the non-immobilized component is added to the coated surface containing the anchored component. After the reaction is complete, unreacted components are removed (e.g., by washing) under conditions such that any complexes formed will remain immobilized on the solid surface. The detection of complexes anchored on the solid surface can be accomplished in a number of ways. Where the previously non-immobilized component is pre-labeled, the detection of label immobilized on the surface indicates that complexes were formed. Where the previously non-immobilized component is not pre-labeled, an indirect label can be used to detect complexes anchored on the surface; e.g., using a labeled antibody specific for the immobilized component (the antibody, in turn, can be directly labeled or indirectly labeled with, e.g., a labeled anti-Ig antibody).
In one embodiment, a p75 receptor/TNFR2 antibody is identified that reacts with an epitope on the p75 receptor/TNFR2. Methods for detecting binding of expression antibody to the receptor are known in the art and include immunodetection of complexes, as well as enzyme-linked assays which rely on detecting an enzymatic activity associated with the channel. Antibodies that bind the p75 receptor/TNFR2 are then tested for the ability to activate the receptor. Such antibodies may tested for angiogenesis promoting activity as, described herein.
Alternatively, cell free assays can be conducted that assay the interaction of a compound with a p75 receptor/TNFR2 or that assay the activity of a p75 receptor/TNFR2. In such an assay, the reaction products are separated from unreacted components, by any of a number of standard techniques, including but not limited to: differential centrifugation (see, for example, Rivas, G., and Minton, A. P., Trends Biochem Sci 18:284-7, 1993); chromatography (gel filtration chromatography, ion-exchange chromatography); electrophoresis and immunoprecipitation (see, for example, Ausubel, F. et al., eds. (1999) Current Protocols in Molecular Biology, J. Wiley: New York). Such resins and chromatographic techniques are known to one skilled in the art (see, e.g., Heegaard, N. H., J Mol Recognit 11:141-8, 1998; Hage, D. S., and Tweed, S. A., J Chromatogr B Biomed Sci Appl. 699:499-525, 1997). Further, fluorescence energy transfer may also be conveniently utilized, as described herein, to detect binding without further purification of the complex from solution. Preferably, cell free assays preserve the structure of the the p75 receptor/TNFR2, e.g., by including a membrane component or synthetic membrane components.
In a specific embodiment, the assay includes contacting the p75 receptor/TNFR2 polypeptide or biologically active portion thereof with a known compound which binds the p75 receptor/TNFR2 polypeptide to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with a the p75 receptor/TNFR2, wherein determining the ability of the test compound to interact with a the p75 receptor/TNFR2 includes determining the ability of the test compound to preferentially bind to the p75 receptor/TNFR2, or to modulate the activity of the p75 receptor/TNFR2, as compared to the known compound.
Compounds isolated by this method (or any other appropriate method) may, if desired, be further purified (e.g., by high performance liquid chromatography). In addition, these candidate compounds may be tested for their ability to increase the activity of a p75 receptor/TNFR2 (e.g., as described herein). Compounds isolated by this approach may also be used, for example, as therapeutics to treat or prevent ischemia in a subject. Compounds that are identified as binding to the p75 receptor/TNFR2 with an affinity constant less than or equal to 10 mM are considered particularly useful in the invention. Alternatively, any in vivo protein interaction detection system, for example, any two-hybrid assay may be utilized.
In another embodiment, a the p75 receptor/TNFR2 nucleic acid described herein is expressed as a transcriptional or translational fusion with a detectable reporter, and expressed in an isolated cell (e.g., mammalian or insect cell) under the control of an endogenous or a heterologous promoter. The cell expressing the fusion protein is then contacted with a candidate compound, and the expression of the detectable reporter in that cell is compared to the expression of the detectable reporter in an untreated control cell. A candidate compound that increases the expression of the detectable reporter is a compound that is useful for the treatment of ischemia. In preferred embodiments, the candidate compound increases the expression of a reporter gene fused to a the p75 receptor/TNFR2 nucleic acid molecule.
Each of the DNA sequences listed herein may also be used in the discovery and development of a therapeutic compound for the treatment of intestinal inflammation or an inflammatory bowel disease. The encoded protein, upon expression, can be used as a target for the screening of drugs. Additionally, the DNA sequences encoding the amino terminal regions of the encoded protein or Shine-Delgarno or other translation facilitating sequences of the respective mRNA can be used to construct sequences that promote the expression of the coding sequence of interest. Such sequences may be isolated by standard techniques (Ausubel et al., supra).
Small molecules of the invention preferably have a molecular weight below 2,000 daltons, more preferably between 300 and 1,000 daltons, and most preferably between 400 and 700 daltons. It is preferred that these small molecules are organic molecules.

Test Compounds and Extracts

In general, compounds capable of increasing the activity of the p75 receptor/TNFR2 are identified from large libraries of both natural product or synthetic (or semi-synthetic) extracts or chemical libraries or from polypeptide or nucleic acid libraries, according to methods known in the art. Those skilled in the field of drug discovery and development will understand that the precise source of test extracts or compounds is not critical to the screening procedure(s) of the invention. Compounds used in screens may include known compounds (for example, known therapeutics used for other diseases or disorders). Alternatively, virtually any number of unknown chemical extracts or compounds can be screened using the methods described herein. Examples of such extracts or compounds include, but are not limited to, plant-, fungal-, prokaryotic- or animal-based extracts, fermentation broths, and synthetic compounds, as well as modification of existing compounds.
Numerous methods are also available for generating random or directed synthesis (e.g., semi-synthesis or total synthesis) of any number of chemical compounds, including, but not limited to, saccharide-, lipid-, peptide-, and nucleic acid-based compounds. Synthetic compound libraries are commercially available from Brandon Associates (Merrimack, N.H.) and Aldrich Chemical (Milwaukee, Wis.). Alternatively, chemical compounds to be used as candidate compounds can be synthesized from readily available starting materials using standard synthetic techniques and methodologies known to those of ordinary skill in the art.
Synthetic chemistry transformations and protecting group methodologies (protection and deprotection) useful in synthesizing the compounds identified by the methods described herein are known in the art and include, for example, those such as described in R. Larock, Comprehensive Organic Transformations, VCH Publishers (1989); T. W. Greene and P. G. M. Wuts, Protective Groups in Organic Synthesis, 2nd ed., John Wiley and Sons (1991); L. Fieser and M. Fieser, Fieser and Fieser's Reagents for Organic Synthesis, John Wiley and Sons (1994); and L. Paquette, ed., Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons (1995), and subsequent editions thereof.
Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant, and animal extracts are commercially available from a number of sources, including Biotics (Sussex, UK), Xenova (Slough, UK), Harbor Branch Oceangraphics Institute (Ft. Pierce, Fla.), and PharmaMar, U.S.A. (Cambridge, Mass.). In addition, natural and synthetically produced libraries are produced, if desired, according to methods known in the art, e.g., by standard extraction and fractionation methods. Examples of methods for the synthesis of molecular libraries can be found in the art, for example in: DeWitt et al., Proc. Natl. Acad. Sci. U.S.A. 90:6909, 1993; Erb et al., Proc. Natl. Acad. Sci. USA 91:11422, 1994; Zuckermann et al., J. Med. Chem. 37:2678, 1994; Cho et al., Science 261:1303, 1993; Carrell et al., Angew. Chem. Int. Ed. Engl. 33:2059, 1994; Carell et al., Angew. Chem. Int. Ed. Engl. 33:2061, 1994; and Gallop et al., J. Med. Chem. 37:1233, 1994. Furthermore, if desired, any library or compound is readily modified using standard chemical, physical, or biochemical methods.
Libraries of compounds may be presented in solution (e.g., Houghten, Biotechniques 13:412-421, 1992), or on beads (Lam, Nature 354:82-84, 1991), chips (Fodor, Nature 364:555-556, 1993), bacteria (Ladner, U.S. Pat. No. 5,223,409), spores (Ladner U.S. Pat. No. 5,223,409), plasmids (Cull et al., Proc Natl Acad Sci USA 89:1865-1869, 1992) or on phage (Scott and Smith, Science 249:386-390, 1990; Devlin, Science 249:404-406, 1990; Cwirla et al. Proc. Natl. Acad. Sci. 87:6378-6382, 1990; Felici, J. Mol. Biol. 222:301-310, 1991; Ladner supra.).
In addition, those skilled in the art of drug discovery and development readily understand that methods for dereplication (e.g., taxonomic dereplication, biological dereplication, and chemical dereplication, or any combination thereof) or the elimination of replicates or repeats of materials already known for their activity should be employed whenever possible.
When a crude extract is found to increase the activity of a p75 receptor/TNFR2, or to bind the p75 receptor/TNFR2, further fractionation of the positive lead extract is necessary to isolate chemical constituents responsible for the observed effect. Thus, the goal of the extraction, fractionation, and purification process is the careful characterization and identification of a chemical entity within the crude extract that increases the activity of a the p75 receptor/TNFR2. Methods of fractionation and purification of such heterogenous extracts are known in the art. If desired, compounds shown to be useful as therapeutics for the treatment of ischemia are chemically modified according to methods known in the art.

Pharmaceutical Therapeutics

The invention provides a simple means for identifying compositions (including nucleic acids, peptides, small molecule inhibitors, and mimetics) capable of acting as therapeutics for the treatment of ischemia. Accordingly, a chemical entity discovered to have medicinal value using the methods described herein is useful as a drug or as information for structural modification of existing compounds, e.g., by rational drug design. Such methods are useful for screening compounds having that treat or prevent ischemia.
For therapeutic uses, the compositions or agents of the invention described herein may be administered systemically, for example, formulated in a pharmaceutically-acceptable buffer such as physiological saline. Alternatively, compositions or agents of the invention described herein are delivered directly to a tissue in need of such treatment, such a tissue suffering from or at risk of ischemia. Preferable routes of administration include, for example, subcutaneous, intravenous, interperitoneally, intramuscular, or intradermal injections that provide continuous, sustained levels of the drug in the subject. Treatment of human subjects or other animals will be carried out using a therapeutically effective amount of an ischemia therapeutic in a physiologically-acceptable carrier. Suitable carriers and their formulation are described, for example, in Remington's Pharmaceutical Sciences by E. W. Martin. The amount of the therapeutic agent to be administered varies depending upon the manner of administration, the age and body weight of the subject, and with the clinical symptoms of ischemia. Generally, amounts will be in the range of those used for other agents used in the treatment of other diseases associated with ischemia, although in certain instances lower amounts will be needed because of the increased specificity of the compound. A compound is administered at a dosage that controls the clinical or physiological symptoms of ischemia or that reduces tissue damage associated with ischemia as determined by a diagnostic method known to one skilled in the art, or using any that assay that measures the expression or the biological activity of a the p75 receptor/TNFR2 polypeptide.

Formulation of Pharmaceutical Compositions

The administration of a compound for the treatment of ischemia may be by any suitable means that results in a concentration of the therapeutic that, combined with other components, is effective in preventing, ameliorating, reducing, or stabilizing an ischemic disease. The compound may be contained in any appropriate amount in any suitable carrier substance, and is generally present in an amount of 1-95% by weight of the total weight of the composition. The composition may be provided in a dosage form that is suitable for parenteral (e.g., subcutaneously, intravenously, intramuscularly, or intraperitoneally) administration route. The pharmaceutical compositions may be formulated according to conventional pharmaceutical practice (see, e.g., Remington: The Science and Practice of Pharmacy (20th ed.), ed. A. R. Gennaro, Lippincott Williams & Wilkins, 2000 and Encyclopedia of Pharmaceutical Technology, eds. J. Swarbrick and J. C. Boylan, 1988-1999, Marcel Dekker, New York).
Pharmaceutical compositions according to the invention may be formulated to release the active compound substantially immediately upon administration or at any predetermined time or time period after administration. The latter types of compositions are generally known as controlled release formulations, which include (i) formulations that create a substantially constant concentration of the drug within the body over an extended period of time; (ii) formulations that after a predetermined lag time create a substantially constant concentration of the drug within the body over an extended period of time; (iii) formulations that sustain action during a predetermined time period by maintaining a relatively, constant, effective level in the body with concomitant minimization of undesirable side effects associated with fluctuations in the plasma level of the active substance (sawtooth kinetic pattern); (iv) formulations that localize action by, e.g., spatial placement of a controlled release composition adjacent to or in the central nervous system or cerebrospinal fluid; (v) formulations that allow for convenient dosing, such that doses are administered, for example, once every one or two weeks; and (vi) formulations that target an ischemic disease by using carriers or chemical derivatives to deliver the therapeutic agent to a particular cell type (e.g., cell subject to reduced blood flow related to ischemia) whose function is perturbed in an ischemia disease. For some applications, controlled release formulations obviate the need for frequent dosing during the day to sustain the plasma level at a therapeutic level.
Any of a number of strategies can be pursued in order to obtain controlled release in which the rate of release outweighs the rate of metabolism of the compound in question. In one example, controlled release is obtained by appropriate selection of various formulation parameters and ingredients, including, e.g., various types of controlled release compositions and coatings. Thus, the therapeutic is formulated with appropriate excipients into a pharmaceutical composition that, upon administration, releases the therapeutic in a controlled manner. Examples include single or multiple unit tablet or capsule compositions, oil solutions, suspensions, emulsions, microcapsules, microspheres, molecular complexes, nanoparticles, patches, and liposomes.
Human dosage amounts for any therapy described herein can initially be determined by extrapolating from the amount of compound used in mice, as a skilled artisan recognizes it is routine in the art to modify the dosage for humans compared to animal models. In certain embodiments it is envisioned that the dosage may vary from between about 1 mg compound/Kg body weight to about 5000 mg compound/Kg body weight; or from about 5 mg/Kg body weight to about 4000 mg/Kg body weight or from about 10 mg/Kg body weight to about 3000 mg/Kg body weight; or from about 50 mg/Kg body weight to about 2000 mg/Kg body weight; or from about 100 mg/Kg body weight to about 1000 mg/Kg body weight; or from about 150 mg/Kg body weight to about 500 mg/Kg body weight. In other embodiments this dose may be about 1, 5, 10, 25, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1600, 1700, 1800, 1900, 2000, 2500, 3000, 3500, 4000, 4500, 5000 mg/Kg body weight. In other embodiments, it is envisaged that higher does may be used, such doses may be in the range of about 5 mg compound/Kg body to about 20 mg compound/Kg body. In other embodiments the doses may be about 8, 10, 12, 14, 16 or 18 mg/Kg body weight. Of course, a dosage amount may be adjusted upward or downward, as is routinely done in such treatment protocols, depending on the results of the initial clinical trials and the needs of a particular patient.

Patient Treatment and Monitoring

The present invention provides methods of treating an ischemic disease and/or disorders or symptoms thereof (e.g., tissue damage related to ischemia) by enhancing angiogenesis or reducing apoptosis. The methods comprise administering a therapeutically effective amount of a pharmaceutical composition comprising a polypeptide, nucleic acid molecule or compound that enhances the expression or activity of p75/TNFR2 as described herein to a subject (e.g., a mammal such as a human). Thus, one embodiment is a method of treating a subject suffering from or susceptible to an ischemic disease or disorder or symptom thereof. The method includes the step of administering to the mammal a therapeutic amount of an amount of a polypeptide, nucleic acid molecule or compound herein sufficient to treat the disease or disorder or symptom thereof, under conditions such that the disease or disorder is treated.
The methods herein include administering to the subject (including a subject identified as in need of such treatment) an effective amount of a compound described herein, or a composition described herein to produce such effect. Identifying a subject in need of such treatment can be in the judgment of a subject or a health care professional and can be subjective (e.g. opinion) or objective (e.g. measurable by a test or diagnostic method).
The therapeutic methods of the invention, which include prophylactic treatment, in general comprise administration of a therapeutically effective amount of the compounds herein, such as a compound of the formulae herein to a subject (e.g., animal, human) in need thereof, including a mammal, particularly a human. Such treatment will be suitably administered to subjects, particularly humans, suffering from, having, susceptible to, or at risk for a disease, disorder, or symptom thereof. Determination of those subjects “at risk” can be made by any objective or subjective determination by a diagnostic test or opinion of a subject or health care provider (e.g., genetic test, enzyme or protein marker, Marker (as defined herein), family history, and the like). The compounds herein may be also used in the treatment of any other disorders in which ischemia may be implicated.
In one embodiment, the invention provides a method of monitoring treatment progress. The method includes the step of determining a level of diagnostic marker (Marker) (e.g., any target delineated herein modulated by a compound herein, a protein or indicator thereof, etc.) or diagnostic measurement (e.g.,angiogenesis or tissue perfusion) in a subject suffering from or susceptible to a disorder or symptoms thereof associated with ischemia, in which the subject has been administered a therapeutic amount of a compound herein sufficient to treat the disease or symptoms thereof. The level of Marker or diagnostic measurement determined in the method can be compared to known levels of Marker or diagnostic measurement in either healthy normal controls or in other afflicted patients to establish the subject's disease status. In preferred embodiments, a second level of Marker or diagnostic measurement in the subject is determined at a time point later than the determination of the first level, and the two levels are compared to monitor the course of disease or the efficacy of the therapy. In certain preferred embodiments, a pre-treatment level of Marker in the subject is determined prior to beginning treatment according to this invention; this pre-treatment level of Marker can then be compared to the level of Marker in the subject after the treatment commences, to determine the efficacy of the treatment.

EXAMPLES

The TNF receptor p55 is largely believed to mediate cytotoxic effects of TNF-α, whereas p75 (TNFR2) is thought to mediate the protective effects of the cytokine.^1,2Since aging is associated with the increased expression of p55 and the decreased expression of p75 receptor/TNFR2 in lymphocytes,³p75 receptor/TNFR2 may be needed in angiogenic signaling in adults. To test this hypothesis neovascularization in the hind limb ischemia model was studied in young and old mice lacking the p75 receptor (p75KO) mice and age-matched wild-type controls. With advanced age, signaling through p75 receptor/TNFR2 was required for collateral vessel development in ischemia-induced neovascularization.
In animal models of ischemia^4-7and in limited human clinical trials,²³transplantation of bone marrow cells or bone marrow-derived endothelial progenitor cells (EPCs) leads to the migration and homing of these cells to areas of ischemia. In the ischemic tissue, these cells contribute to the process of neovascularization by locally releasing angiogenic growth factors and cytokines⁶and further differentiation of EPCs into mature endothelial cells. These activities lead to the development of collateral vessels that then contribute to the faster recovery of blood flow in the ischemic areas^4-8.

Example 1

Neovascularization and EPC Mobilization is Mediated by p75 TNFR2 Signaling

To evaluate the effect of bone marrow transplantation on neovascularization in old mice and to examine the role of functional p75 receptor/TNFR2 in post-ischemic recovery, two bone marrow transplantation murine models were established. The first model was used to evaluate whether the replacement of old p75KO bone marrow with young wild-type marrow would prevent limb autoamputation in old p75KO mice. This model provides for the evaluation of the contribution of wild-type bone marrow-derived cells to the processes of post-ischemic recovery in p75KO tissue. To evaluate the contribution of bone marrow-derived p75KO cells to post-ischemic recovery in otherwise wild-type tissue, a second murine model was established. In this model, old wild type mice were treated with lethal irradiation to destroy their endogenous bone marrow and the old wild-type mice were subsequently transplanted with bone marrow from young p75KO mice. Transplantation of young wild-type bone marrow into old p75KO mice rescued the limbs of these old animals from autoamputation. This result was not observed when bone marrow mononuclear cells from young p75KO mice was used. Furthermore, in the old wild-type bone marrow transplantation model functional p75 receptor was necessary for the bone marrow-derived EPCs to properly contribute to the post-ischemic recovery. These results indicated that ischemia-induced neovascularization and mobilization of EPCs from bone marrow is mediated, at least in part, by p75 TNFR2 signaling.

Example 2

Ischemia-Induced Angiogenesis is Impaired in Old TNFR2 KO Mice

When assessing the relationship between age and TNFR1 and 2 levels in EPCs, a 25-30% statistically non-significant decrease in p55 levels was found in EPCs from elderly mice compared with younger animals. In contrast, there was a more than 55% (P<0.01) decrease in p75 mRNA levels in EPCs from elderly mice compared with younger animals.
Mean blood flow in young wild-type mice twenty-eight days after hind limb surgery reached 80% of the pre-ischemic flow (FIG. 1A, black bars). In contrast, recovery of blood flow was delayed up to fourteen days in old wild-type (gray bars) and young p75KO (clear bars) mice (40% of pre-ischemic value vs. 80% in young wild-type mice, P<0.03), but was similar to the recovery in young wild-type mice thereafter (FIG. 1A, days 21 and 28). These results suggested that old wild-type and young p75KO mice exhibit a partial and temporal insufficiency of post-ischemic recovery compared with young wild-type mice.
All of the old p75KO mice (n=12, FIG. 1B, black bars) experienced autoamputation of the operated limb between days 7-10 after hind limb surgery, suggesting an absolute requirement of TNFR2 p75 for post-ischemic blood flow recovery in adult mice. Surprisingly, at day 14 after surgery post-ischemic blood flow in young p55KO mice was 75% of the pre-surgery level (FIG. 1C, black bars), approaching the rate of recovery of age-matched young wild-type controls (FIG. 1A, black bars). In contrast, old p55KO mice demonstrated significantly slower recovery of post-ischemic blood flow between days 7-14 compared with young p55KO mice (FIG. 1C, black bars=young p55KO mice, gray bars=old p55KO mice). The old p55KO mice also demonstrated no improvement in blood flow recovery from immediately post-surgery blood flow for the first 7 to 21 days after surgery.
On day 21 after surgery, only 60% (n=7) of old p55KO mice preserved the ischemic limb (FIG. 1F) (with two animals exhibiting distal toe necrosis) (FIG. 1G). At this time, old p55KO mice showed a 60% lower rate of blood flow recovery than the young p55KO mice (P<0.04), whereas about 40% (n=5) of the old TNFR1 p55KO mice lost an ischemic limb between days 14-21 after hind limb surgery (approximately 1-2 weeks later than age-matched old p75KOs) (FIG. 1H), suggesting that the p55 receptor may make a smaller contribution to the processes of ischemia-induced neovascularization than the p75 receptor in old mice.
There was a 50% decrease in capillary density at 28 days after hind limb ischemia in young p55KO mice compared to young wild-type mice. Similarly, capillary density in young p75KO mice was also 50% lower than in young wild-type mice (1.83±0.3 vs. 0.92±0.1 and 0.99±0.14, respectively, P<0.05; FIG. 1D). There was a statistically non-significant 27% lower capillary density in young wild-type mice compared with old wild-type mice (1.83±0.3 vs. 1.34±0.42, P=NS). The difference in capillary density between old p55KO mice and old wild-type mice was also not statistically significant (1.34±0.42 vs. 0.75±0.25, P=NS). Due to autoamputation, there was insufficient tissue remaining to assess capillary density in old p75KO mice (FIG. 1B). Because of the severity of the post-ischemic outcome in old p75KO mice and reported age-associated decrease in p75 receptor expression, subsequent studies focused on elucidating the role of TNFR2 p75 in the processes of post-ischemic recovery.
Fluorescence activated cell sorter (FACS) analysis of circulating bone marrow-derived EPCs in peripheral blood after hind limb surgery in wild-type and P75 KO mice revealed a greater increase in circulating bone marrow-derived EPCs in the wild-type mice than in the p75KO mice between days 1 and 3, with a maximal 4-fold increase by day 3 (11.1±3.1 vs 2.6±1.8, P<0.05; FIG. 1E). Circulating EPCs remained about two-fold higher (7.6±0.5 vs 3.21±0.7, P<0.01) in wild-type than in p75KO mice up to seven days after hind limb surgery, suggesting that ischemia-induced mobilization of EPC from bone marrow depends, at least in part, on TNFR2 p75 signaling. By day ten the EPC levels were similar in wild-type and P75KO mice (4.75±3.3 vs 4.5±0.9, P=NS; FIG. 1E).

Example 3

Ischemia-Induced VEGF Expression is Lower in the Limbs of p75KO Mice

When VEGF expression in the muscles of operated limbs was assessed by immunofluorescence, it was highly expressed in the muscle tissue of wild-type mice between days 3 and 7 post-surgery (FIG. 2A, upper panel). In contrast, VEGF expression was minimal in the tissues of p75KO mice between days 3 and 7 (FIG. 2A, lower panel), suggesting that ischemia-induced VEGF expression is impaired in p75KO mice.
Densitometric analysis of total RNA from hind limb muscle revealed that VEGF expression was not detectable in p75KO mice 3 days after hind limb surgery (FIG. 2B, upper panel), whereas VEGF was highly expressed in wild-type tissue (FIG. 2B, upper panel, clear bars), confirming the immunofluorescent staining results (FIG. 2A). By day 10 VEGF was also detectable in p75KO mice (FIG. 2B, upper panel, black bars), but was about half the level of wild-type controls.
These results differed from those obtained when mRNA expression of two other known angiogenic factors, bFGF and angiopoietin-1 was examined. In p75KO mice by day 3 expression of bFGF mRNA was 50% of bFGF levels compared to wild-type controls (FIG. 2B, middle panel, clear bars). By day 10 the level of bFGF was similar in both genotypes (FIG. 2B, middle panel). Interestingly, mRNA levels of angiopoeitin-1 were consistently and substantially higher in p75KO mice (FIG. 2B, bottom panel, black bars) than in wild-type mice up to day 10 after surgery, suggesting that ischemia-induced angiopoeitin-1 gene expression does not require signaling through p75 TNFR2.
Real-time PCR analysis of homogenized hind limb muscle on days 3 and 10 after hind limb surgery revealed that, compared to wild-type tissue, VEGF expression was decreased about 40 to 50% (p<0.05) in p75KO mice between days 3 and 10 after HL surgery (FIG. 3A). The mRNA expression of bFGF was also decreased by 15 and 36% ( day 3 and 10, respectively) in p75KO compared to wild-type tissue, but differences were not significant statistically. Interestingly, mRNA levels of angiopoeitin-1 were comparable in p75KO than in wild-type mice up to day 10 after surgery, suggesting that ischemia-induced angiopoeitin-1 gene expression does not require signaling through p75 TNFR2.

Example 4

Ischemia-Induced Apoptosis is Greater in the Limbs of p75KO Mice

To evaluate the viability of ECs, sections of operated hind limb muscle were triple stained for TUNEL (TdT-dUTP terminal nick-end labeling) a marker of apoptosis, for Isolectin B4, a marker of endothelial cells, and TopRo3 to visualize nuclei. When sections of operated hind limb muscle were stained for TUNEL, no apoptosis was detectable in wild-type mice before hind limb surgery and up to day 3 post-surgery (FIG. 4A, upper panel). A few TUNEL-positive cells were detected at day 7 post-surgery. In contrast, p75KO mice demonstrated extensive apoptosis by day 1 after hind limb surgery (FIG. 4A, lower panel). The extent of apoptosis remained greater in the p75KO mice compared with wild-type controls up to day 7 post-surgery, suggesting that ischemia-induced apoptosis was attenuated by p75 expression. There was a statistically significant increase in the number of TUNEL positive endothelial cells in p75KO mice following hind limb surgery, representing a 67% and 93% increase in endothelial cell apoptosis in p75KO mice on days 1 and 10 respectively (FIG. 4B). Representative images of TUNEL positive endothelial cells that are also stained for isolectin B4 and TopRo3 (to visualize nuclei) are shown in FIG. 4C.

Example 5

Loss of p75 Impairs the Function of Cultured EPCs

Our in vivo studies showed that loss of p75 TNFR2 impairs post-ischemic recovery, by affecting angiogenesis (FIGS. 1A and B) and ischemia-induced mobilization of EPCs from bone marrow is decreased in p75 KO mice. To examine whether endothelial cell functions may be altered in p75KO cells ex-vivo expanded cultures of BM-derived EPCs from wild-type and p75KO mice were used. The identity of ex-vivo expanded cells was confirmed before functional experiments. In addition, RT-PCR expression of TNF receptors p55R1 and p75R2 was confirmed in ex-vivo expanded endothelial progenitor cells.
Up to 14 days after initial plating of ex vivo expanded cultures of bone marrow-derived EPCs from wild-type and p75KO mice, no difference was detected between the two genotypes in morphologic phenotype or rate of proliferation of ex vivo expanded EPCs. Seven days after initial plating and 2 days after re-plating almost 100% of cells were identified as EPCs by Isolectin B4-positive staining and by incorporation of DiI-labeled acetylated low density lipoprotein (LDL). These characteristics were indicative of the endothelial origin of these cells. No differences were detected in cells from mice of different genotypes.
One of the functional features of endothelial cells is their ability to migrate towards chemotactic stimuli⁹. No difference in chemotactic activity was found between wild-type and p75KO EPCs in in vitro migration toward TNF, VEGF, and GM-CSF (FIG. 5A). The ability to form tube-like structures on VEGF-enriched matrigel is another functional characteristic of ECs. EPCs from wild-type mice formed tube-like structures in control chambers and those treated with TNF, whereas EPCs from p75KO mice failed to form tube-like structures in either type of chamber, indicating a functional loss in the bone marrow-derived EPCs of p75KO mice (FIG. 5B).
Because induction of endothelial growth factors and cytokines (i.e., VEGF, bFGF, platelet-derived growth factor (PDGF), IL-8, TNF-α etc) is required for initiation of angiogenesis and neovascularization,^10-13the effect of TNF treatment on the expression mRNA transcripts using custom-made multiprobe ribonuclease protection assay (RPA) for bFGF, VEGF, angiopoeitin-1 and actin (FIG. 5C) was evaluated. Densitometric analysis revealed that TNF-induced expression of bFGF mRNA was counteracted in p75KO mice up to 16 hours after treatment and at 24 hours after treatment was only a third the level of the bFGF mRNA in wild-type cells (FIG. 5D, black bar=P75KO; clear bar=WT). The difference between wild-type and P75KO mice in VEGF expression was even more dramatic. VEGF expression was nearly undetectable in p75KO cells up to 8 hours after TNF treatment, and remained 8-fold lower at 24 hours after surgery in p75KO compared with wild-type EPCs (FIG. 5D). Interestingly, TNF-induced mRNA levels of angiopoeitin-1 were comparable at 8 and 16 hours in EPCs from p75KO and wild-type cells, but by twenty-four hours the mRNA levels in the cells from the p75KO mice were reduced to half the levels observed in wild-type cells (FIG. 5D). This paralleled the in vivo observation of angiogenic factor expression in tissue homogenates of wild-type and p75KO mice after hind limb surgery (FIG. 2B) and confirming the loss of several functions characteristic of endothelial cells in p75KO cells.

Example 6

Signaling Through TNFR2 p75 is Required for NFκB-Mediated VEGF Gene Expression

Because treatment with TNF activates transcription factor NFBκand NFκB is known to regulate VEGF expression^10,14, NFκB nuclear translocation and DNA binding activity was compared in wild-type and p75KO cells. Thirty minutes after TNF treatment, no NFκB nuclear translocation was observed in EPCs from p75KO mice, whereas NFκB was translocated in the nucleus in 100% of EPCs from wild-type mice (FIG. 6A). Electrophoretic Mobility Shift Assay (EMSA) using NFκB consensus sequence probe showed that constitutive NFκB DNA binding was higher in p75KO EPCs (FIG. 6B). TNF treatment failed to activate (and in fact decreased) NFκB DNA binding activity in p75KO EPCs up to 120 minutes post-stimulation. In contrast, TNF treatment increased (3- to −4-fold) the NFκB DNA binding activity in wild-type cells from 30 to 120 minutes post-stimulation. These findings suggested that in the absence of TNF stimulation, signaling via the p55 receptor maintained a slightly higher NFκB DNA binding activity in p75KO cells than in wild-type cells. Following TNF stimulation, NFκB signaling in p75KO EPCs is counteracted.
To further investigate the molecular mechanisms of TNF signaling in the regulation of VEGF expression, serial VEGF promoter-luciferase reporter constructs were transfected into EPCs from wild-type and p75KO mice. When the effects of 1 ng/ml TNF (a known angiogenic concentration of TNF) were examined, a 2- to −3-fold increase in the activity of full-length (2.6 kb) VEGF promoter was observed in p75KO EPCs compared with wild-type EPCs (FIG. 6C). A schematic diagram of the VEGF luciferase constructs is provided at FIG. 6D. Interestingly, in wild-type cells transfected with an NFκB construct (0.35 kb) there was a 3-fold increase in VEGF promoter activity compared with wild-type cells transfected with a full-length construct. A schematic diagram of the VEGF/luciferase reporter deletion constructs is provided at FIG. 6D. This suggested that in wild-type cells under similar TNF treatment conditions, NFκB alone could activate VEGF promoter to the same degree as the full length promoter (FIG. 6C). In contrast, VEGF promoter activity was completely counteracted in p75KO cells transfected with an NFκB construct, indicating that signaling through p75 receptor was required for TNF-induced activation of VEGF promoter (FIG. 6E). In addition, NFκB may mediate the induction of VEGF expression through the TNFα/p75 pathway. VEGF/Luciferase activity is quantitated in FIG. 6C. No difference was observed between wild-type and P75KO EPCs transfected with an inactive (0.07 kb) VEGF promoter construct.

Example 7

Transplantation of Bone Marrow Cells from Young Wild-Type Mice Preserved Limbs of Old Mice from Ischemia-Induced Autoamputation

A schematic diagram of the bone marrow transplantation models described herein is provided at FIG. 7. Lethally irradiated old p75KO mice with BM MNCs isolated from WT GFP (+) and control p75KO DiI-labeled cells from young (4 weeks old) mice (for detailed BMT protocol please refer to the diagram in FIG. 7). Four weeks after BMT, to allow for complete engraftment of transplanted BM (by day 28 recipients BM was completely reconstituted with donor marrow and no difference between wild-type or p75KO engraftment was observed and the number BM-derived PB EPCs were also similar in recipient-mice of both genotypes, as evaluated by FACS analysis (FIGS. 8A and 8B). Evaluation of bone marrow engraftment and peripheral blood mononuclear cells in wild-type and p75KO mice is provided at FIGS. 8A and 8B.
Because bone marrow-derived mononuclear cells contributed to post-natal neovascularization, and because post-ischemic recovery was substantially impaired in p75KO mice, the question of whether restoration of p75 receptor expression in bone marrow of old p75KO mice could augment ischemic recovery was examined. Twenty-eight days after hind limb surgery, 100% of old p75KO mice transplanted with wild-type bone marrow had a preserved ischemic limb, whereas only half the old P75KO mice transplanted with bone marrow from young p75KO mice had a preserved ischemic limb (FIGS. 9A and 9C). These results suggested that bone marrow-derived EPCs play an important role in to post-ischemic recovery.

Example 7

p75/TNFR2 is Required for Efficient Contribution of Bone Marrow-Derived EPCs in Post-Ischemic Recovery

To explore further the contribution of bone marrow-derived EPCs old wild-type mice were transplanted with bone marrow mononuclear cells from young wild-type or p75KO mice. Although, old wild-type mice that received either wild-type or P75KO bone marrow avoided post-ischemic limb loss at 28 days after hind limb surgery, the mice that received P75KO bone marrow experienced a greater loss of total muscle tissue in the ischemic limb (50% vs. 23%; FIGS. 9B and 9D). These data indicated that even in wild-type ischemic tissue, bone marrow-derived EPCs with functional p75 TNFR2 are required for efficient post-ischemic recovery.
Homing of bone marrow-derived EPCs to ischemic tissue was also examined. Confocal microscopy of hind limb tissue from the operated limbs of GFP-labeled bone marrow-transplanted mice showed that bone marrow-derived cells homed only into ischemic areas of operated limbs (FIGS. 10A-10C). In addition, homing of endothelial lineage cells into the areas of ischemia was examined. Twenty-eight days after hind limb surgery, more that 60-70% of GFP-positive cells in the ischemic limbs were identified as bone marrow-derived endothelial lineage cells (FIGS. 11A and 11B, merged image: double-positive cells), strongly suggesting a substantial contribution of bone marrow-derived EPCs to post-ischemic recovery.
Age-related impairment of angiogenesis has been documented in previous research^11,15-18. Specifically, investigators have delineated deficiencies in several components of ischemia-induced neovascularization, including inhibition of endothelial cell proliferation and function^11,19-21; impaired expression of angiogenic growth factors, such as VEGF, bFGF, transforming growth factor-beta (TGF-β), and PDGF ^11,22-24. In addition, some studies have also demonstrated a significant contribution of ischemia-induced inflammatory responses to the delayed cutaneous wound healing associated with age^25,26.
Interestingly, impaired signaling by TNF-α and other cytokines in endothelial cells has been correlated with enhanced apoptotic responses in cutaneous microvasculature in adult tissue²⁷. It is well known that TNF-α can induce the expression of many important immune- and angiogenesis-related genes^10,28, through two different TNF-α receptors: p55 and p75^29-31. In various vascular endothelial cells, TNF-α increased the expression of the well-known angiogenic factors VEGF, bFGF, IL-6¹⁰, and PDGF.³²The role of two distinct TNF-α receptors in mediating these responses remains obscure. Although the distribution of p55 is more widespread than that of p75, p75 is present in greater amounts on cells of endothelial and hematopoietic lineage than is p55. Also, expression of p55 is constitutive in most of the cells, whereas expression of p75 appears to be inducible³³. The p55 receptor is largely believed to mediate the cytotoxic effects of TNF-α, whereas signaling through p75 is thought to mediate the protective effects of TNF-α.^1,2Aging is associated with increased expression of p55 and decreased expression of p75 in human cells,³and a decrease in the expression of p75 receptor in peripheral blood EPCs from adult donors, as reported herein. The p75 may be needed in angiogenic signaling and post-ischemic recovery in adults.
To test this hypothesis neovascularization and the underlying molecular, cellular and tissue repair processes in the hind limb ischemia models of young and old p55 and p75KO mice and age-matched wild-type controls was studied. TNF-α-mediated cell signaling pathways were examined in wild-type and p75KO bone marrow-derived mouse EPCs in vitro physiologic post-ischemia recovery after hind limb surgery in vivo, and wild-type and p75KO EPC function in vitro.
These results demonstrated that a deficiency in p75 TNFR2 expression lead to the failure of post-ischemic recovery in adult mice, manifested by 100% limb loss in old p75KO, compared with only 40% limb loss in old p55 mice, which occurred one to two weeks later than that observed in p75KO mice. This suggested that the p55 receptor contributed less than the p75 receptor to the processes of ischemia-induced neovascularization in old p55KO mice. The negative effect of p75 receptor deficiency on post-ischemic recovery was also evident in young animals. Recovery in young p55 mice after hind limb surgery was similar to recovery in age-matched young wild-type controls, whereas recovery in young p75KO mice was delayed up to 14 days and was similar to the recovery in old wild-type mice. These data strongly suggested that decreased angiogenesis in aging^11,17,18is due to impaired signaling through TNFR2 p75. Likewise, the number of capillaries per muscle fiber in young p75KO, old p55KO, and old wild-type mice were significantly lower than in young wild-type mice.
Numerous reports suggest that VEGF is a useful growth factors in therapeutic angiogenesis.^34-37In the present study, VEGF expression in muscle tissue from ischemic limbs of p75KO mice was shown to be lower in both mRNA and protein levels than in wild-type mice. In addition, expression of bFGF, another potent angiogenic growth factor,^13,38was lower in p75KO than in wild-type mice. VEGF, which is present early in the response to ischemia,³⁹has been shown to mobilize bone marrow-derived EPCs in murine models and in humans^9,40. It is conceivable that significantly lower mobilization of bone marrow-derived EPCs into peripheral blood that was observed in p75KO mice was a direct result of decreased VEGF and bFGF expression.
Aging is associated with alterations in cytokine signaling pathways that result in enhanced apoptosis^27,41,42. The reduced expression of TNFR2 p75 associated with increasing age, coupled with post-ischemic increases in the systemic levels of TNF-α, favor apoptosis in adult endothelial cells,⁴²which could subsequently lead to inhibition of angiogenesis. The present studies observed an exaggerated apoptotic response in the hind limbs of p75KO mice. It is possible that unopposed signaling through the p55 receptor in p75KO mice, and conceivably in human adult tissue with decreased p75 receptor expression,⁴¹increased a predominantly proapoptotic cascade via increases in Fas-associating protein with death domain (FADD), TNFR1-associated death domain protein (TRADD), and Fas death domain (FASDD),^3,41as impaired p75-mediated anti-apoptotic signaling via NFκB potentiated further apoptotic responses in the same tissue.
Aged endothelials cells show impaired proliferation and migration in response to various cytokines^12,43. As reported herein, while bone marrow-derived EPCs from wild-type and p75KO mice, did not show impaired in vitro proliferation or migration in response to several cytokine and growth factor stimuli, suggesting that these endothelial cell functions may not depend on TNFR2 p75 signaling, a substantial decrease in VEGF and fibroblast growth factor (FGF) mRNA expression was observed following TNF-α stimulation in vitro of bone marrow-derived EPCs from wild-type and p75KO mice. These results strongly suggest that ischemia-induced expression of the angiogenic growth factors VEGF and FGF, depends, at least in part, on signaling through p75 TNFR2. Similar to previous findings in adult tissue,¹¹a bifactorial reduction in VEGF expression was observed in the p75KO mouse model. First, lower VEGF protein expression by immunostaining in the ischemic hind limb was observed in p75KO mice as compared with wild-type mice; and second, VEGF promoter activity was lower in bone marrow-derived EPCs from p75KO than in wild-type EPCs, strongly suggesting that previously reported decreases in VEGF reporter activity in adult cells may be a direct consequence of age-associated decreases in the expression/signaling via p75 TNFR2.
To support these findings and to identify the molecular mechanisms by which TNFR2 p75 mediates decreased VEGF promoter activity, NFκB nuclear translocation and DNA binding activity was studied in wild-type and p75KO EPCs. TNF-induced NFκB nuclear translocation and DNA binding activity were inhibited in p75KO EPCs, whereas NFκB was readily translocated to the nucleus in wild-type cells, where it showed enhanced DNA binding activity. To further elucidate the role of TNF signaling in NFκB-mediated VEGF gene expression, serial transfection assays were performed using VEGF promoter deletion constructs. In wild-type EPCs transfected with full-length promoter, a 3-fold increase in VEGF promoter activity was observed, and a 2-fold increase in p75KO EPCs transfected with full length-promoter.
Interestingly, in cells transfected with a VEGF promoter deletion construct that contained only NFκB response elements, the VEGF promoter was completely counteracted in p75KO EPCs. In contrast, there was a 3-fold increase in VEGF promoter activity in wild-type EPCs that had been transfected with the same deletion construct. These findings suggest that intact signaling through both p55 and p75 receptors assures proper NFκB activation and subsequent transcriptional activation of VEGF in cells from young donors, whereas signaling through p55 receptor alone is not sufficient for proper NFκB activation and regulation of VEGF transcription in cells from adult donors having decreased p75 receptor expression^3,41. These findings may explain, at least in part, the impaired angiogenic response observed in adult tissue^{11,15,16,44,18}.
Research in animal models of ischemia^4-7and in limited human clinical trials²⁸has shown that transplantation of bone marrow cells or bone marrow-derived EPCs significantly augmented ischemia-induced neovascularization by recruitment and retention of these cells to the areas of ischemia. Further, researchers have shown that transplanted bone marrow cells or bone marrow-derived EPCs contribute to the processes of neovascularization through local release of angiogenic growth factors and cytokines⁶and further differentiation of EPCs into mature endothelial cells, which leads to the development of collateral vessels that contribute to more rapid recovery of blood flow in the ischemic areas^4-8.
In an attempt to prevent total loss of the ischemic limb of old p75KO mice and to further confirm the role of EPCs in neovascularization, a series of bone marrow-derived mononuclear cell transplantation experiments was performed in which bone marrow from young wild-type and young p75KO was transplanted into old p75KO mice. Bone marrow transplant was followed by hind limb surgery, hind limb blood flow recovery was evaluated one month after hind limb surgery. Transplantation of young wild-type but not p75KO bone marrow-derived mononuclear cells into old p75KO mice rescued ischemic limbs from autoamputation, strongly suggesting that ischemia-induced neovascularization and mobilization of EPCs from bone marrow is mediated, at least in part, by p75 TNFR2 signaling.
Since numerous component of the neovascular response may be affected in the tissue of old p75KO mice (i.e. the proliferation, survival, migration, and altered angiogenic growth factor supply of endothelial cells and vascular smooth muscle cell (VSMC) in the local hind limb tissue), a second bone marrow transplant model was established that allowed the evaluation of the contribution of bone marrow-derived tissue from young wild-type and young p75KO mice in post-ischemic recovery in old wild-type tissue. While neither wild-type nor p75KO bone marrow transplanted mice experienced post-ischemic limb loss after hind limb surgery, there was a statistically significant (50%; P<0.0001) loss of total muscle tissue in old wild-type mice that had been transplanted with young p75KO cells, compared to insignificant (P=ns) 23% muscle loss in old wild-type mice transplanted with cells from young wild-type mice. These data suggest that bone marrow-derived EPCs with functional p75 TNFR2 are required even in wild-type ischemic tissue for efficient post-ischemic recovery. In addition, this finding clearly substantiates the importance of bone marrow- and peripheral blood-derived EPCs in ischemia-induced neovascularization.
These results indicate, first, that the loss of p75/TNFR2 expression impairs post-ischemic recovery; second, p75/TNFR2 is important for ischemic recovery in part via up-regulation of VEGF gene expression; third, post-ischemic apoptotic responses are exaggerated in the absence of p75/TNFR2; fourth, ischemia-induced neovascularization and mobilization of EPCs from bone marrow is mediated, at least in part, via p75/TNFR2; fifth, p75/TNFR2 expression by bone marrow-derived mononuclear cells is important for ischemia-induced neovascularization; sixth, expression of p75/TNFR2 is decreased in EPC from elderly donors; and seventh, signaling through p75/TNFR2 is required for collateral vessel development in models of advanced age. Moreover, based on these results it is likely that augmentation of p75/TNFR2 in older subjects will improve recovery after ischemia and prevent the development of severe ischemia-induced damage in adult coronary and peripheral vascular disease.
These experiments were carried out using the following materials and methods.

Mouse Strains

Wild-type and mutant mice strains that lack TNFR1 (p55) and p75 (TNFR2), as described herein, are commercially available from The Jackson Laboratory, Bar Harbor, Me.

EPC Culture and In Vitro Functional Assays

Bone marrow-derived EPCs from young wild-type p75KO mice were isolated and expanded ex vivo as previously described⁵. Cells were grown on 1.5% gelatin-coated plates (Sigma, St. Louis, Mo.) and cultured with commerciall available cell culture media, endothelial cell basal medium 2 (EBM-2) with 10% fetal bovine serum (Clonetics, San Diego, Calif.). EBM-2 was supplemented with growth factors provided by the manufacturer of the gelatin-coated plates. For in vitro studies bone marrow-derived ex vivo expanded EPCs were used 6 to 8 days after initial plating as described^5,9.
Chemotaxis and chemokinesis of wild-type and TNFR2 EPCs in response to TNF-α (1 and 10 ng/ml), rmVEGF (20 ng/ml) and GM-CSF (50 ng/ml) were evaluated using a modified checkerboard assay with commercially available microplates, Costar Transwell chambers (6.5 mm diameter, 5 μm pore) as described previously⁹. Cells migrating into the lower chamber were collected in 50 μl of buffer and counted manually using hemocytometer and Coulter Counter.
Circulating peripheral blood EPCs in wild-type and TNFR2 KO mice were evaluated using EPC culture assay as described previously⁹. Briefly, mononuclear cells isolated from 500 μl of peripheral blood were cultured in EBM2 medium (Clonetics, San Diego, Calif.) conditioned with growth factor on 4-well glass slides coated with 0.5% gelatin solution (Sigma, St. Louis, Mo.). After 4 days in culture, EPCs were identified morphologically and were stained with antibodies against acetylated low density lipoprotein-1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate-labeled (DiI-AcLDL) (Molecular Probes, Eugene, Oreg.) and fluorescein isothiocyanate (FITC)-labeled Isolectin B4 (Vector Laboratories, Inc, Burlingame, Calif.). Double-positive staining is characteristic for cells of the endothelial lineage. On fluorescent microscopy, double-positive cells were identified as EPCs. In addition, the kinetics of EPC mobilization into peripheral blood was evaluated by Fluorescence Activated Cell Sorting (FACS) analysis as described⁴⁵.
To examine the formation of tube-like structures, ex vivo expanded wild-type and p75KO EPCs were seeded on 4-well chamber slides (at 5×10⁴cells/well), which were coated with a commercially available solubilized basement membrane preparation, MATRIGEL (Collaborative Biomedical Products, Bedford, Mass.). Cells were incubated for 12 hours in medium containing 5% fetal bovine serum and supplemented with medium alone, 1 ng/ml, or 10 ng/ml of recombinant (rm)TNF-α (BD PharMingen, Los Angeles, Calif.). Cells in the chambers were examined and photographs were taken 12 hours post-stimulation.
To examine mRNA expression of angiogenic factors in vivo in the homogenized tissue of wild-type and p75KO mice, ribonuclease protection assay (RPA) was performed using commercially available multiprobe custom-made angiogenic factor (VEGF, bFGF and angiopoeitin DNA templates (PharMingen, Los Angeles, Calif.). The operated limbs were examined on days 3 and 10 after hind limb surgery in ex vivo expanded wild-type and p75KO EPCs treated with 10 ng/ml of mrTNF-α. Total cellular RNA was isolated using Trizol reagent (Life Technologies, Inc., St. Paul, Minn.). [α ³²P]UTP (NEN) was used to synthesize in vitro transcribed antisense riboprobes. RNAse Protection Assays were performed using commercially available hybridization and digestion buffer reagents provided in the RPA III TM kit (Ambion, Austin, Tex.) following manufacturer instructions.
To examine the effect of TNF on NFκB nuclear translocation and NFκB DNA binding activity, EPCs (grown in 4-well chamber slides) from wild-type and p75KO mice were treated with TNF (10 ng/ml) for 30, 60, and 120 min. EPCs were then processed for immunostaining with NFκB p65 (Santa Cruz Biotechnology, Santa Cruz, Calif.) to evaluate nuclear translocation and electromobility shift assay with NFκB consensus sequence to evaluate NFκB DNA binding activity (Santa Cruz Biotechnology, Santa Cruz, Calif.) as described²⁶.
To examine the role of TNF on NFκB-mediated VEGF promoter activity, EPCs from wild-type and p75KO mice were transfected with full length (2.6 kb) VEGF promoter-reporter construct (in basic pGL2 plasmid backbone), a deletion construct (0.35 kb) containing two putative NFκB sites and one Sp1 cluster, and an inactive deletion construct. The inactive deletion construct spanned up to −70 from transcription origination site and containing an incomplete Sp1 cluster. (All constructs were kind gift from Dr. Debabrata Mukhopadhyay, Beth Israel Medical Center, Boston, Mass.), which are described in Mukhopadhyay et al., Mol Cell Biol. 1997 September; 17(9):5629-39). Twenty-four hours after transfection cells were treated with TNF (1 ng/ml) and evaluated the VEGF promoter activity 18 hours later by measuring luciferase activity as described⁴⁶.
To evaluate the constitutive mRNA expression of TNF receptors p75 and p55 in peripheral blood EPCs from adults, human peripheral blood EPCs were isolated from peripheral blood of donors of various ages first by separating mononuclear cell fraction using density centrifugation over Histopaque-1083 (Sigma, St. Louis, Mo.) and then growing mononuclear cells on selective medium as described previously⁹. After 7 days in culture (standard time for selection of EPC), cells were harvested and processed for evaluation of mRNA expression of TNF receptors p75 and p55 by RPA using a custom-made (p75, p55 and actin) multiprobe DNA template (BD PharMingen, Los Angeles, Calif.).

Ischemia Studies

Ligation and removal of femoral artery was used as previously described to induce unilateral hind limb ischemia in male young and old wild-type C57BL/6J and p75KO mice²⁷. Serial assessments of hind limb blood flow was performed with a PIM 2.0 Laser Doppler perfusion imager (LDPI) (Lisca) as previously described²⁷. Perfusion was expressed as the ratio of left (ischemic) to right (control) limb. Results represent the mean±SEM of at least 10-15 mice per group.
To determine capillary density, on day 28 post-hind limb surgery whole nonischemic and ischemic limbs of at least 5 mice from each group were immediately fixed in methanol overnight and processed for histology. Endothelial cells were identified by histochemical staining with biotinylated isolectin B4 according to the manufacturer's directions (Vector Labs, Burlingame, Calif.)⁴⁷. Capillary networks were evaluated as described previously⁴⁷.
To evaluate the kinetics of VEGF expression in operated limbs of wild-type and p75KO mice after hind limb surgery, tissue from 5 mice per group was collected before, and 1, 3, and 7 days after surgery, immediately fixed in methanol overnight and then processed for immunofluorescent staining with VEGF (Santa Cruz Biotechnology, Santa Cruz, Calif.). In addition, continuous sections of hind limb tissue from the same animals were immunostained with TUNEL to assess the viability of hind limb tissue in the operated limbs in wild-type and p75KO mice. Both VEGF- and TUNEL-stained slides were evaluated using laser scanning confocal microscopy (Zeiss, Axiovert 100, Thornwood, N.Y.).

Murine Bone Marrow Transplantation Studies

To evaluate the effect of bone marrow-derived cells in ischemic recovery in p75KO tissue, a bone marrow transplantation model was established in which bone marrow mononuclear cells from young (3 to 4 week old) wild-type and young p75KO mice were injected into old (12 to 14 month old) p75KO animals. To evaluate the effect of bone marrow-derived p75KO mononuclear cells in ischemic recovery in wild-type tissue, bone marrow-derived mononuclear cells from young wild-type and young p75KO mice were transplanted into old wild-type mice. Bone marrow cells were obtained by flushing the tibias and femurs of young (3-4 weeks) donor wild-type (C57BL/6J)/GFP (green fluorescent protein) mice and p75KO (on C57BL/6J background) mice (both from The Jackson Laboratory, Bar Harbor, Me.). Low-density bone marrow mononuclear cells were isolated by density centrifugation using a commercially available medium containing polysucrose and sodium diatrizoate that facilitates the recovery of large numbers of viable mononuclear cells, Histopaque-1083 (Sigma, St. Louis, Mo.). A schematic diagram showing murine bone marrow transplantation models is provided at FIG. 6.
To evaluate the impact of bone marrow-derived mononuclear cell transplantation on therapeutic neovascularization, old TNFR2/p75KO mice were lethally irradiated with 9˜11 Gy and subsequently received intracardiac injections of 3×10⁶of donor bone marrow mononuclear cells. These mononuclear cells were either WT/GFP or TNFR2, and were labeled prior to intracardiac injection with 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanide (DiI) (Molecular Probes, Eugene, Oreg.). At 4 weeks post-bone marrow transplantation, by which time the bone marrow of the recipient mice has usually regenerated with donor bone marrow cells, hind limb surgery was performed. Animals were evaluated at different times post-hind limb surgery for physiologic recovery using LDPI, histologic assessment of capillary network (CD31 and/or isolectin B4 staining)²⁸, evaluation of total muscle loss using the ratio of operated vs. non-operated limbs on day 28, and homing of WT/GFP and TNFR2/DiI-labeled bone marrow-derived mononuclear cells in the areas of ischemia by confocal microscopy²⁶.
To evaluate the recipient bone marrow engraftment before hind limb surgery and at the end of the post-hind limb surgery evaluation period (4 weeks after surgery), 5 animals from each transplantation group were sacrificed. Bone marrow from tibias and femurs were flushed and after density centrifugation and methanol fixation, cells were processed for FACS analysis²⁵. In addition, 500 μl of peripheral blood was drawn from the same animals. Circulating peripheral blood mononuclear cells were isolated by lysing red blood cells with an ammonium chloride solution (Stem Cell Technologies, Seattle, Wash.) then using density centrifugation with Histopaque-1083 (Sigma, St. Louis, Mo.). After centrifugation, mononuclear cells were fixed with 1% PFA and processed for FACS analysis⁴⁵.

Statistical Analysis

Results are expressed as mean±SEM. Differences among groups were evaluated by ANOVA and Fisher's PLSD post hoc test using StatView software (SAS Institute Inc., Gary, N.C.). Differences were considered significant at P<0.05.

Other Embodiments

From the foregoing description, it will be apparent that variations and modifications may be made to the invention described herein to adopt it to various usages and conditions. Such embodiments are also within the scope of the following claims.
The recitation of a listing of elements in any definition of a variable herein includes definitions of that variable as any single element or combination (or subcombination) of listed elements. The recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.
All patents and publications mentioned in this specification are herein incorporated by reference to the same extent as if each independent patent and publication was specifically and individually indicated to be incorporated by reference.
A review of the following specific references will help advance appreciation of the present invention.

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Claims

1. A method of treating, reducing the severity of, or preventing ischemia in a subject having or at risk of developing ischemia, the method comprising,

a) contacting a cell of the subject with a nucleic acid molecule encoding a p75/TNFR2 polypeptide or a fragment thereof; and

b) expressing the p75/TNFR2 polypeptide in the cell, wherein the method treats or prevents ischemia in the subject.

2. A method of enhancing angiogenesis in a tissue before, during, or after an ischemic event, the method comprising

a) contacting a cell with a nucleic acid molecule encoding a p75/TNFR2 polypeptide or a fragment thereof; and

b) expressing the p75/TNFR2 polypeptide in the cell, wherein the method enhances angiogenesis in the tissue.

3. The method of claim 1,

further comprising administering the cell to the subject, wherein the method enhances angiogenesis.

4. The method of claim 1, wherein

the method reduces apoptosis in the subject.

5. The method of claim 1, wherein the method enhances the local release of angiogenic growth factors and cytokines in the tissue.

6. The method of claim 1, further comprising the step of administering to the subject an angiogenic factor selected from the group consisting of: vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), basic fibroblast growth factor (bFGF), angiopoietin 1, angiopoietin 2 and monocyte chemotactic protein-1 (MCP-1).

7. The method of claim 1, further comprising the step of administering to the subject an endothelial cell mitogen selected from the group consisting of acidic and basic fibroblast growth factors, vascular endothelial growth factor, epidermal growth factor, transforming growth factor a and 13, platelet-derived endothelial growth factor, platelet-derived growth factor, tumor necrosis factor a, hepatocyte growth factor, insulin like growth factor, erythropoietin, colony stimulating factor, macrophage-CSF, granulocyte/macrophage CSF and nitric oxide synthase.

8-10. (canceled)

11. The method of claim 3, wherein the cell is delivered directly to an ischemic tissue or is delivered systemically.

12. (canceled)

13. The method of claim 1, wherein the nucleic acid molecule is present in a vector.

14-15. (canceled)

16. A method of enhancing p75/TNFR2 expression in a cell, the method comprising

b) expressing the p75/TNFR2 polypeptide in the cell.

17-21. (canceled)

22. The method of claim 20, wherein the cell is an endothelial progenitor cell or bone marrow derived cell.

23. An expression vector comprising a nucleic acid molecule encoding a mammalian p75/TNFR2 polypeptide or a fragment thereof operably linked to a promoter sufficient to direct expression of the p75 TNFR2 receptor polypeptide in a cell.

24-26. (canceled)

27. The vector of claim 26, wherein the vector is selected from the group consisting of adenoviral vectors, adeno-associated viral vectors, retroviral vectors, lentiviral vectors, alphaviral vectors, and herpes virus vectors.

28-30. (canceled)

31. A host cell comprising the vector of claim 23.

32-34. (canceled)

35. The host cell of claim 31, wherein the cell is an endothelial progenitor cell or a bone marrow-derived cell.

36. (canceled)

37. The host cell of claim 31, wherein the cell is in vitro or in vivo.

38. A pharmaceutical composition comprising an effective amount of an expression vector encoding a human p75/TNFR2 polypeptide or a fragment thereof in a pharmaceutically acceptable excipient, wherein the p75/TNFR2 polypeptide is operably linked to a promoter sufficient to drive expression of the p75/TNFR2 polypeptide in a mammalian cell.

39. (canceled)

40. The pharmaceutical composition of claim 38, wherein the promoter is sufficient to drive expression in an endothelial progenitor cell or a bone marrow-derived cell.

41. A kit for the treatment or prevention of ischemia, the kit comprising an effective amount of an expression vector encoding a human p75/TNFR2 polypeptide or a fragment thereof in a pharmaceutically acceptable excipient, wherein the p75/TNFR2 polypeptide is operably linked to a promoter sufficient to drive expression of the p75/TNFR2 polypeptide in a mammalian cell.

42. (canceled)

43. A method of monitoring a subject being treated for ischemia, the method comprising

a) administering a treatment that enhances the expression of a p75/TNFR2 polypeptide in a cell of the subject; and

b) measuring apoptosis or an increase in angiogenesis in a tissue of the subject relative to a reference, wherein a decrease in apoptosis or an increase in angiogenesis indicates a reduced severity of ischemia in the subject.

44-50. (canceled)

51. A method for identifying a candidate compound a candidate compound useful for the treatment of ischemia, the method comprising the steps of:

(a) contacting a cell expressing p75/TNFR2 polypeptide with a candidate compound; and

(b) detecting an increase in the level or the biological activity of a p75/TNFR2 polypeptide or nucleic acid molecule in the cell relative to a reference, wherein an increase in the level or the biological activity of p75/TNFR2 polypeptide identifies a candidate compound useful for the treatment of ischemia.

52. (canceled)