WO2010052715A2

WO2010052715A2 - Devices and methods for local treatment of cardiovascular diseases

Info

Publication number: WO2010052715A2
Application number: PCT/IL2009/001046
Authority: WO
Inventors: Britta Hardy; Alexander Battler; Annat Raiter
Original assignee: Ramot at Tel Aviv University Ltd; Mor Research Applications Ltd
Current assignee: Ramot at Tel Aviv University Ltd; Mor Research Applications Ltd
Priority date: 2008-11-07
Filing date: 2009-11-05
Publication date: 2010-05-14
Anticipated expiration: 2011-05-07
Also published as: WO2010052715A3

Abstract

Implantable medical devices and methods for local treatment of cardiovascular diseases are disclosed. The devices and methods utilize peptides capable of chemoattracting endothelial progenitor cells (EPCs), of promoting angiogenesis and/or of inhibiting apoptosis, which are locally administered to a bodily site afflicted by the cardiovascular disease. The peptides can be deposited on a surface of an implantable medical device configured for treating the cardiovascular disease and/or can be locally administered to a desired bodily site by means of an implantable medical device configured for treating the cardiovascular disease.

Description

DEVICES AND METHODS FOR LOCAL TREATMENT OF CARDIOVASCULAR DISEASES

FIELD AND BACKGROUND OF THE INVENTION

The present invention, in some embodiments thereof, relates to the field of therapy and, more particularly, but not exclusively, to methods, compositions and implantable devices which utilize peptides that are capable of chemoattracting endothelial progenitor cells and of inhibiting apoptosis, for treating cardiovascular diseases.

Cardiovascular diseases include a wide spectrum of both acute and chronic conditions associated with significant morbidity and mortality world-wide. Cardiovascular disease includes any condition that is associated with and/or affects the circulatory system and the organs supplied by them. This ranges from diseases of the heart, arteries, veins and lymph vessels to blood disorders that affect circulation. Vascular diseases include of example, Coronary disorders, Peripheral Artery Disease, Aneurysm, Renal (Kidney) Artery Disease, Raynaud's phenomenon, Buerger's Disease, Peripheral Venous Disease, Varicose Veins, Blood Clots in the Veins, hypertension and cerebrovascular diseases, as detailed hereinbelow. Coronary disorders can be categorized into at least two groups. Acute coronary disorders include acute myocardial infarction, and chronic coronary disorders include chronic myocardial ischemia and dysfunction, arteriosclerosis, congestive heart failure, angina pectoris, atherosclerosis, arrhythmia, valvular regurgitation and pulmonary hypertension. Other coronary disorders following coronary angioplasty include restenosis and stent thrombosis.

Ischemia is an acute condition that results from insufficient flow of oxygenated blood to a part of the body. During an ischemic event, there is a gradation of injury that arises from the ischemic site. Cells at the site of blood flow restriction undergo necrosis and apoptosis. The reduced flow is typically caused by blockage of a vessel by an thrombus (blood clot); the blockage of a vessel due to atherosclerosis; the breakage of a blood vessel (a bleeding stroke); the blockage of a blood vessel due to vasoconstriction such as occurs during vasospasms and possibly, during transient ischemic attacks (TIA), and following subarachnoid hemorrhage. Conditions in which ischemia may continue include myocardial infarction; trauma; and during cardiac and thoracic surgery and neurosurgery (blood flow needs to be reduced or stopped to achieve the aims of surgery). Procedures that are used to prevent ischemia include coronary thrombolysis, coronary angioplasty (with or without stent placement), and coronary artery bypass grafts. During myocardial infarction, the damaged myocardium may decrease myocardial function resulting reduced flow of blood to other organs and ischemia. Cardiac tissue itself is also subjected to ischemic damage.

Atherosclerosis is one of the leading causes of death and disability in the world. Atherosclerosis involves the deposition of fatty plaques on the luminal surface of arteries. The deposition of fatty plaques on the luminal surface of the artery causes narrowing of the cross-sectional area of the artery. Ultimately, this deposition blocks blood flow distal to the lesion causing ischemic damage to the tissues supplied by the artery. Coronary arteries supply the myocardium with blood. Coronary artery atherosclerosis disease (CAD) is the most common, serious, chronic life-threatening illness in the United States, affecting more than 11 million persons each year. The social and economic costs of coronary atherosclerosis vastly exceed that of most other diseases. Narrowing of the coronary artery lumen may be presented as angina, myocardial infarction or sudden death. There are over 1.5 million myocardial infarctions in the United States each year.

Current therapies for coronary artery disease fall into two main categories: drug treatment or revascularization. Gene therapy and cell therapy are being investigated in clinical trials. Drug treatment includes aspirin, statins, beta blockers, nitrates, calcium channel blockers and angiotensin receptor inhibitors; Revascularization includes angioplasty or bypass surgery. Delivery of novel drugs intended to address specific modes of action, such as anti-apoptosis, modulation of differentiation, or cardio- protection; or delivery of potent peptide growth factors such as the pro-angiogenic VEGF or the anti-apoptotic IGF-I. Gene therapy includes delivery of DNA or viruses encoding genes, intended to induce angiogenesis or myogenesis. Cell therapy includes surgically introducing cells directly into the heart muscle; injecting cells from inside the heart via catheter-mounted syringes using standard percutaneous catheterization interventional techniques; injecting cells into a coronary artery, whence they would be carried by the blood downstream into the injured zone; or introducing cells intravenously or mobilization of bone marrow cells by GCSF. Other techniques include, for example, the enhancement of the healing properties of blood through super- oxygenization or other ex-vivo blood treatment.

One of the common treatments of CAD is percutaneous coronary intervention (PCI). More than million PCI procedures are performed each year in the United States. In PCI, a catheter is inserted into a peripheral artery and threaded through the arterial system into the blocked coronary artery. Restenosis can involve recoil and shrinkage of the vessel, which are followed by proliferation of medial smooth muscle cells (SMC) in response to injury of the artery from PCI, the latter causing a narrowing of the inner lumen diameter of the blood vessel and thereby causing a decrease in blood flow distal to the injury. In response to blood vessel injury, SMCs in the tunica media and fibroblasts of the adventitial layer undergo phenotypic change which results in the secretion of metallopro teases into the surrounding matrix, luminal migration, proliferation and protein secretion. Various other inflammatory factors are also released into the injured area, including thromboxane A2, platelet derived growth factor (PDGF) and fibroblast growth factor (FGF). A number of different techniques have been used to overcome the problem of restenosis, including treatment of patients with various pharmacological agents or mechanically holding the artery open with a stent.

An angioplastic procedure using a metallic stent is a standard of percutaneous therapy of coronary artery disease. However, the coronary stent implantation provokes a cascade of cellular and biochemical events that induce pathophysiologic process such as thrombus formation and release of cytokines which trigger the proliferation of SMCs.

The occurrence of sudden occlusion may be due to an initial, in-stent thrombus formation and, at a later stage, in-stent restenosis, and is the principal drawback of coronary angioplasty. Drug-coated (i.e. drug-eluting) stents (DES) are frequently used in the treatment of coronary artery disease given their anti-restenotic effect. DES are stents coated with anti-proliferative agents that reduce or prevent inflammation and exaggerated SMCs proliferation and accumulation, and thereby, reduce restenosis. Examples of such drug eluting stents (DES) are paclitaxel-eluting stent (TAXUS, Boston Scientific), which inhibits the proliferation of SMCs, and sirolimus-eluting stent (Cypher, Cordis Corporation), which inhibits the inflammation response of the arterial wall. Currently available DES therefore do not address the in-stent thrombosis adverse phenomenon.

Re-endotheliazation is an intraluminal delivery of a sufficient number of endothelial cells (ECs) to diseased arterial sites, which provides an inherent non- thrombogenic potential, interrupts cytokine-driven activation of SMCs in vascular medial tissues and accelerates normal wound healing at diseased sites. The use of Drug Eluting Stents (DES) to overcome in-stent restenonsis with antiproliferative drugs that prevent cell growth results in delayed endothelization of the stent-treated coronary segment and leads to excess in-stent thrombotic events. Re-endothelization upon DES implantation can take many months and even years, during which the patient must be treated in order to prevent in-stent thrombosis. Such a treatment typically involves antiplatelet therapy for at least a year after DES implantation.

One possible approach to speed re-endothelization is seeding of cells either by a specially designed catheter or by in-stent cell delivery.

The catheter-based EC delivery involves transluminally delivering endothelial cells to angioplasty sites using a double balloon catheter with a central instillation port. Inflation of the balloon allows a segment of artery to be isolated from the circulation which is then incubated with infused endothelial cells. So far, this technique has been tested only in rabbits and is limited by the short period of cell infusion and the resulting detachment of cells that are not fully adhered. In-stent cell delivery technique involves adhering endothelial cells to stents which, following implantation of the stent in arteries, migrate onto the injured vascular surface and proliferate, thus inhibiting thrombus formation as well as SMC stimulation. Experimental attempts using EC-seeded stent struts were reported (Shirota et al. Biomateήals 2003; 24:2295-2302). However, irrespective of whether an in-stent or catheter-infusion delivery system is used, difficulty of harvesting EC from patients with atherosclerotic diseases has hampered clinical usage of these methods. In the last decade, endothelial progenitor cells (EPCs) were characterized

(Hristov et al. Thromb Haemost 2007; 98:274-277), showing a significant proliferation capacity and differentiation into mature endothelial cells (ECs). EPCs -augment vasculogenesis and differentiate into functional endothelial cells and hence are considered promising therapeutic agents to be transplanted.

EPCs are a heterogeneous group of cells that can be characterized by the expression of surface markers, such as CD34, CD133, and VEGFR-2 (KDR or FIk-I), by the uptake of DiI-Ac-LDL and by binding lectins such as Ulex europaeus, which are thought to be endothelial specific. Expression of the stem cell marker CD34 is also found, on a lower level, on mature endothelial cells, and the search for more specific stem cell markers led to the discovery of CD133 (also termed AC133), which is highly expressed on immature stem cells but whose expression is lost during the differentiation to mature endothelial cells. CD347CD133+/VEGFR-2+ EPC are precursors of CD34+/CD133+/VEGFR-2+ EPC with a higher potential for vascular repair.

The most commonly used method to obtain EPCs is from cultures of unprocessed mononuclear cells (MNCs) by adherence of the cells to fibronectin coated culture plate.

It has been shown that following tissue ischemia or endothelial damage, EPCs, originating in the bone marrow enter the blood circulation and incorporate into the foci of the injured endothelium, thereby inhibiting thrombus formation and improving blood flow and tissue recovery.

Since interventional procedures such as stenting causes damage to the endothelial lining of coronary arteries, EPCs capture technology enhances this process and facilitates the natural healing process of damage arterial segments. This accelerated healing lessens the chance of sub acute thrombosis and reduces the restenotic response. The prospect of vascular endothelium healing using EPCs injected directly to the site of damaged arterial segments or, applied onto a stent device implanted in the site, in the prevention of thrombosis and restenosis, has therefore been studied. The direct injection of EPCs, to sites of ischemia have been reported, for example, in U.S. Patent Applications having Publication Nos. 2009/142306 and 2009/0136460, and in Schachinger et al., [2006 N. Engl. J. Med., 355:1210-1221] and Bartunek et al., [2007 Nat. Clin. Pract. Cardiovasc. Med., 4:S100-S105]. However, the number of EPCs retained in the site of injury upon injection is low, limiting the effectiveness of therapy.

U.S. Patent Application having Publication No. 2009/142296 teaches a method of increasing trafficking of endothelial progenitor cells to ischemia-damaged tissue in a subject, which is effected by the direct injection of interleukin-8, a cytokine that attracts endothelial progenitor cells to the ischemia-damaged tissue.

Shirotta et al. [Biomaterials 2003; 24:2295-2302] addressed fabrication procedures of cell delivery-stent devices of endothelial progenitor cells (EPCs) seeded using either the surface of a stent strut or the surface of a covered stent to promote rapid re-endothelialization .

Genous stent (OrbusNeich, Fort Lauderdale, FL) is a commercially available endothelial progenitor cell (EPC)-capture stent. The stent is coated with murine monoclonal antihuman CD34 antibodies designed to attract circulating EPCs. It has been shown that in the first few hours of acute myocardial infarction (AMI), the implantation of the CD34-antibodies containing stent leads to an increase in the number of circulating EPCs in the site of the injured endothelium and a functional endothelial layer is established upon the damage arterial segments. , [Aoki et al. JACC 2005; 45:1575-1579, Co et al. Am Heart J 2008; 155:128-132]. The Genous stent and related aspects are disclosed in WO 03/065881.

U.S. Patent Application having Publication No. 2007/123977 teaches a medical device, such as a stent and a synthetic graft, identified for use in balloon angioplasty procedures for preventing or inhibiting restenosis.

U.S. Patent Application having Publication No. 2008/147176 teaches a bioabsorbable stent including a chemoattractant for EPCs, for the treatment, prevention, mitigation or reduction of vascular medical conditions.

U.S. Patent Application having Publication No. 2007/264306 teaches the use of homing factors in the generation of scaffolds of implantable devices, wherein the homing factors are capable of binding stem cells and EPCs. U.S. Patent applications having Publication Nos. 2007/166350 and 2007/160644 teach compositions and methods for promoting attachment of cells of endothelial cell lineage to medical devices. Peptides capable of binding endothelial cells and uses thereof in the treatment of angiogenesis-related pathologies have been described in WO 2005/039616 and WO 2008/047370. These peptides were reported to bind endothelial cells via glucose regulated protein 78 (GRP78) found on the surface of the cells. Glucose regulated protein 78 (GRP78) belongs to the heat shock protein 70 group (HSP70) and has been widely used as a marker for Endoplasmic Reticulum

' stress. GRP78 can enhance cell survival through either repair or degradation of misfolded proteins under the conditions of stress. It has been recently demonstrated that intracellular GRP78 induction occurs in cardiomyocytes under hypoxic conditions [Reddy et al. 2003 J Biol Chem. 278(23):20915-20924; Fu et al. 2008 Cardiovasc /tes.79(4):547-548; and Thuerauf et al. 2006 Circ Res. 99(3):275-282]. Studies have also shown that cardiac GRP78 is highly responsive to hypoxia and ischemia [Shintani- Ishida et al. 2006 Biochem Biophys Res Commun. 345(4): 1600-1605; Hayashi et al. 2003 J. Cereb. Blood Flow Metab. 23:949-961] and that induction of GRP78 expression by pharmacological agents might protect cardiomyocytes from hypoxic/ischemic injury and oxidative stress, suggesting a cytoprotective characteristic of GRP78 in the heart [Martindale et al. 2006 Circulation Research 98:1186-1193; Pan et al. 2004 Journal of Cardiovascular Pharmacology 44 (Suppl 1): S117-S120; Zhang et al. 2004 Annals of Clinical and Laboratory Science; 34 (4):449-457]. Membrane GRP78 was found to protect tumor cells from hypoxia-induced apoptosis by blocking procaspase-7 activation [Reddy et al. 2003 J Biol Chem. 278(23):20915-20924]. It has been also demonstrated that membrane GRP78 is implicated in angiogenic processes [Bruneel et al. 2005 fVøteom/c.s.;5(15):3876-3884; Hardy et al. 2008 Biochem Pharmacol 75(4):891-899; McFarland et al. 2009 Cancer Res. 69(13):5537-5545].

SUMMARY OF THE INVENTION

The present inventors have surprisingly uncovered that small peptide molecules, which were previously shown to promote angiogenesis, are characterized by the capability of promoting endothelial progenitor cells (EPCs) propagation and recruitment from cord and adult human peripheral blood and thus, upon attachment to implantable medical devices, can serve as potent scavengers for the recruitment and attachment of EPCs onto the device. The present invention therefore relates, in some embodiments thereof, to a medical device, having deposited on its surface such peptides, and to its use in the treatment and prevention of coronary and other cardiovascular artery diseases.

The present inventors have further surprisingly uncovered that the same small peptide molecules exhibit anti-apoptosis activity. These peptides were shown to inhibit hypoxia-induced apoptosis in cardiomyocytes, and can thus serve as cardioprotecting agents and as protecting agents in other ischemic conditions.

The present invention therefore relates, in some embodiments thereof, to methods of treating cardiovascular diseases by local administration of a peptide- containing solution to a vascular organ to be treated.

Accordingly, broadly stated, embodiments of the present invention relate to the use of small peptide molecules in treatment of cardiovascular diseases which involves local administration of the peptides to a blood vessel. The local administration can be via implantation of a medical device which has a peptide deposited on its surface, and/or via a medical device that delivers the peptide to a vascular bodily site.

According to an aspect of some embodiments of the present invention there is provided an article of manufacture comprising an implantable medical device and at least one peptide deposited on at least a portion of a surface of the medical device, the peptide being capable of chemoattracting endothelial progenitor cells and is being selected from the group consisting of:

(a) a peptide capable of binding Glucose-Regulated Protein, 78-kD (GRP78; SEQ ID NO:9)

(b) a peptide which comprises an amino acid sequence selected from the group consisting of HWRRAHLLPRLP (ADOPEPl; SEQ ID NO:1), HWRRP (MOTIFA; SEO ID NO:2), and YPHIDSLGHWRR (RoY; SEQ ID NO:3);

(c) a peptide which comprises an amino acid sequence selected from the group consisting of amino acid sequences as set forth by SEQ ID NOs :4 (VPWMEPAYQRFL), 5 (LLADTTHHRPWT), 6 (QPWLE0AYYSTF), 7 (SAHGTSTGVPWP) , 3 (YPHIDSLGHWRR) and 9 (TLPWLEESYWRP); (d) a peptide which comprises an amino acid sequence selected from the group consisting of amino acid sequences as set forth by SEQ ID NOs: 10, 11 and 12, the peptide being at least 6 and no more than 50 amino acid residues in length; and (e) a peptide which comprises an amino acid sequence HWRR as set forth by SEQ ID NO: 13.

According to some embodiments of the invention, the peptide which comprises an amino acid sequence HWRR as set forth by SEQ ID NO: 13 consists of 4 or 5 amino acids.

According to some embodiments of the invention, the peptide has an amino acid sequence selected from the group consisting of the amino acid sequences as set forth by SEQ ID NOs: 4, 6, 7 and 9.

According to some embodiments of the invention, the peptide has an amino acid sequence selected from the group consisting of the amino acid sequences as set forth by SEQ ID NOs: 4, 6 and 9.

According to some embodiments of the invention, the amino acid sequence is HWRRP (SEQ ID NO:2) or HWRRA (SEQ ID NO: 15).

According to some embodiments of the invention, the peptide has an amino acid sequence as set forth by SEQ ID NO: 1.

According to some embodiments of the invention, the peptide has an amino acid sequence as set forth by SEQ ID NO:3.

According to some embodiments of the invention, the peptide has an amino acid sequence selected from the group consisting of the amino acid sequences as set forth by SEQ ID NOs:l, 16 and 17.

According to some embodiments of the invention, the peptide is a linear peptide.

According to some embodiments of the invention, the peptide consists of no more than 12 amino acids.

According to some embodiments of the invention, the peptide is capable of selectively chemoattracting endothelial progenitor cells, such that the amount of endothelial progenitor cells that migrate towards the peptide is at least twice the amount of non-progenitor endothelial cells.

According to some embodiments of the invention, the implantable medical device is a blood-contacting medical device. According to some embodiments of the invention, the medical device is selected from the group consisting of a stent, a stent graft, a vascular graft, a synthetic vascular graft, a heart valve, a vascular prosthetic filter, a pacemaker, a pacemaker lead, a defibrillator, a patent foramen ovule septal closure device, a vascular clip, a vascular aneurysm occluder, a hemodialysis graft, an atrioventricular shunt, an aortic aneurysm graft device or components, a venous valve, a sensor, a suture, a vascular anastomosis clip, an indwelling venous catheter, an indwelling arterial catheter, a vascular occluder and a vascular sheath.

According to some embodiments of the invention, the medical device is a stent.

According to some embodiments of the invention, the medical device further comprises a therapeutically active agent for treating a vascular disease, the therapeutically active agent being deposited onto at least a portion of the surface of the medical device.

According to some embodiments of the invention, the therapeutically active agent is an anti-proliferative agent.

According to some embodiments of the invention, the article of manufacturing is further comprising an agent that stimulates a formation of an endothelium on the surface of the medical device by the endothelial progenitor cells, the agent being deposited on at least a portion of the surface of the medical device.

According to some embodiments of the invention, the medical device is a transiently insertable medical device.

According to some embodiments of the invention, the transiently insertable medical device is a balloon catheter.

According to some embodiments of the invention, the medical device further comprises a biocompatible matrix deposited on the surface.

According to some embodiments of the invention, the peptide is linked to the biocompatible matrix. According to some embodiments of the invention, the peptide is deposited directly on the surface of the medical device directly.

According to some embodiments of the invention, the peptide is linked to the surface via a spacer.

According to some embodiments of the invention, the article of manufacture is being identified for use in a method of treating a cardiovascular disease.

According to some embodiments of the invention, the article of manufacture of is being packaged in a packaging material and identified in print, in or on the packaging material, for use in a method of treating a cardiovascular disease in a subject in need thereof.

According to some embodiments of the invention, the method comprises implanting the medical device in a bodily site afflicted by the cardiovascular disease in the subject.

According to some embodiments of the invention, the method further comprises locally administering to the bodily site a solution of the peptide, the solution of the peptide being for reducing or preventing apoptosis in the bodily site and/or for inducing angiogenesis in the bodily site. According to some embodiments of the invention, the bodily site is a cardiac lumen.

According to some embodiments of the invention, the bodily site is selected from the group consisting of a cardiac lumen, a renal artery, a renal vein, a coronary artery, a coronary vein, a cerebral artery, a cerebral vein, a cervical artery, a cervical vein , a stomach artery, a stomach vein, a hepatic artery, a portal vein, a mesenteric artery, a mesenteric vein, an arm artery, an arm vein, a hand artery, a hand vein, a leg artery, a leg vein, a foot artery, and a foot vein.

According to some embodiments of the invention, implanting the medical device and locally administering the solution of the peptide are effected via the same technique. According to some embodiments of the invention, implanting the medical device and co-administering the solution of the peptide are both effected via catheterization.

According to another aspect of embodiments of the invention there is provided a method of treating a vascular disease in a subject in need thereof, the method comprising: (i) contacting a bodily site afflicted by the cardiovascular disease with an implantable medical device configured for treating the vascular disease; and

(ii) contacting the bodily site with a peptide as described in any of claims 1-11, by means of the implantable medical device.

According to some embodiments of the invention, the implantable medical device has the peptide deposited on at least a portion of a surface thereof.

According to some embodiments of the invention, contacting the bodily site with the peptide is effected by implanting the implantable medical device. According to some embodiments of the invention, the implantable medical device is a transient insertable medical device, and contacting the bodily site with the peptide comprises delivering the peptide to the bodily site via the transient insertable device. According to some embodiments of the invention, the bodily site is a cardiac lumen.

According to another aspect of embodiments of the invention there is provided a peptide as described herein, identified for use in a method of treating a cardiovascular disease in a subject undergoing an implantation of an implantable medical device configured for treating the vascular disease.

According to another aspect of embodiments of the invention there is provided a method of preparing an enriched, isolated population of endothelial progenitor cells, the method comprising: implanting an article comprising an implantable medical device as described herein in a body, to thereby chemoattract the cells to a surface of the device; retrieving the medical device from the body; and isolating the endothelial progenitor cells from the medical device, thereby obtaining an enriched, isolated population of endothelial progenitor cells.

According to another aspect of embodiments of the invention there is provided a process for preparing an article of manufacturing which comprises an implantable medical device and at least one peptide capable of chemoattracting endothelial progenitor cells deposited on at least a portion of the surface of the medical device, the process comprising contacting the device with at least one peptide as described herein, thereby obtaining the article of manufacture.

Unless otherwise defined, all technical and/or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the invention, exemplary methods and/or materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

Some embodiments of the invention are herein described, by way of example only, with reference to the accompanying drawings. With specific reference now to the drawings in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of embodiments of the invention. In this regard, the description taken with the drawings makes apparent to those skilled in the art how embodiments of the invention may be practiced. In the drawings:

FIGs. IA-B are bar graphs depicting the binding of peptide-presenting phages at a concentration of 10 (Figure IA) or 10¹ (Figure IB) phage per well, to ECs under normoxic conditions and following 3, 6, and 24 hours of hypoxia. The bars represent the binding to ECs of 15 different peptide-presenting phage (VL, LP, TR, ST, QF, NS, SP, YR, LT, HR, HY, SV, TP, NR, and SA) and the control (NO, unmodified M13 phage) following a 2-hour incubation. Absorbance at 450 nm, produced by anti-M13 HRP antibody, which detects peptide-presenting phages attached to ECs in the presence of tetramethyl benzidine liquid substrate, was measured using an ELISA reader. FIG. 2 is a graph depicting the inhibition of binding of ADOPepl (2 μg/ml) to endothelial cells by specific peptides corresponding to conserved motifs from the ADOPeps. Endothelial cells were incubated for 2 hours under normoxia conditions in the presence of increasing concentrations (0.01, 0.1 or 1 ng/ml) of peptides having the amino acid sequences corresponding to Motif A (HWRRP; SEQ ID NO:2), Motif B (HWRRA; SEQ ID NO:15), Motif C (AHLLP; SEQ ID NO:18) or non-biotinylated ADOPepl (SEQ ID NO:1) and then were incubated for 1 hour with biotinylated ADOPepl (2 μg/ml) and the binding of biotinylated ADOPepl to endothelial cells was determined using HRP-streptavidin. Note that the peptides having amino acid sequence corresponding to Motif A and B exhibited a significant inhibition of the binding of ADOPepl^Blot to endothelial cells.

FIGs. 3A-D are FACS analyses depicting hypoxia induced apoptosis. Endothelial cells were incubated for 24 hours under hypoxia conditions in the absence (Figure 3A) or presence of 10 ng/ml of ADOPepel (Figure 3B), peptide of Motif A (Figure 3C) or peptide of motif C (Figure 3D) and the level of apoptosis was determined using FACS analysis and the PI (shown on the Y axis) /Annexin V (shown on the X axis) markers. Note that while ADOPepl and Motif A peptides were capable of inhibiting the hypoxia induced apoptosis from 79.3% under hypoxia to 28.6% (ADOPepl) or 35.8% (Motif A), the motif C peptide exhibited no effect of hypoxia induced apoptosis (81.1%).

FIG. 4 is a bar graph depicting the inhibition of hypoxia induced apoptosis by ADOPep 1, Motif A and Motif B. Endothelial cells were incubated for 24 hours under hypoxia conditions in the absence or presence of 10 ng/ml of ADOPepel, Motif A, Motif B or motif C and the level of apoptosis was determined using FACS analysis and quantified as the percent of Annexin V and PI positive cells. The results represent average ± standard deviation of 4 independent experiments. Note that while ADOPepl and Motif A and B peptides exhibited a significant inhibition of hypoxia induced apoptosis, the Motif C peptide exhibited no effect on apoptosis.

FIGs. 5A-B are graphs depicting induction of endothelial cell migration under hypoxia by the ADOPEP motifs. Endothelial cells were incubated for 5 hours under hypoxia conditions in the presence of increasing concentrations of ADOPepl (red line), peptide motif A (dark blue line), peptide motif B (green line) or peptide motif C (light blue line) and the migration of endothelial cells was detected. Figure 5A- experiment 1; Figure 5B - experiment 2. Note that while the ADOPepl, Motif A and B peptides induced endothelial cell migration at a concentration of about 10 ng/ml, motif C peptide exhibited no significant effect on endothelial cell migration.

FIGs. 6(A-B) are images of Coomassie blue staining of polyacrylamide gel elecrophoresis (PAGE) of endothelial cell lysates obtained from cells under hypoxia conditions and analyzed before (Figure 7A) and after (Figure 7A) immunoprecipitation (IP) of the cells with biotinylated ADOPepl, an exemplary peptide according to embodiments of the present invention. Figure 6A - lane 1 - endothelial cell lysate before IP; lane 2 - molecular weight markers; Figure 6A - lane 3 - IP endothelial cell lysate with ADOPepl. Note the major single protein band of 78 kDa following IP with biotinylated ADOPepl.

FIG. 7 is a Western Blot analysis of immune precipitation of endothelial cells lysate with biotinylated ADOPepl, an exemplary peptide according to embodiments of the present invention, under normoxia and hypoxia conditions. Staining of the nitrocellulose membrane with biotinylated ADOPepl followed by Chemiluminescent Substrate revealed a band at 78 kDa which was further identified by Mass spectrometry as GRP78 protein (GenBank Accession No. CAB71335; SEQ ID NO:9). Lane 1 - IP of endothelial cells lysate under normoxia with biotinylated ADOPepl; lane 2 - IP of endothelial cells lysate under hypoxia with biotinylated ADOPepl.

FIG. 8 is Western Blot analysis of immune precipitation of endothelial cells lysate under hypoxia and normoxia with biotinylated ADOPepl an exemplary peptide according to embodiments of the present invention. Staining of the nitrocellulose membrane with anti GRP78 antibody (Santa Cruz Biotechnologies, CA, USA) followed by Chemiluminescent Substrate confirmed the identity of the GRP78 protein in the major 78 kDa protein band. Lane 1 - IP of endothelial cells lysate under hypoxia with biotinylated ADOPepl; lane 2 - IP of endothelial cells lysate under normoxia with biotinylated ADOPepl.

FIG. 9 is a graph depicting inhibition of binding (in percentages) of anti-GRP78 antibody to endothelial cells by increasing concentrations of ADOPepl, 2, and 3, exemplary peptides according to embodiments of the present invention. Endothelial cells were incubated for 2 hours with increasing concentrations of ADOPeps, following which the cells were incubated for another 1 hour with the 2 μg/ml of anti-GRP78 antibody and the amount of bound antibody on EC was detected using an ELISA reader. Note that while increasing concentrations of ADOPepl, 2 or 3 resulted in up to 50% inhibition of binding of the anti-GRP78 antibody to endothelial cells, the sRoY scrambled peptide (SEQ ID No. 8) exhibited no specific inhibition effect on the binding of anti-GRP78 to endothelial cells.

FIGs. 10 (A-D) are FACS histograms (Figures lOA-C) and a graph (Figure 10D) demonstrating that AdoPepl and Motif A, exemplary peptides according to embodiments of the present invention, compete on the binding to the same receptor on endothelial cells. Endothelial cells were cultured for 24 hours under hypoxic conditions in endothelial cell growth medium. Cells were removed by trypsin and incubated for 1 hour on ice with increasing concentrations of AdoPepl, Motif A and Motif C peptides. GRP78 polyclonal antibody (2 μg/100,000) was added to the cells for 2 hours on ice. Anti-goat FITC (Jackson ImmunoResearch) was added for 30 minutes on ice. IgGl- FITC was used as the isotype control. The samples were analyzed with a FACScan (Beckton Dickinson). Note that AdoPepl peptide (Figure 10A) and Motif A peptide (Figure 10B) but not Motif C peptide (Figure 10C) inhibit the binding of GRP78 to endothelial cells. Figure 1OD depicts the average percent binding of GRP78 to endothelial cells as a function of peptide amount as determined by 2 independent experiments.

FIG. 11 presents bar graphs showing the percentage (on a scale of 0-50%) of CD34, CD31 and CD133/31 positive cells in a population of cord blood mononuclear cells incubated with exemplary peptides according to embodiments of the present invention: RoY (red), MotifA (light blue), as compared to control cells (not incubated with any peptide; dark blue). The percents of positive cells were detected by staining the cells with the fluorescently labeled anti-CD34-phycoerythrin, anti-CD133- phycoerythrin and anti-CD31-FITC and subjecting the cells to FACS analysis. Quantitative fluorescence analysis was performed with a FACS-Calibur instrument and Cellquest Software (Becton-Dickinson). FIGs. 12(A-B) present bar graphs showing the percentage (on a 0-10% scale) of

CD34/KDR (figure 12A) and CD 133 (figure 12B) positive cells in a population of cord blood mononuclear cells incubated for a week, with 0, 10, 50 and 100 ng/ml of peptide ADoPep, an exemplary peptide according to embodiments of the present invention. The percents of positive cells was detected by staining the cells with the fluorescently labeled anti-CD34-phycoerythrin, anti-CD133-phycoerythrin and anti-KDR-FITC and subjecting the cells to FACS analysis. Quantitative fluorescence analysis was performed with a FACS-Calibur instrument and Cellquest Software (Becton- Dickinson).

FIG. 13 presents bar graphs depicting the ability of Cord Blood Mononuclear cells to migrate towards the peptides AdoPepl, RoY, and MotifA, exemplary peptides according to embodiments of the present invention, as compared to the migration of cells not exposed to any peptide (marked control). The percentage (on a 0.0-1.8% scale) of EPCs in the migrating cell population was determined by the percentage of CD34 positive cells as detected using anti-CD34-phycoerythrin, using FACS analysis. The percentage of CD34 positive cells in cord blood mononuclear cells before migration is also shown. FIGs. 14(A-B) present bar graphs depicting the ability of peripheral blood mononuclear cells to migrate towards the peptides AdoPepl, RoY, and MotifA, exemplary peptides according to embodiments of the present invention, as compared to the migration of cells not exposed to any peptide (marked control). The percentage of endothelial cells, in the migrating cell population, was determined by the percentage of CD34 positive cells as detected using anti-CD34-phycoerythrin and subjecting the cells to FACS analysis (Figure 14A, 0.0-1.2% scale). The percentage of CD34 positive cells in peripheral blood mononuclear cells before migration is also shown. Due to the selective expression of the antigen CD 133 by immature endothelial cells (i.e. EPCs), the percentage of immature endothelial cells was determined by the percentage of CD133/CD31 positive cells, as detected using anti-CD133-phycoerythrin and anti- CD31-FITC, and subjecting the cells to FACS analysis (Figure 14B, 0-16% scale). The percentage of CD133/CD31 positive cells in peripheral blood mononuclear cells before migration is also shown.

FIG. 15 presents bar graphs showing the ability of AdoPepl and MotifA, exemplary peptides according to embodiments of the present invention, to bind EPCs when attached to a nitrocellulose membrane sheet. Peptides AdoPep 1 or Motif A at 2 micrograms per ml were incubated overnight with a piece of 0.5 cm² nitrocellulose membrane sheet followed by incubation with cord blood mononuclear cells for 24 hours. The number of membrane bound cells (purple) and unbound cells (blue) was quantified and compared using the XTT reagent test wherein the number of cells corresponded to level of reading using an ELISA reader at a wavelength of 450nm. Cells incubated with nitrocellulose membrane sheet which did not contain any peptide, served as control (two separate control groups were used: one control group was used in the binding experiments of the EPCs to AdoPepl (control 1) and one group in the binding experiments of the EPCs to Motif A).

FIGs.16(A-C) present FACS analyses (FIGs. 16A and 16B) and a FACS histogram (FIG. 16C) depicting membranal GRP78 expression in human smooth muscle cells. The level of membranal GRP78 expression was determined using FACS analysis and staining with polyclonal rabbit anti GRP78 followed by staining with anti rabbit FITC (FIG. 16A). Anti-rabbit-FITC was used as isotype control (FIG. 16B and black line in FIG. 16C). A FACS histogram shown in FIG. 16C, demonstrates that 30 % of the smooth muscle cell population expressed membranal GRP78 (red line). The samples were analyzed with a FACScan (Beckton Dickinson).

FIG. 17 presents FACS histograms showing membranal GRP78 expression in human fibroblasts under normal conditions (green line); and upon incubation under hypoxic conditions (red line), as determined using FACS analysis and staining with polyclonal rabbit anti GRP78 followed by staining with anti rabbit FITC. Also shown is the fluorescence detected using Anti-rabbit-FITC as the isotype control (black line). The histograms indicate lack of membranal GRP78 expression in human fibroblasts both at normal and under hypoxic conditions. The samples were analyzed with a FACScan (Beckton Dickinson). FIG. 18 presents bar graphs showing the number of smooth muscle cells that migrate towards the peptide AdoPepl, an exemplary peptide according to embodiments of the present invention (pink bar, marked AdoPepl), compared with the migration of cells not exposed to any peptide (gray bar, marked none), as determined by an ELISA reader, at a wave length of 450 nm, using the XTT assay, and demonstrating the inability of smooth muscle cells to migrate towards the peptide AdoPepl.

FIGs. 19(A-C) present data indicating the level of GRP78 expression in immature stem cells (EPCs). Human peripheral blood mononuclear cells (PBMC) were obtained from four healthy donors (PMBCl, PMBC2, PMBC3 and PMBC4) and double labeled with anti-CD133 (marker for EPCs) and anti-GRP78-FTTC fluorescent antibodies. FIG. 19A presents a bar graphpresenting the fraction of cells in each of the four PMBC groups that express CD133 /GRP78⁺ (purple bars; i.e. mature stem cells) and CD133⁺/GRP78⁺ (blue bars; i.e. immature stem cells). The CD133 positive cells were then isolated from each of the four PMBC using anti-CD133 magnetic beads in 90 % purity. FIG. 19B presents a bar graph demonstrating the fraction of CD1337GRP78⁺ (purple bars) and CD133⁺/GRP78⁺ (blue bars) expression in the CD133⁺ isolated population. FIG. 19C presents representative FACS analysis dot plots for the anti- GRP78 (bottom axis)/anti-CD133 (left axis) staining of PMBCl and PMBC2 groups. FIGs. 20(A-C) present data demonstrating CD133⁺ cell migration toward AdoPepl, an exemplary peptide according to embodiments of the present invention. Human peripheral blood mononuclear cells (PBMC) from two healthy donors were separated to CD133 positive and CD133 negative cell subpopulations using anti CD133 magnetic beads. FIGs. 2OA and 2OB presents bar graphs showing the percent of GRP78⁺ cells that migrated toward AdoPepl (FIG. 20A), and the fraction of CD133⁺ cells as compared to CD133^" cells that migrated toward AdopPepl peptide (FIG. 20B), in CD133 positive and CD133 negative cell subpopulations (blue bars) as compared to control cells that migrated toward a medium without any peptide (purple bars; marked as "without chemoatt"). FIG. 2OC presents representative FACS analysis dot plots for the anti-GRP78-FITC (bottom axis)/anti-CD133 (left axis) staining of the CD133 positive and CD133 negative cell subpopulations that migrated toward the AdoPepl peptide or toward the control (marked "without chemoatt").

FIGs. 21(A-B) present FACS analysis of the membranal GRP78 expression level in cardiomyocytes exposed to hypoxia and incubated with AdoPepl, an exemplary peptide according to embodiments of the present invention. Cultures of cardiomyocytes were incubated under normoxic conditions (marked normoxia) or under hypoxic conditions (marked hypoxia). Three groups of cells exposed to hypoxic conditions were further incubated with the 20 ng/ml of ADoPepl, either 14 hours before hypoxia (marked peptide pre-hypoxia), during hypoxia (marked peptide during hypoxia) or 14 hours post hypoxia (marked peptide post hypoxia). FIG. 21A presents a bar graph showing the percent of membrane GRP78 positive cells in each of the experimental groups (5 experiments). FIG. 21B presents FACS analysis of a representative experiment, showing the cardiomyocytes membrane GRP78 staining from each of the cell groups.

FIGs. 22(A-B) present a bar graph (FIG. 22A) and an image of a Western blot analysis (FIG. 22B) showing the total GRP78 expression in cardiomyocytes exposed to hypoxia and incubated with AdoPepl, an exemplary peptide according to embodiments of the present invention. Cultures of cardiomyocytes were incubated under normoxic conditions (marked normoxia) or under hypoxic conditions (marked hypoxia). Cardiomyocytes from each group were further incubated with 20 ng/ml ADoPepl,. Total GRP78 protein was determined using Western blotting and GRP78 polyclonal antibody (Santa Cruz, Biotechnologies, CA, USA). To verify equal protein loading, blots were also stained with mouse monoclonal anti-Actin (MP Biomedicals, Illkirch, France) and densitometry ratio (GRP78/Actin) was determined in 3 different experiments. FIG. 22A presents a bar graph showing the total GRP78 levels in cardiomyocytes from each experimental group. FIG. 22B presents a representative western blot membrane stained with anti-GRP78 and anti-Actin from a representative experiment.

FIGs. 23(A-B)_present a bar graph (FIG. 23A) and an image of a PCR analysis (FIG. 23B) demonstrating GRP78 gene expression in cardiomyocytes exposed to hypoxia. Cultures of cardiomyocytes were incubated under normoxic conditions (marked normoxia) or under hypoxic conditions ((marked hypoxia). Cardiomyocytes from each group were further incubated with 20 ng/ml ADoPepl. Semi quantative RT- PCR was performed using specific primers to target GRP78 gene. The amount of PCR product was quantitatively determined and normalized with GAPDH as reference genes in 3 different experiments. FIG. 23A presents a bar graph showing the GRP78 PCR product in each of the cardiomyocyte experimental groups. FIG. 23B presents a representative PCR analysis.

FIGs. 24(A-B) present a bar graph (FIG. 24A) and FACS analysis demonstrating the level of apoptosis in cardiomyocytes exposed to hypoxia. Cultures of cardiomyocytes were incubated under normoxic conditions (marked normoxia) or under hypoxic conditions (marked hypoxia). Three groups of cells exposed to hypoxic conditions were further incubated with the 20 ng/ml of ADoPepl, an exemplary peptide according to embodiments of the present invention, either 14 hours before hypoxia (marked peptide pre-hypoxia), during hypoxia (marked peptide during hypoxia) or 14 hours post hypoxia (marked peptide post hypoxia). The cells were double stained with Annexin V-FTTC and Propidium iodide (PI) and analyzed with the Annexin-V-positive and Pi-negative cells scored as apoptotic cells. FIG. 24A presents a bar graph showing the percent of Annexin V positive cells in each of the five experimental groups (five different experiments). FIG. 24B presents a representative FACS analysis of the double labeling of cardiomyocytes with PI and Annexin V.

FIGs. 25 (A-C) present data showing the effect of silencing the expression of GRP78 by siRNA on GRP78 expression in cardiomyocytes. RNA interference of GRP78 was induced with small interfering RNA (siRNA) directed against the mouse GRP78 mRNA. A scrambled siRNA was used as negative control. Cardiomyocytes were transfected with either 5, 20 or 50 nmol/liter positive or 50 nM scramble oligonucleotides. FIG. 25A presents a bar graph showing the total GRP78 levels in cardiomyocytes exposed to the different siRNA concentration and control scramble siRNA group (3 different experiments). FIG. 25B presents an image of a representative gel from one of the three experiments conducted. FIG. 25C presents a FACS analysis of the level of membrane GRP78 in cardiomyocytes transfected with siRNA or control scramble siRNA when incubated with or without AdoPepl, using anti-GRP78 antibody. FIGs. 26(A-C) present data demonstrating the effect of silencing the expression of GRP78 by siRNA on apoptosis levels in cells. Transfected cardiomyocytes with 5OnM GRP78 siRNA were exposed to 4 hours hypoxia without or with AdoPepl peptide (marked "hypoxia" and "with peptide" respectively). Cardiomyocytes transfected with scrambled siRNA and incubated with or without the AdoPepl peptide served as a negative control. Cells were double stained with Annexin-FITC and Propidium Iodide for assessment of the level of apoptosis. The activity Caspase 3/7 as a measure of apoptosis level was also determined. FIG. 26A presents bar graph showing the level of Annexin V positive cells in each of the experimental groups (average of three separate experiments). FIG. 26B presents representative FACS dot plots, of the apoptosis levels as assessed by the Annexin V (lower axis) / PI (left axis) staining. FIG. 26C presents FACS analysis showing the level of Caspase 3/7 activity in each of the experimental groups.

FIGs. 27(A-C) present data showing the extent of capillary formation in sites of myocardial infraction (MI) following administration of RoY, a peptide according to some embodiments of the present invention. Histological section were taken from mice 15-60 days after myocardial infraction from the sites of the heart infract and subjected to anti-CD31 staining for detection of capillary density in that region. FIG. 27A presents a bar plot comparing the number of capillaries at the site of the infract from mice that received 10 micrograms of RoY peptide one day after the MI (purple bars) as compared to control (blue bars), at different time points following the MI. FIG. 27B presents a graph of the percent difference in capillary density in mice that received the RoY treatment, as a function of time post the MI. FIG. 27C presents representative histological section taken from the infract zone of a mouse myocardium, 60 days after the MI, showing the level of CD31 staining in a mouse that received RoY treatment (right two pictures) as compared to a control mouse (left two pictures).

DESCRIPTION OF EMBODIMENTS OF THE INVENTION

The present invention, in some embodiments thereof, relates to the field of therapy and, more particularly, but not exclusively, to methods, compositions and implantable devices which utilize peptides that are capable of chemoattracting endothelial progenitor cells, and of exhibiting a cardioprotective effect, for treating cardiovascular diseases.

The principles and operation of the peptides, article of manufacture, devices, compositions, use and methods according to the invention may be better understood with reference to the drawings and accompanying descriptions.

Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not limited in its application to the details set forth in the following description or exemplified by the Examples. The invention is capable of other embodiments or of being practiced or carried out in various ways. Also, it is to be understood that the phraseology and terminology employed herein is for the purpose of description and should not be regarded as limiting. Endothelial cells (ECs) line the interior surface of blood vessels, forming an interface between circulating blood in the lumen and the rest of the vessel wall. Endothelial cells line the entire circulatory system (referred to herein as the cardiovascular system), from the heart to the smallest capillary. These cells reduce turbulence of the flow of blood, allowing the fluid to be pumped farther. Endothelial progenitor cells (EPCs) are bone marrow-derived cells that circulate in the blood and have the ability to differentiate into endothelial cells.

The most commonly used method to obtain EPCs is from cultures of unprocessed mononuclear cells (MNCs), obtained either from cord blood or adult peripheral blood, by adherence of the cells to fibronectin coated culture plate.

Cardiovascular diseases are the leading cause of death in developing countries, and the magnitude of an acute myocardial infarction (MI) or more specifically the number of dead cardiomyocytes, is a vital factor of subsequent heart function. Rational design of therapeutic interventions that protect the myocardium from cell death during ischemia has been a research priority for many years.

Myocardial ischemia is a condition in which oxygen deprivation to the heart muscle is accompanied by inadequate removal of metabolites because of reduced blood flow or perfusion. In contrast, mere oxygen deprivation without reduction in the clearance of metabolites is termed hypoxia or anoxia. Ischemia and hypoxia are relevant to pathologic cardiomyocyte (CM) cell death because this decrease in oxygen leads to a rapid decline in energy and ATP levels which are needed for the high energy requirements of these cells. Glycolysis is increased in these states leading to the formation of harmful byproducts such as lactate. Although prolonged myocardial ischemia has deleterious effects on mitochondrial function, evidence suggests that the generation of ROS during reperfusion (re-establishment of oxygen and blood flow after MI) is even more harmful through reaction of oxygen with reduced mitochondrial ubisemiquinone to produce high amounts of superoxides. Until quite recently, cardiomyocyte death from ischemia was attributed to necrosis, or "accidental cell death". However recently, collective evidence from experimental and clinical studies has demonstrated that cardiomyocyte cell death following ischemia and/or reperfusion involves apoptosis, oncosis, autophagy (and necrosis) in ischemic cardiac conditions. These conditions are thought to lead at later stages to necrosis.

Many cardiovascular diseases such as myocardial ischemia are treated via invasive techniques involving the implantation of medical devices, optionally via PCI. Such procedures include for example the implantation of a stent in order to treat coronary artery stenosis or the implantation of an artificial valve.

The present inventors envisioned that cardiovascular diseases may be treated more effectively if during these invasive procedures a therapeutically active agent having cardioprotective activity and EC proliferation propagation activity would be administered concomitantly to the site of medical device implantation or be deposited on the medical device itself. As discussed hereinabove, a number of peptide families, which have previously been isolated and/or synthesized, were found to exhibit angiogenic activity. These peptide families were each characterized by certain sequence motifs.

An exemplary such peptide family was uncovered using a 12-mer phage display peptide library (see, WO2005/039616, incorporated by reference as if set forth herein). The amino acid sequences of some exemplary peptides in this family are set forth in SEQ ID NOs: 3 (referred to herein as Roy), 4, 5, 6, 7 and 9. Another peptide family includes peptides which were found to include a certain short amino acid sequence: Histidine-Tryptophan-Arginine-Arginine (HWRR) motif (derived from the ADAM 15 protein) (see, WO 2008/047370, incorporated by reference as if fully set forth herein). Such exemplary peptides are set forth in SEO ID NOs: 1 (also referred to herein as AdoPepl), 2 (also referred to herein as MotifA), 3, 15, 16 and 17. Interestingly, the sequence of Roy (SEQ ID NOs: 3), an exemplary peptide discovered using the phage display peptide library technique, also includes the HWRR motif. While reducing the invention to practice, the present inventors have surprisingly uncovered that exemplary small peptides, belonging to, or sharing structural motifs as, the above-described peptide families, are capable of promoting endothelial progenitor cells (EPCs) propagation and recruitment from cord and adult human peripheral blood and thus, upon attachment to implantable medical devices, can serve as potent scavengers for the recruitment and attachment of EPCs onto the device.

As presented in the Examples section that follows, it has been demonstrated that such peptides are able to bind and/or chemoattract EPCs. Thus, it has been shown, for example, that the peptides RoY (SEQ ID NO: 3), AdoPepl (SEQ ID NO: 1) and MotifA (SEQ ID NO: 2), induce a substantial increase in the percentage of EPCs in a population of cord mononuclear cell population, after one week incubation therewith (See, Figures 11 and 12). Induction of migration of EPCs from cord blood was obtained by both RoY and AdoPepl peptides (see, Figure 13). In addition, ADoPepl also exhibited selective recruitment of EPCs from adult human peripheral blood, which led to an EPC enrichment of the cell population (see, Figures 14 and 20). It has also been demonstrated that the ability of Motif A and AdoPepl to bind EPCs, when attached to a solid matrix, was preserved (see, Figure 15).

These findings demonstrate that structurally and/or functionally related peptides, as described herein, when deposited onto an implantable medical device, can serve as recruiters and chemoattractants of EPCs. Thus, implantable medical devices having these peptides deposited onto their surface can be beneficially utilized in the treatment and prevention of coronary and other vascular artery diseases in which the recruitment and proliferation of EPCs is beneficial.

While further reducing the invention to practice, the present inventors have surprisingly uncovered that the peptides described herein also exhibit an anti-apoptotic effect in cardiomyocytes, indicating a role for these peptides as cardioprotective agents that can be utilized in the treatment of cardiovascular diseases where such cardioprotective effect, or an anti-apoptotic effect, is beneficial.

As presented in the Examples section that follows, it has been demonstrated that such peptides inhibit hypoxia-induced apoptosis in cardiomyocytes. Specifically, it has been shown that the percent of hypoxia-induced apoptosis was significantly inhibited when ADoPepl, a peptide according to some embodiments of the present invention, was added before or during the hypoxia insult (see, Figure 24). The reversal of the hypoxia dependent apoptosis by AdoPepl was found to be mediated through the binding of the peptide to GRP78. This could be deduced from the inability of the AdoPepl peptide to reverse the hypoxia-induced apoptosis in cardiomyocytes in which the GRP78 gene expression was silenced using siRNA techniques (see, Figure 26). The promotion of angjogenesis after myocardial infract by RoY, a peptide according to some embodiments of the present invention, was also shown (see, Figure 27).

These results suggest that the peptides described herein may be further utilized for the treatment of cardiovascular disease conditions characterized by cardiomyocyte apoptosis. In such disease conditions, the local administration of a peptide as described herein to the site of disease may be therapeutically beneficial.

The term "apoptosis" describes a form of programmed cell death and involves a series of biochemical events that lead to a characteristic cell morphology, such as cytoplasmic condensation, blebbing of the plasma membrane, condensed chromatin, and DNA fragmentation. Apoptosis is an active process requiring metabolic activity and protein synthesis by the dying cell. Apoptosis stands in contrast to "necrosis", which is a form of traumatic cell death that results from acute cellular injury.

The peptides described herein, by exhibiting an anti-apoptotic effect, may act as cardioprotective agents when contacted the site of injury, by arresting the sequence of events that lead to apoptosis, and thus by limiting the damage caused to the site of injury.

Thus, taken together the surprising findings described herein, the peptides described herein exhibit the following therapeutic effects: (1) recruitment of EPCs thereto; (2) an anti-apoptotic effect; and (3) a pro-angiogenesis effect.

These activities are therefore utilized, according to various embodiments of the invention, in procedures for locally treating cardiovascular diseases. The EPCs recruitment effect is utilized, for example, for endothelization of an implantable device onto which the peptide is deposited, or for endothelization of an impaired blood vessel upon direct application of the peptide to the blood vessel. The anti-apoptotic effect is utilized, for example, for treating impaired tissues downstream or upstream the cardiovascular system by locally administering the peptide to a respective a blood vessel. The pro-angiogenesis effect can be utilized, for example, for locally promoting angiogenesis at bodily sites in need thereof. The methods and articles of manufacturing described in the following description utilize the above features of the peptides, either alone or in combination, as is detailed hereinbelow.

In some embodiments, the methods and articles of manufacturing presented herein, in which a peptide as described herein is contacted with an afflicted bodily site such as a cardiac lumen, both while being deposited on an implantable device and while being concomitantly administered in a solution to the bodily site during implantation of the medical device, results in improved myocardiac function, stemming from the combined effected exhibited by the peptide(s) and the device.

Thus, the articles of manufacture, uses and methods described herein may be utilized for the treatment of cardiovascular diseases in which EPCs recruitment and propergation and/or inhibition of apoptosis and/or promotion of angiogenesis is therapeutically beneficial.

Accordingly, according to an aspect of some embodiments of the present invention there is provided an article of manufacture comprising an implantable medical device and at least one peptide capable of chemoattracting endothelial progenitor cells, the peptide being deposited on at least a portion of a surface of the medical device, wherein the peptide comprises an amino acid sequence selected from the group consisting of HWRRAHLLPRLP (AdoPepl; SEQ ID NO 1), HWRRP (Motif A; SEQ ID NO 2), and YPHIDSLGHWRR (RoY; SEQ ID NO 3).

The term "chemoattracting" and its grammatical divergences, as used herein, describes a functionality of a substance, herein a peptide, to attract another substance, herein EPCs, namely, cause migration of the cells towards the peptide, by means of chemical interactions. The migration of the cells towards the peptide is typically effected when present in a gradient (i.e. during chemoattraction the cells move in the direction from a low to a high concentration of the chemoattractant).

The chemical interactions can include, for example, covalent interactions, electrostatic interactions, hydrophobic interactions, aromatic interactions, Van der Waals interactions, and more.

Hence, the term "chemoattracting" also encompasses binding the cells to the peptide.

As discussed hereinabove, some of the peptides utilized in embodiments of the ' invention were uncovered using a 12-mer phage display peptide library. The amino acid sequences of these exemplary peptides are set forth in SEQ ID NOs: 3 (RoY), 4, 5, 6, 7 and 9.

Thus, according to embodiments of the invention there is provided an article of manufacture comprising an implantable medical device and at least one peptide capable of chemoattracting endothelial progenitor cells deposited on at least a portion of the surface of the medical device, wherein the peptide comprises an amino acid sequence selected from the group consisting of amino acid sequences set forth by SEQ ID NOs: 4 (VPWMEPAYQRFL), 5 (LLADTTHHRPWT), 6 (QPWLEQAYYSTF), 7 (SAHGTSTGVPWP), 3 (YPHIDSLGHWRR) and 9 (TLPWLEESYWRP). Some of the peptides utilized in embodiments of this aspect of the invention share a conserved amino acid sequence (SEQ ID NO: 10, 11 or 12) with mammalian vascular endothelial growth factor B (VEGF-B, SwissProt/TrEMBL Accession: VEGB-MOUSE).

Thus, according to embodiments of the invention there is provided an article of manufacture comprising an implantable medical device and at least one peptide capable of chemoattracting endothelial progenitor cells, the peptide being deposited on at least a portion of the surface of the medical device, wherein the peptide comprises an amino acid sequence selected from the group consisting of amino acid sequences as set forth by SEQ ID NO: 10, 11 or 12, the peptide being at least 6 and no more than 50 amino acid residues in length.

Thus, peptides utilized in these embodiments have a length of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48 or 49 amino acid residues.

In some embodiments, the peptide utilized in these embodiments has an amino acid sequence as set forth by SEQ ID NO: 4, 6, 7 or 9.

In some embodiments, the peptide utilized in these embodiments has an amino acid sequence as set forth by SEQ ID NO: 4, 6 or 9.

In some embodiments, the peptides utilized in embodiments of the invention belong to a peptide family, which includes a certain short amino acid sequence: Histidine-Tryptophan-Arginine-Arginine (HWRR, SEQ ID NO: 13) motif (derived from the ADAM15 protein). Exemplary peptides in this family have an amino acid sequence as set forth in SEQ ID NOs: 1 (herein also refereed to as AdoPepl), 2 (herein also referred to as MotifA), 3, 15, 16 and 17.

Thus, according to embodiments of the invention there is provided an article of manufacture comprising an implantable medical device and at least one peptide capable of chemoattracting endothelial progenitor cells, the peptide being deposited on at least a portion of the surface of the medical device, wherein the peptide comprises an amino acid sequence HWRR as set forth by SEQ ID NO: 13.

According to some embodiments, the peptides utilized in this context of the invention consist of no more than 12 amino acids.

According to some embodiments, peptides comprising the amino acid sequence HWRR (motif) and consisting of 4 or 5 amino acids are utilized. Thus, a peptide which consists of 4 amino acids comprises only the conserved Motif as set forth in SEQ ID No. 13. A peptide which consists of 5 amino acids can be, for example, Motif A having the amino acid sequence HWRRP (SEQ ID NO:2) or Motif B having the amino acid sequence HWRRA (SEQ ID NO: 15). As should be recognized by any person skilled in the art, small peptides are highly advantageous for therapeutic application, due to their relative stability, solubility, increased bio-availability and lack of immune response thereto in the host cell. Furthermore, short peptides are not subjected to substantial folding, which may result in an un-ideal confirmation with regard to the interaction of the active site (e.g., the HWRR

, motif) with the target site on the EPCs, but rather remain conformationally stable such that the binding site is not hidden. In addition, short peptides are readily and cost- effectively prepared and purified, and moreover, are easy for use in terms of their application onto devices.

As demonstrated in the Examples section that follows, the short peptides Motif A and Motif B exhibited high activity in binding to ECs as well as to EPCs (see, Figure 11) and in inducing endothelial cell migration (see, Figures 5, 13 and 14). The high activity of these short peptides, along with the advantageous features thereof, render these peptides highly potent agents for application onto surfaces of implantable medical devices as chemoattractants of EPCs.

Exemplary peptides which comprise the conserved motif and consist of 12 amino acids include, but are not limited to, AdoPep 1, AdoPep 2 AdoPep3 as well as RoY (set forth in SEQ ID NOs: 1, 16, 17 and 3).

As demonstrated in the Examples section that follows, AdoPep-1 was found to exhibit high activity is bindings to ECs as well as to EPCs (see, Figure 11 and 12) and AdoPep-1 and RoY were able to induce endothelial cell migration (see, Figures 5, 13, 14 and 20). As discussed hereinabove, the peptides describe herein were shown to chemoattract EPCs to a much greater extent as compared to mature endothelial cells. This feature in highly beneficial in the context of some embodiments of the invention, since EPCs are able to proliferate to a much greater extent than mature endothelial cells and thus enable the formation of a large, wide spread and coherent population of endothelial cells where desired (e.g., a site of vascular injury and/or on the surface of an implantable medical device). The selective peptide-dependent chemoattraction of EPCs is further achieved, by the greater interaction of the peptide with circulating EPCs since these are present in the blood, whereby mature, differentiated endothelial cells are not.

Without being bound to any particular theory, it is suggested herein that the peptides described herein chemoattract EPCs through the binding of Glucose regulated protein 78 (GRP78) located on the EPCs membrane.

GRP78 belongs to the heat shock protein 70 group (HSP70) and has been shown to be widely expressed on the surface of tumor cells as well as endothelial cells. As demonstrated in the Example section hereinbelow, the peptides described herein bind to GRP78 in ECs (see Figures 7-10). It has been also shown that membranal GRP78 is also expressed in EPCs (see Figure 19) suggesting the involvement of GRP78 in the chemoattraction ability of the peptides.

Thus, according to embodiments of the invention there is provided an article of manufacture comprising an implantable medical device and at least one peptide capable of chemoattracting endothelial progenitor cells, the peptide being deposited on at least a portion of the surface of the medical device, wherein the peptide is capable of binding Glucose-Regulated Protein, 78-kD (GRP78; SEQ ID NO:9).

As further demonstrated in the Examples section that follows, while GRP78 is expressed both in ECs as well as in EPCs, the latter were shown to be chemoattracted by the peptides described herein to a much greater extent (see, Figures 11, 12, 14 and 20). Furthermore, while smooth muscle cells also express membranal GRP78 (see, Figure 16), these cells are not chemoattracted by AdoPep 1 (see, Figure 18).

The surprising findings that the peptides described herein chemoattract EPCs via interaction of GRP78, and to a greater extent than do mature endothelial cells and SMCs, render these peptides highly suitable for being deposited on implantable medical devices as described herein. While it could have been expected that ECs and EPCs would compete on chemoattraction to the deposited peptides, thus reducing the therapeutic effect of the deposited peptides, due to the inferior effect of ECs as compared to EPCs discussed supra, the present findings unexpectedly show a beneficial role for the presented peptides when deposited on an implantable medical device.

As discussed hereinabove, the peptides described herein possess anti-apoptotic activity in cardiomyocytes. Without being bound by theory it is suggested herein that the anti-apoptotic activity of the peptide is mediated through a mechanism involving GRP78 as could be deduced by the loss of anti-apoptotic activity of the peptide upon silencing of the GRP78 expression using siRNA techniques.

The concentration of peptides deposited onto the medical device should be less than the level at which the peptide produces potential toxic effects and greater than the level at which non-therapeutic results are obtained. The concentration of the peptide can depend upon factors such as the particular circumstances of the patient, the nature of the trauma, the nature of the therapy desired, the time over which the device implanted resides at the vascular site, and if other active agents are employed, the nature and type of the substance or combination of substances. Therapeutically effective dosages can be determined empirically, for example by infusing vessels from suitable animal model systems and using immunohistochemical, fluorescent or electron microscopy methods to detect the peptide and its effects, or by conducting suitable in vitro studies. Standard pharmacological test procedures to determine dosages are understood by those of ordinary skill in the art.

In some cases, the deposition of a combination of different peptides having a different amino acid sequence may be beneficial. For example, longer peptides may be beneficial in attracting circulating EPCs to the surface of the medical device, thereby anchoring the EPC to the medical device. Once the EPC is in proximity to the device, shorter peptides may further effectively bind the EPC, thereby ensuring stable attachment of the EPC to the device. An additional example may be the use of a combination of peptides described herein in order to obtain a high level of coating on the surface of the device.

Thus, according to some embodiments of the invention, the article of manufacture comprises more than one peptide of the peptides described herein, wherein each peptide is of a different amino acid sequence. In some embodiments, as detailed hereinafter, during the cardiovascular invasive procedure a solution of the peptide is injected to the site of medical device implantation thereby enhancing the concentration of the peptide in the site of implantation. In such cases where the procedure is used to treat cardiovascular diseases characterized by cardiomyocyte apoptosis, a high concentration of the peptide in the medical device implantation site is beneficial due to a higher level of penetration of the peptide through the blood vessel wall into the site of myocardial ischemia.

The term "peptide" as used herein encompasses native peptides (either degradation products, synthetically synthesized peptides or recombinant peptides) and peptidomimetics (typically, synthetically synthesized peptides), as well as peptoids and semipeptoids which are peptide analogs, which may have, for example, modifications rendering the peptides more stable while in a body or more capable of penetrating into cells. Such modifications include, but are not limited to, N-terminus modification, C- terminus modification, peptide bond modification, including, but not limited to, CH₂-

NH, CH₂-S, CH₂-S=O, O=C-NH, CH₂-O, CH₂-CH₂, S=C-NH, CH=CH or CF=CH, backbone modifications, and residue modification. Methods for preparing peptidomimetic compounds are well known in the art and are specified, for example, in Quantitative Drug Design, CA. Ramsden Gd., Chapter 17.2, F. Choplin Pergamon

Press (1992), which is incorporated by reference as if fully set forth herein.

Further details in this respect are provided hereinunder. Peptide bonds (-CO-

NH-) within the peptide may be substituted, for example, by N-methylated bonds (-

N(CH₃)-C0-), ester bonds (-C(R)H-C-O-O-C(R)-N-), ketomethylen bonds (-CO-CH₂-), α-aza bonds (-NH-N(R)-CO-), wherein R is any alkyl, e.g., methyl, carba bonds (-CH₂- NH-), hydroxyethylene bonds (-CH(OH)-CH₂-), thioamide bonds (-CS-NH-), olefinic double bonds (-CH=CH-), retro amide bonds (-NH-C0-), peptide derivatives (-N(R)- CH₂-CO-), wherein R is the "normal" side chain, naturally presented on the carbon atom. These modifications can occur at any of the bonds along the peptide chain and even at several (2-3) at the same time.

Natural aromatic amino acids, Trp, Tyr and Phe, may be substituted for synthetic non-natural acid, such as Phenylglycine, TIC, naphthylelanine (NoI), ring-methylated derivatives of Phe, halogenated derivatives of Phe or o-methyl-Tyr.

In addition to the above, the peptides described herein may also include one or more modified amino acids or one or more non-amino acid monomers (e.g. fatty acids, complex carbohydrates etc).

As used herein, the phrase "amino acid" or "amino acids" is understood to include the 20 naturally occurring amino acids; those amino acids often modified post- translationally in vivo, including, for example, hydroxyproline, phosphoserine and phosphothreonine; and other less common amino acids including, but not limited to, 2- aminoadipic acid, hydroxylysine, isodesmosine, nor-valine, nor-leucine and ornithine. Furthermore, the term "amino acid" includes both D- and L-amino acids.

The peptides described herein are preferably utilized in a linear form, although it will be appreciated that in cases where cyclization does not severely interfere with peptide characteristics, cyclic forms of the peptide can also be utilized. Cyclic peptides can either be synthesized in a cyclic form or configured so as to assume a cyclic form under desired conditions (e.g., physiological conditions). The peptides described herein can be synthesized from D-isomers of natural amino acids [Le., inverso peptide analogues, Tjernberg (1997) J. Biol. Chem. 272:12601-5, Gazit (2002) Curr. Med. Chem. 9:1667-1675].

Additionally, the peptides described herein include retro, inverso, and retro- inverso analogues thereof. It will be appreciated that complete or extended partial retro- inverso analogues of hormones have generally been found to retain or enhance biological activity. Retro-inversion has also found application in the area of rational design of enzyme inhibitors (see U.S. Pat. No. 6,261,569).

As used herein a "retro peptide" refers to peptides that are made up of L-amino acid residues which are assembled in opposite direction to the native peptide sequence.

Retro-inverso modification of naturally occurring polypeptides involves the synthetic assembly of amino acids with α-carbon stereochemistry opposite to that of the corresponding L-amino acids, i.e., D- or D-allo-amino acids in inverse order to the native peptide sequence. A retro inverso analogue, thus, has reversed termini and reversed direction of peptide bonds, while essentially maintaining the topology of the side chains as in the native peptide sequence.

It will be appreciated that incorporation of any of the above-mentioned amino acid modifications, including conserved changes in amino acid residues of the peptides described herein, can be effected, as long as the EPC and EC recruiting/chemoattraction functionality of the peptides is retained. To test this, any of the binding/migration assays described hereinbelow and in the Examples section which follows, or otherwise known in the art, can be effected.

Any of the peptides described herein may be synthesized by any of the techniques that are known to those skilled in the art of peptide synthesis. For solid phase peptide synthesis, a summary of the many techniques may be found in: Stewart, J.

M. and Young, J. D. (1963), "Solid Phase Peptide Synthesis," W. H. Freeman Co. (San

Francisco); and Meienhofer, J (1973). "Hormonal Proteins and Peptides," vol. 2, p. 46,

Academic Press (New York). For a review of classical solution synthesis, see Schroder,

G. and Lupke, K. (1965). The Peptides, vol. 1, Academic Press (New York). In general, peptide synthesis methods comprise the sequential addition of one or more amino acids or suitably protected amino acids to a growing peptide chain.

Normally, either the amino or the carboxyl group of the first amino acid is protected by a suitable protecting group. The protected or derivatized amino acid can then either be attached to an inert solid support or utilized in solution by adding the next amino acid in the sequence having the complimentary (amino or carboxyl) group suitably protected, under conditions suitable for forming the amide linkage. The protecting group is then removed from this newly added amino acid residue and the next amino acid (suitably protected) is then added, and so forth; traditionally this process is accompanied by wash steps as well. After all of the desired amino acids have been linked in the proper sequence, any remaining protecting groups (and any solid support) are removed sequentially or concurrently, to afford the final peptide compound. By simple modification of this general procedure, it is possible to add more than one amino acid at a time to a growing chain, for example, by coupling (under conditions which do not racemize chiral centers) a protected tripeptide with a properly protected dipeptide to form, after deprotection, a pentapeptide, and so forth.

Further description of peptide synthesis is disclosed in U.S. Pat. No. 6,472,505. One method involves solid-phase peptide synthesis, utilizing a solid support. Large- scale peptide synthesis is described by Andersson Biopolymers 2000, 55(3), 227-50.

Recombinant techniques can be used when large amounts of the peptides are required (can also be used when long peptides are required). Such recombinant techniques are described by Bitter et al., (1987) Methods in Enzymol. 153:516-544, Studier et al. (1990) Methods in Enzymol. 185:60-89, Brisson et al. (1984) Nature 310:511-514, Takamatsu et al. (1987) EMBO J. 6:307-311, Coruzzi et al. (1984) EMBO J. 3:1671-1680 and Brogli et al., (1984) Science 224:838-843, Gurley et al. (1986) MoI. Cell. Biol. 6:559-565 and Weissbach & Weissbach, 1988, Methods for Plant Molecular Biology, Academic Press, NY, Section VIII, pp 421-463. To produce a peptide as described herein, using recombinant technology, a polynucleotide encoding a selected peptide is ligated into a nucleic acid expression construct, which includes the polynucleotide sequence under the transcriptional control of a promoter sequence suitable for directing constitutive tissue specific or inducible transcription in the host cells, as further described hereinbelow. Other then containing the necessary elements for the transcription and translation of the inserted coding sequence, the expression construct can also include sequences engineered to enhance stability, production, purification, yield or toxicity of the expressed polypeptide. Such a fusion protein can be designed so that the fusion protein can be readily isolated by affinity chromatography; e.g., by immobilization on a column specific for the heterologous protein. Where a cleavage site is engineered between the peptide moiety and the heterologous protein, the peptide can be released from the chromatographic column by treatment with an appropriate enzyme or agent that disrupts the cleavage site [e.g., see Booth et al. (1988) Immunol. Lett. 19:65-70; and Gardella et al., (1990) J. Biol. Chem. 265:15854-15859].

A variety of prokaryotic or eukaryotic cells can be used as host-expression systems to express the peptide coding sequence. These include, but are not limited to, microorganisms, such as bacteria transformed with a recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vector containing the peptide coding sequence; yeast transformed with recombinant yeast expression vectors containing the peptide coding sequence; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors, such as Ti plasmid, containing the peptide coding sequence. Mammalian expression systems can also be used to express the peptides of the present invention. Bacterial systems are preferably used to produce recombinant peptides, according to the present invention, thereby enabling a high production volume at low cost. Other expression systems, such as insects and mammalian host cell systems, which are well known in the art, can also be used.

In any case, transformed cells are cultured under effective conditions, which allow for the expression of high amounts of recombinant peptides. Effective culture conditions include, but are not limited to, effective media, bioreactor, temperature, pH and oxygen conditions that permit protein production. An effective medium refers to any medium in which a cell is cultured to produce the recombinant peptides of the present invention. Such a medium typically includes an aqueous solution having assimilable carbon, nitrogen and phosphate sources, and appropriate salts, minerals, metals and other nutrients, such as vitamins. Cells can be cultured in conventional fermentation bioreactors, shake flasks, test tubes, microtiter dishes, and petri plates. Culturing can be carried out at a temperature, pH and oxygen content appropriate for a recombinant cell. Such culturing conditions are within the expertise of one of ordinary skill in the art.

Depending on the vector and host system used for production, the resultant peptide may either remain within the recombinant cell; be secreted into the fermentation medium; be secreted into a space between two cellular membranes, such as the periplasmic space in E. coli; or be retained on the outer surface of a cell or viral membrane.

Following a certain time in culture, recovery of the recombinant peptide is effected. The phrase "recovering the recombinant peptide" refers to collecting the whole fermentation medium containing the peptide and need not imply additional steps of separation or purification. Peptides can be purified using a variety of standard peptide purification techniques, such as, but not limited to, affinity chromatography, ion exchange chromatography, filtration, electrophoresis, hydrophobic interaction chromatography, gel filtration chromatography, reverse phase chromatography, concanavalin A chromatography, chromatofocusing and differential solubilization. The peptides can be retrieved in "substantially pure" form. As used herein,

"substantially pure" refers to a purity that allows for the effective use of the peptide in the diverse applications, as described herein (e.g., for therapeutic purposes).

As discussed hereinabove, and demonstrated in the Examples section that follows, the peptides described herein chemoattract EPCs to a greater extent as compared to ECs. This preferential chemoattraction was observed by the enrichment of blood mononuclear population with cells expressing EPC associated proteins surface markers, such as CD31, CD34, CD133 and VEGFR-2 (KDR or FIk-I), either alone or in combination. It has been shown, for example, that the peptide RoY (SEQ ID NO: 3) induced a 51 %, 67 % and 39 % increase in the EPC markers CD34, CD31 and CD133/CD31 positive cells, respectively, in the population of cord blood mononuclear cells (see, Figure 11). AdoPepl (SEQ ID NO: 1) also induces a substantial increase in the percentage of EPCs in a population of cord mononuclear cell population, after one week incubation therewith (a 86 % increase in CD34/KDR positive cells and 80 % increase in the CD 133 positive cells; see, Figure 12). Induction of migration of EPCs from cord blood was obtained by both RoY and AdoPepl peptides (see, Figure 13). In addition, ADoPepl (SEQ ID NO: 1) also exhibited selective recruitment of EPCs from adult human peripheral blood, which led to a EPC enrichment of the cell population (4 folds increase in the percent of CD34 positive cells, 9 folds increase in the percent of CD133/CD31 positive cells (see Figures 14) and 2.3 fold increase in the percent of CD133/GRP78 positive cells (see Figure 20).

Hence, according to some embodiments of the present invention the peptide described herein is capable of chemoattracting endothelial progenitor cells (EPCs) to a greater extent as compared to non-progenitor endothelial cells (non-EPCs).

In some embodiments the peptide is capable of selectively chemoattracting endothelial progenitor cells, such that the amount of endothelial progenitor cells that migrate towards the peptide is at least twice the amount of non-progenitor endothelial cells. For example, the peptide may be capable of chemoattracting EPCs more than 2, 3, 4, 5, 6, 7, 8, 9, 10 times as much as non-EPCs. This ability of the peptide may be evaluated by common migration assays such as the Boyden chamber assay described in the Example section hereinbelow. In such migration assays a population of EPCs and non-EPCs are tested for migration toward the peptide described herein, as the chemoattractant, and toward a control solution (without any added chemoattractant). The amount of EPCs in the migrated population of cells is then quantified by assessing the amount of cells expressing EPC associated proteins (such as CD31, CD34, CD133 and VEGFR-2 (KDR or Rk-I or a combination thereof), each alone or in combination, and compared to the amount of cells expressing these protein in the control group or in the cell population before the migration assay was performed. The detection of the amount of EPCs associated protein may be effected by means well known to those skilled in the art such as immunopercipitation and Western blotting using antibodies specific for the EPC associated protein. For example, the peptide ability to chemoattract EPCs more than twice that of non-EPCs may be concluded from the number of cells expressing CD 133 in the migrated cell population being twice that of the CD 133 expressing cells in the control group and/or in the cell population before the migration assay was performed.

Hence, in some embodiments, the peptide promotes the migration of endothelial cells expressing a protein selected from the group consisting of CD31, CD34, CD133 and VEGFR-2 or a combination thereof, more than twice than endothelial cells not expressing the protein. In some embodiments the peptide promotes the migration of endothelial cells expressing CD133 more than twice than endothelial cells not expressing the protein.

In some embodiments the peptide chemoattracts endothelial cells expressing a protein such as CD31, CD34, CD133 and VEGFR-2 or a combination thereof, more than twice than endothelial cells not expressing the protein. In some embodiments, the peptide chemoattracts endothelial cells expressing CD133 more than twice than endothelial cells not expressing the protein.

In some embodiments the peptide is capable of enriching a population of endothelial cells with endothelial progenitor cells, upon incubation of the cells with the peptide, said enrichment is determined by a higher fraction of endothelial progenitor cells detected in said population after the incubation with the peptide as compared to before said incubation. In some embodiments, the detection is effected by determining the amount of cells expressing a protein selected from the group consisting of CD31, CD34, CD133 and VEGFR-2 or a combination thereof on their membrane.

The implantable medical device described herein may further comprise another therapeutically active agent, being other than the peptide described herein, wherein the therapeutically active agent may act additively or in synergy with the peptide for treating the disease being treated by the device.

Hence, in some embodiments, the implantable medical device further comprises an additional therapeutically active agent capable of treating the disease being treated by the device, as detailed hereinbelow, the agent being deposited on at least a portion of a surface of the device.

As discussed hereinabove, the anti-apoptotic and angiogenesis activity of the peptide may be utilized in procedures for treating cardiovascular conditions characterized by cardiomyocyte apoptosis such as myocardial ischemia (i.e. reduced blood flow to the heart muscle for various reasons). In such cases the incorporation of an additional therapeutically active agent for treating the cardiovascular disease can be beneficial. For example, the incorporation of another agent (other than the peptide), known to induce angiogenesis and/or inhibit apoptosis, may lead to improved therapeutic results following the procedure. As further discussed hereinabove, upon implantation of a medical device into a natural orifice of a patient such as, for example, a blood vessel, proliferation of cells such as medial smooth muscle cells (SMC) in the site of implantation may cause a narrowing of the orifice. For example, in coronary arterial atherosclerosis, the narrowing of the coronary artery lumen may be treated by holding the artery open with a stent. However, the coronary stent implantation provokes a cascade of cellular and biochemical events that induce pathophysiologic processes such as thrombus formation and release of cytokines which trigger the proliferation of SMCs (restenosis). Therefore, while the EPCs propagation and recruitment onto the implantable device reduces and prevents thrombus formation, and while, as discussed hereinabove, the peptides of the present invention do not chemoattract SMCs, incorporation of agents which are capable of further inhibiting thrombosis formation and SMCs proliferation can be beneficial.

For example, incorporation of antiproliferative agents onto the implantable medical device described herein may inhibit/prevent restenosis by reducing or preventing inflammation and exaggerated SMCs proliferation and accumulation.

Hence, according to some embodiments of the present invention the implantable medical device described herein further comprises a therapeutically active agent other than the peptide described herein, for example, an anti-proliferative agent, being deposited on at least a portion of the device's surface.

Such a therapeutically active agent is also referred to herein interchangeably as a drug. An "anti-proliferative agent" refers to any agent which acts to reduce the rate or level of cellular proliferation. The subject agent may function by any suitable mechanism including, but not limited to, inducing apoptosis, modulating cellular microtubule structure (e.g., promoting microtubule polymerisation), inhibiting tyrosine kinase mediated signaling, antagonizing cell surface receptor binding (e.g., EGFR & VEGFR inhibitors), modulating glucocorticoid receptor functioning, downregulating angiogenesis (e.g,. inhibiting VEGF functioning) or otherwise inducing cell death or reducing cellular proliferation events.

Exemplary anti-proliferative agents which are suitable for use in the context of the these embodiments include, but are not limited to, alkylating agents, antimetabolites, antitumur antibiotics, vinca alkaloids, epipodophyllotoxins, nitrosoureas, hormonal and antihormonal agents, and toxins.

Thus, exemplary anti-proliferative agents include, but are not limited to, paclitaxel, rapamycin, cyclophosphamide, chlorambucil, busulfan, Melphalan, Thiotepa, ifosphamide, Nitrogen mustard, methotrexate, 5-Fluorouracil cyrosine arabinoside, 6- thioguanine, 6-mercaptopurine, doxorubicin, daunorubicin, idorubicin, nimitoxantron, dactinomycin, bleomycin, mitomycin, plicamycin, epipodophyllotoxins vincristin, vinblastin, vinclestin, Etoposide, Teniposide. carmustin, lomustin, semustin, streptozocin, adrenocorticoids, estrogens, antiestrogens, progestins, aromatase inhibitors, androgens, antiandrogens, dacarbazin, hexamethylmelamine, hydroxyurea, mitotane, procarbazide, cisplastin, and carboplatin.

In some embodiments of the invention, the implantable medical device further comprises an agent that stimulates a formation of an endothelium on the surface of the medical device by the endothelial progenitor cells, the agent being deposited onto at least a portion of the device's surface.

Such an agent may be for example, a growth factor such as, but not limited to, vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF) -3, FGF-4, FGF-5, FGF-6, FGF-7, FGF-8, FGF9, basic fibroblast growth factor, platelet-induced growth factor, transforming growth factor beta 1, acidic fibroblast growth factor, osteonectin, angiopoietin 1, angiopoietin 2, insulin-like growth factor, granulocyte- macrophage colony-stimulating factor, platelet-derived growth factor AA, platelet- derived growth factor BB, platelet-derived growth factor AB, endothelial PAS protein 1, trhombospondin, proliferin, Ephrin-Al, E-selectin, leptin, heparin, interleukin 8, thyroxine, and sphingosine 1-phosphate.

The concentration of the drug for the treatment of a cardiovascular disease and the agent for stimulation endothelium formation, as described hereinabove, is a concentration effective to achieve the intended purpose. More specifically, the concentration of the active ingredients (the drug and agent as hereinabove described) is selected so as to effectively prevent, alleviate or ameliorate symptoms of a disorder (e.g., a pathology associated with thrombosis or myocardial ischemia) or prolong the survival of the subject being treated.

Determination the concentration of the active ingredients is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.

As discussed hereinabove, the peptides described herein are capable of chemoattracting endothelial progenitor cells, inhibiting apoptosis and inducing angiogenesis and are deposited on a portion of a surface of an implantable medical device, thereby serving as (i) potent scavengers for the recruitment and attachment of EPCs onto the device; and (ii) cardioprotective agents. The phrase "implantable medical device" as used herein, describes any type of appliance that is totally or partly introduced, surgically or medically, into a patient's bodily site or by medical intervention into a natural orifice. The duration of implantation may be essentially permanent, such that the device is intended to remain in the targeted site after the procedure is completed, i.e., intended to remain in place for the remaining lifespan of the patient; until the device biodegrades; or until it is physically removed, or can be essentially transient, such that the device is withdrawn from the patient's bodily site once the procedure is completed.

Herein throughout, implantable medical devices intended to remain in the targeted site after the procedure is completed are referred to as "permanent" implantable devices; and implantable medical devices intended to be removed from the targeted site once the procedure is completed are referred to as "transient" implantable devices.

As used herein, the phrase "bodily site" includes any organ, tissue, membrane, biological surface or muscle, to which the medical device of the present invention is implanted.

Exemplary bodily sites include, but are not limited to, a cardiac lumen, a renal artery, a renal vein, a coronary artery, a coronary vein, a cerebral artery, a cerebral vein, a cervical artery, a cervical vein , a stomach artery, a stomach vein, a hepatic artery, a portal vein, a mesenteric artery, a mesenteric vein, an arm artery, an arm vein, a hand artery, a hand vein, a leg artery, a leg vein, a foot artery, and a foot vein. As used herein, the term "natural orifice" encompasses a bodily cavity. The bodily cavity can be, for example, a heart cavity, an organ cavity, a blood vessel, a pulmonary cavity, an artery, a vein, a kidney, a capillary, the space between dermal layers, an organ of the female or male reproductive system, an organ of the digestive tract and any other visceral organs or cavity. In some embodiments, the implantable medical device described herein is an intraluminal device.

As used herein, the term "intraluminal device" describes a medical device that is suitable for being implanted in a lumen of a subject. Thus, an intraluminal device may be, for example, a vascular device such as a vascular stent, a cardiac device such as an artificial valve, a catheter, a cerebrospinal fluid shunt, or an intrauterine device.

According to some embodiments of the invention, the implantable medical device is a blood-contacting medical device. The term "blood-contacting medical device" describes a medical device wherein one or more surfaces of the medical device comes in contact with blood and/or is introduced into vasculature of an individual; and hence, such surface is susceptible to any one or more of thrombosis, neointima formation, and restenosis. Preferably, the medical device is a cardiovascular device. The terms

"cardiovascular device", "intravascular device" and "vascular device" are used interchangeably herein, to describe a structure that is introduced into a human or animal vasculature to restore function of damaged, diseased, or blocked tissue, and includes prosthetic devices, and vascular grafts. The term "cardiovascular device" as used herein, encompasses devices introduced to the cardiac system organs, such as the heart.

The term "cardiovascular device" as used herein also includes device-related materials that are associated with the device and are also introduced into a human or animal body in conjunction with the device. Exemplary medical devices (both permanent and transient) according to embodiments of the invention include, but are no limited to, a stent, a stent graft, a vascular graft, a synthetic vascular graft, a heart valve, a catheter, a vascular prosthetic filter, a pacemaker, a pacemaker lead, a defibrillator, a patent foramen ovule septal closure device, a vascular clip, a vascular aneurysm occluder, a hemodialysis graft, a hemodialysis catheter, an atrioventricular shunt, an aortic aneurysm graft device or components, a venous valve, a sensor, a suture, a vascular anastomosis clip, an indwelling venous catheter, an indwelling arterial catheter, a vascular sheath, a vascular occluder and a drug delivery port.

The device may be implanted either permanently, or for a prolonged time period, or, alternatively, can be a transient device, utilized, for example, only for performing a procedure.

In some embodiments, the medical device is configured in accordance with the disease to be treated and the desired procedure for treating the disease, as detailed hereinbelow.

In some embodiments the medical device is a stent. As known in the art, a stent is a metallic and/or polymeric cage-like or tubular support device that is used to hold vessels (e.g., blood vessels) open.

The medical device according to this embodiment of the invention typically includes a device structure onto which a peptide according to the present embodiments is deposited.

By "deposited" it is meant that peptide can be applied on, entrapped in or attached to the device structure, via chemical and/or physical interactions.

Chemical interactions include, for example, covalent bonds, electrostatic interactions, hydrogen bonds, van der Waals interactions, donor-acceptor interactions, aromatic (e.g., π- π) interactions, cation- π interactions and metal-ligand interactions. Physical interactions include, for example, swelling, adsorption, encapsulation, entrapment, entanglement and the likes.

The medical device may be comprised of, and hence have one or more surfaces comprised of, a variety of materials including, but not limited to, a metal, a metal oxide, a non-metal oxide, a metal alloy, a ceramic, a rubber, a plastic, an acrylic, a silicone, a polymer, and combinations thereof. The above-described interactions via which the peptide is deposited onto the device's surface depend, at least in part, on the chemical, physical and mechanical properties of the device's surface.

The phrase "surface" as used herein encompasses both inner and outer surfaces of the device. Preferably, the peptides are applied to the outer surface of the device. As used herein, an "outer surface" of an implantable medical device refers to the part of the surface that is directly in contact with the external environment.

By "at least a portion" of the surface it is meant that the peptide(s) may be applied onto all of the surface's area or, alternatively, only onto a portion or some portions of the surface. The underlying structure of the device can be of virtually any shape and design.

Exemplary device shapes which are suitable for use in the context of the present embodiments include, but are not limited to, cylindrically shaped structures (for example vascular stents); tube shaped structures (for example, catheters) and long thin filaments (for example sutures) etc.

The device can be produced using any biocompatible material; however, because of the difficulties with biocompatibilities in the vasculature, it is preferred that the biocompatible material be relatively inert. Such devices are made of a variety of materials that are known in the art, but most typically are biologically inert polymers or metals.

Metals used in the manufacture of medical devices are known in the art to include, without limitation, stainless steel, tantalum, gold, platinum, silver, tungsten, chromium, alumina, titanium, titanium alloys (for example, memory titanium alloys such as nitinol), a transition metal, alkali metals, and alkaline earth metals (each of the latter three comprise metals related in structure and function, as classified in the Periodic Table). Metal alloys (e.g., cobalt-chromium alloy) and metal oxides of each of these groups, individually and separately, are included. Alternatively, the device structure may be comprised of other biocompatible materials. These include, for example, plastics, silicon, polymers, resins, and may include at least one component such as, for example, polyurethane, cellulose ester, polyethylene glycol, polyvinyl acetate, dextran, gelatin, collagen, elastin, laminin, fibronectin, vitronectin, heparin, segmented polyurethane-urea/heparin, poly-L-lactic acid, fibrin, cellulose and amorphous or structured carbon such as in fullerenes, and any combination thereof.

In cases where a biodegradable implantable device is desired, the device structure can be comprised of a biocompatible material that is biodegradable. Biodegradable material can include, for example, biodegradable polymers such as poly- L-lactic acid.

The medical device may further comprise a biocompatible matrix deposited on the surface of the device.

A "biocompatible matrix", as defined herein, describes a matrix that is deposited on the surface of the device and does not induce a significant allergic, inflammatory or any other adverse reaction in the host subject into which it is administered (implanted). The biocompatible matrix can be biodegradable or nondegradable.

The biocompatible matrix form is selected based on the device shape and intended use. For example, the matrix may be in a form of a jacket when the device is tubular shaped. Alternatively, when the device is, for example, a valve, the matrix may be in the form of a covering. Stents may be encapsulated by the biocompatible matrix.

Thus, according to some embodiments of the invention, the matrix is in a form of a jacket, a covering or an encapsulation.

The biocompatible matrix may comprise a synthetic material and/or a naturally- occurring material. Exemplary synthetic materials include, but are not limited to, a polyurethane, a segmented polyurethane-urea/heparin, a poly-L-lactic acid, cellulose ester, polyethylene glycol, polyvinyl acetate, dextran, gelatin, PVA hydrogel, ePTFE, PTFE, porous HDPE, and polyethyleneterephthalate. Naturally-occurring material may be, for example, collagen, elastin, laminin, fibronectin, vitronectin, heparin, fibrin, cellulose and amorphous carbon.

According to some embodiments, the peptides described herein are deposited on the implantable medical device via linkage to the surface of the device or, if present, to the biocompatible matrix coating the surface. The linkage may be formed via chemical interactions which can include, for example, covalent interactions (i.e. covalent bonds), electrostatic interactions, hydrophobic interactions, aromatic interactions or Van der

Waals interactions. Alternatively, the peptides may be entrapped within pores of a porous surface of the device or of a porous biocompatible matrix deposited on the medical device, swelled or soaked within the surface or the biocompatible matrix, or physically adhered to the surface/matrix.

In some embodiments of the invention the peptide is deposited directly to the surface of the implantable medical device or to the biocompatible matrix deposited onto the surface of the medical device. In other embodiments of the present invention the peptide linkage is through a spacer.

A spacer is used in order to facilitate the linkage of the peptide to the surface and/or matrix, as described herein, and/or to improve the peptide's adherence to the surface and/or matrix. The spacer is preferably a biocompatible moiety.

The spacer can be linked at one end to the surface or to a matrix deposited on the surface, if present, and may further be capable of interacting with the peptide.

Exemplary spacers include, without limitation, amino acid spacers (typically, a short peptide of between 3 and 15 amino acids, and often containing amino acids such as glycine, and/or serine), polymers (e.g., polyethylene glycol), functionalized fatty acids, and any other biocompatible material.

Suitable polymeric spacers are known in the art, and can comprise a synthetic polymer or a natural polymer. Representative synthetic polymers include, but are not limited to, polyethers (e.g., poly(ethylene glycol) ("PEG")), polyesters (e.g., polylactic acid (PLA) and polyglycolic acid (PGA)), polyamines, polyamides (e.g., nylon), polyurethanes, polymethacrylates (e.g., polymethylmethacrylate; PMMA), polyacrylic acids, polystyrenes, polyhexanoic acid, flexible chelators such as EDTA, EGTA, and other synthetic polymers which preferably have a molecular weight of about 20 daltons to about 1,000 kilodaltons. Representative natural polymers include but are not limited to hyaluronic acid, alginate, chondroitin sulfate, fibrinogen, fibronectin, albumin, collagen, calmodulin, and other natural polymers which preferably have a molecular weight of about 200 daltons to about 20,000 kilodaltons (for the constituent monomers).

Depending on such factors as the peptide to be linked, and the conditions in which the linking is performed, the spacer may vary in length and composition for optimizing such properties as preservation of biological function, stability, resistance to certain chemical and/or temperature parameters, and of sufficient stereo-selectivity or size. The peptide can be attached to the spacer compound described prior to or after the spacer compound is applied to the surface of the medical device. As discussed hereinabove, the peptides described herein are capable of promoting EPCs propagation and recruitment from cord and adult human peripheral blood and thus, upon attachment to the implantable medical device according to embodiments of the invention, can serve as potent scavengers for the recruitment and attachment of EPCs onto the device. Furthermore, due to the anti-apoptotic and angiogenesis promoting activity of the peptides, upon release from the device or concomitant injection of a solution of the peptide in the device implantation site, during the procedure, the peptides can exert cardioprotective activity and thus contribute further to the success of the medical procedure.

The EPC scavenging capacity, the anti-apoptotic activity and angiogenesis enhancing capability of the peptides described herein may be utilized for the treatment of a medical condition in which the treatment involves implantation of a medical device in the body of the subject suffering from the medical condition and wherein the reduction in apoptosis and/or enhancement of angiogenesis and/or attachment of EPCs onto the device is beneficial.

As used herein, the term "subject" refers to any animal (e.g., a mammal), including, but not limited to, humans, non-human primates, rodents, and the like, which is to be the recipient of a particular treatment. Typically, the terms "subject" and "patient" are used interchangeably herein in reference to a human subject.

The treatment of many cardiovascular diseases involves the implantation of medical devices in the body of a subject suffering from the disease. For example, as discussed hereinabove, in coronary arterial atherosclerosis, the narrowing of the coronary artery lumen may be treated by holding the artery open with a stent. Another example is severe aortic valve stenosis disease wherein a new artificial valve may be implanted instead of the old non-functional valve.

As discusses hereinabove, the medical device implantation (e.g. stent or artificial valve) provokes a cascade of cellular and biochemical events that induce pathophysiologic process such as thrombus formation and release of cytokines which trigger the proliferation of SMCs. Endotheliazation (i.e. delivery of a sufficient number of endothelial cells (ECs) to the surface of the medical device) provides an inherent non-thrombogenic potential, interrupts cytokine-driven activation of SMCs in vascular medial tissues and accelerates normal wound healing at diseased sites. Endothelial progenitor cells (EPCs) have proliferation capacity and differentiation into mature endothelial cells (ECs).

In some embodiments, the articles of manufacture described herein are for use in a method of treating a cardiovascular disease in a subject in need thereof.

In some embodiments the medical device is a permanently insertable medical device.

A permanently insertable medical device is a device which is inserted, via a medical procedure, into a bodily site for a time period exceeding that of the procedure time (i.e. is left within the body upon the completion of the procedure). The medical device can be left within the body for a time period which is determined by those skilled in the art as needed in order to achieve the appropriate medical result (i.e. amelioration of the disease condition that necessitated the device implantation).

Non limiting examples of permanent insertable medical devices are a stent implanted in cardiovascular organs for the opening of a blocked or narrow vessel such as in atherosclerosis, myocardial infraction, an inferior vena cava filter for prevention of pulmonary emboli (PEs) in the treatment of deep vain thrombosis (DVT), a pacemaker, an artificial graft such as artificial heart valves for replacement of un-functional valves, intracardiac closure devices or artificial vascular grafts for replacement of injured or ruptured blood vesicles. Other non limiting examples include sutures, sensors and vascular prosthetic filters.

Other permanent insertable medical devices are described, for example, in U.S. Patent No. 5,683,411, which is incorporated by reference as if fully set forth herein. In some embodiments, the implantable medical device used in a method of treating a cardiovascular disease is a stent. As discussed herein, the stent may advantageously comprise, in addition to the peptide(s) described herein, other therapeutically active agents, as described herein.

In some embodiments of the invention, the implantable medical device is utilized in a method which further comprises locally administering to the target bodily site a solution which comprises the peptides described herein.

As noted herein, the peptides described herein, in addition to their capability to chemoattract EPCs, as described herein, were further found to exhibit an anti-apoptotic effect, including an anti-apototic effect of cardiomyocytes. These peptides were further shown previously to promote angiogenesis. Co-administering a solution of these peptides can therefore be for reducing or preventing apoptosis in the target bodily site, where the device is implanted and/or for inducing angiogenesis in the bodily site, as is further detailed hereinbelow.

In some embodiments, implanting the medical device and locally administering the solution of the peptide described herein are effected via the same technique.

Accordingly, a procedure for implanting a medical device as described herein is utilized also for locally administering the peptide in a form of a solution containing same, as is detailed hereinbelow, so as to improve the therapeutic effect of the implant and to provide, for example, an overall treatment of the disease or the impaired bodily site to be treated within the same procedure, as is further detailed hereinbelow.

For example, implantable medical devices for implantation in a cardiac lumen are often implanted via catheterization. In some embodiments, implanting the medical device and co-administering the solution of the peptide are both effected via catheterization.

In another example, an implantable medical device is implanted during an operation procedure. The solution of the peptide(s) can therefore be administered locally to the impaired bodily site during the operation.

A further, detailed discussion of a method which utilizes an implantable device as described herein, in combination with local administration of a solution containing the peptides described herein, is provided hereinbelow.

As discussed hereinabove, the medical device may be implanted in a bodily site permanently (i.e. permanent medical device) or transiently (i.e. transient insertable medical device).

In some embodiments the medical device is a transiently insertable medical device.

A transient insertable medical device is a device which is inserted into a bodily site as part of a medical procedure for treating a disease, and upon the completion of the procedure is withdrawn from the body. A non-limiting example of a transient medical device is a devise comprising a balloon and a catheter used during transluminal coronary balloon angioplasty (PTCA) for treatment of myocardial infraction, thrombosis and coronary atherosclerosis as well as all other cardiovascular interventions. The procedure involves the insertion of a catheter (through, for example, the femoral artery) to vascular site in need of widening (such as a coronary artery) and the balloon is inserted through the catheter and inflated. Upon the completion of the procedure both the catheter and the balloon are removed from the body.

Other examples of transient insertable medical devices are described in U.S. Patents Nos. 5,611,775, 5,573,515, 5,857,464, 6,964,649 and 6,280,414 and in U.S.

Patent Applications having Publication Nos. 2005/0226855, 2004/0048375,

2004/0097905, 2002/0107504 and 2009/0246252, wherein are all incorporated by reference as if all fully set forth herein. These, as well as other transient insertable devices known to be used for implanting a medical device and/or for administering a therapeutically active therethrough, are contemplated herein.

Transient implantable medical devices according to embodiments of the present invention, having a peptide as described herein deposited on a surface thereof, can be used while utilizing the EPCs chemoattraction exhibited by peptides for treating conditions, such as, for example, impaired blood vessels. In some embodiments, transient implantable medical devices according to embodiments of the present invention, having a peptide as described herein deposited on a surface thereof, are formulated for releasing the peptides in the desired bodily site.

In some embodiments the transiently insertable medical device is a balloon catheter.

The term "balloon catheter" describes a thin catheter tube that can be guided through a body conduit of a patient such as a blood vessel, and a distensible balloon located at the distal end of the catheter tube or inserted through the catheter to its distal end. Actuation of the balloon is accomplished through use of a fluid filled syringe or similar device that can inflate the balloon by filling it with fluid (e.g., water or saline solution) to a desired degree of expansion and then deflate the balloon by withdrawing the fluid back into the syringe. By depositing the peptides described herein on the balloon surface (or a portion thereof), in a suitable formulation, upon inflating the balloon within a blood vessel, the peptide is applied to a wall of the blood vessel.

Such a transiently insertable medical device can be used, for example, for repairing an impaired blood vessel or for repairing a wound downstream the cardiovascular system. The articles of manufacture according to the present embodiments may, if desired, be presented in a pack, such as an FDA (the U.S. Food and Drug Administration) approved kit. The pack device may be accompanied by instructions for administration. The pack may also be accompanied by a notice associated with the container in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the compositions for human or veterinary administration. Such notice, for example, may be of labeling approved by the U.S. Food and Drug Administration for prescription drugs or of an approved product insert. The pack may be further labeled for treatment of an indicated condition, as is detailed herein. Thus, according to embodiments of the invention, the article of manufacture described herein is packaged in a packaging material and identified in print, in or on the packaging material, for use in a method of treating a cardiovascular disease, as described herein.

As discussed hereinabove, the findings that the peptides, as described herein, are capable of recruiting EPCs, and to exhibit a cardiomyocyte protective effect, taken together with the activity of such peptides to promote angiogenesis, can advantageously utilized in treating cardiovascular diseases by local administration of these peptides.

The local administration of the peptides described herein, when deposited on a surface of a medical devices used during medical procedures for treating cardiovascular diseases, or when locally administered during the same procedure, conform to the medical device and to the procedure itself superior performance and improved therapeutic results.

Thus, according to as aspect of some embodiments of the invention there is provided a method of treating a vascular disease in a subject in need thereof, which is effected by (i) contacting a bodily site afflicted by the cardiovascular disease with an implantable medical device configured for treating the vascular disease; and (ii) contacting the bodily site with a peptide as described herein, by means of said implantable medical device.

The phrase "cardiovascular disease" is used interchangeably with "vascular disease" and encompasses any disorder of the cardiovascular system.

The phrase "cardiovascular system" refers to the heart itself and to the arteries, veins and capillaries that transport blood throughout the body. This includes, without limitation, the cardiovascular system, the carotid artery system and the peripheral vascular system and the veins that complete the circulatory system between each of the foregoing and the heart. The cardiovascular system is the general circulatory system between the heart and all parts of the body. The carotid system supplies blood to the brain. The peripheral vascular system carries blood to and from the peripheral organs such as, without limitation, the arms, legs, kidneys and liver.

The phrase "by means of the implantable device" means that the peptides are contacted with the target bodily site during implantation of the device, either by being deposited on the device, by being delivered to the target bodily site through the device or by both, being deposited on the device and in addition delivered to the target bodily site during implantation of the device.

The method described herein utilizes the peptides described herein either in solution and/or when deposited on a medical device used in a procedure for treatment of cardiovascular diseases. The peptides are injected to the site of cardiovascular disease or are dispersed within the site when being locally administered through the medical device. The peptides described herein may be utilized for the efficient treatment of cardiovascular diseases either by recruiting EPCs to the medical device surface as discussed hereinabove or, by reducing the level of apoptosis, enhancing the level of angiogenesis and recruiting EPCs to sites of organ damage (such as myocardial damage) and endothelial injury.

It is noted herein that locally administering the peptide by means of an implantable medical device, as detailed hereinbelow, provides for an improved therapeutic effect of the peptides, as compared to administering the peptides via noninvasive procedures.

Hence, in some embodiments, the implantable medical device has a peptide as described herein deposited on at least a portion of a surface thereof. Such an implantable medical device is described in detail hereinabove.

Contacting the bodily site with the peptide when deposited on a surface of an implantable device is effected by implanting the implantable medical device, as discussed hereinbelow.

Hence, according to some embodiments of the invention, there is provided a method of treating a cardiovascular disease in a subject in need thereof, which is effected by contacting a bodily site afflicted by the cardiovascular disease with an implantable medical device configured for treating the cardiovascular disease.

Thus, the article of manufacture described herein may be utilized for the treatment of a cardiovascular disease in which EPCs recruitment and propergation and/or inhibition of apoptosis and/or promotion of angiogenesis is therapeutically beneficial.

In cases where the implantable medical device is a transient insertable medical device, contacting the bodily site with the peptide can be effected by delivering the peptide to the bodily site via the transient insertable device. Exemplary devices that can be utilized for delivering the peptides described herein are provided hereinabove. These include both, a transient insertable device having the peptide deposited thereon and a transient insertable device for delivering a solution containing the peptide therethrough.

Implanting the medical device is performed in a bodily site afflicted by the cardiovascular disease, or in a vascular vessel that reaches an afflicted bodily site.

In some embodiments, the method is effected by implanting the medical device in a bodily site afflicted by the cardiovascular disease in the subject.

The medical device is configured in accordance with the specific procedure applied in the treatment of the cardiovascular disease and in accordance with the treated bodily site.

As discussed herein, the method described herein may be utilized for the treatment of cardiovascular disease conditions in which a permanent insertable medical device is used as part of the medical procedure for treating the disease. Such exemplary cardiovascular diseases include, but are not limited to, valvular stenosis and valvular regurgitation; arteriosclerosis, atherosclerosis, thrombosis such as inferior vena cava thrombosis deep vein thrombosis, and hepatic vein thrombosis, Thrombosis of the portal venous system; restenosis; vascular dissection or perforation; vascular aneurysm; vulnerable plaque; chronic total occlusion; claudication; anastomotic proliferation (for vein and artificial grafts); tumor obstruction; angina pectoris; myocardial infarction; chronic coronary ischemia; cerebrovascular accident (CVA); transient ischemic attack (TIA); Renal (Kidney) Vascular Disease such as renal artery stenosis, renal artery thrombosis; renal artery aneurysm; atheroembolic renal disease and renal vein thrombosis; Peripheral Artery Disease; Peripheral Venous Disease; Blood Clots in the Veins; obstruction of the inferior vena cava and hepatic veins (Budd-Chiari syndrome); occlusion of the small hepatic veins (veno-occlusive disease), artificial vascular grafts, trauma and repair of congenital heart disease or combinations of these. As further discussed herein, the treatment of cardiovascular disease conditions is such that a transiently insertable medical device is used as part of the medical procedure for treating the disease. Such cardiovascular diseases may include, for example, valvular insufficiencies such as valvular stenosis and valvular regurgitation; arteriosclerosis, atherosclerosis, thrombosis such as inferior vena cava thrombosis deep vein thrombosis, and hepatic vein thrombosis, Thrombosis of the portal venous system; restenosis; hemorrhage; vascular dissection or perforation; vascular aneurysm; vulnerable plaque; chronic total occlusion; claudication; anastomotic proliferation (for vein and artificial grafts); tumor obstruction; angina pectoris; myocardial infarction; chronic coronary ischemia; cerebrovascular accident (CVA); transient ischemic attack (TIA); Renal (Kidney) Vascular Disease such as renal artery stenosis, renal artery thrombosis; renal artery aneurysm; atheroembolic renal disease and renal vein thrombosis; Peripheral Artery Disease; Raynaud's Phenomenon; Buerger's Disease; Peripheral Venous Disease; Diabetes mellitus related vascular disease; Varicose Veins; Blood Clots in the Veins; hypertension; obstruction of the inferior vena cava and hepatic veins (Budd-Chiari syndrome); occlusion of the small hepatic veins (veno- occlusive disease), vascular grafts, trauma ; during invasive procedures such as cardiac and thoracic surgery and neurosurgery, radiofrequency catheter ablation of arrhythmia foci, coronary thrombolysis, coronary angioplasty (with or without stent placement); and coronary artery bypass grafts or combinations of these.

In some embodiments the method described herein is effected by implanting a medical device having the peptide deposited thereon in a bodily site afflicted by the cardiovascular disease and by locally administering, concomitantly, to the bodily site a solution of the peptide, the solution of the peptide being for reducing or preventing apoptosis in the bodily site and/or for inducing angiogenesis in said bodily site.

The local administration of the peptide solution is effected, for example, by the same technique utilized for implanting the device, as described hereinabove. For example, in the treatment of atherosclerosis the implantation of a stent in the site of stenosis enables the widening of the narrowed vessel thereby improving blood flow through the vessel. Such a stent, when coated with the peptide described herein will reduce the extent of restenosis for reasons discussed hereinabove, namely the ability of the peptides to recruit EPCs to the stent. The concomitant administration of a solution of the peptide during the procedure, to the site of stent implantation is also beneficial in treating myocardial ischemia since the peptide, upon penetrating the site of ischemic myocardium may reduce the level of cardiomyocyte apoptosis and promote angiogenesis thereby enhancing blood flow to the ischemic site.

Such a treatment is particularly beneficial in treating patients that underwent myocardial infraction, and are thereafter subjected to stentization, since it enables to reduce the hypoxia-induced apoptosis caused during the MI.

As discussed hereinabove, without being bound by theory, it is suggested that the ability of the peptide to induce angiogenesis/inhibit apoptosis and recruit EPCs is mediated through its binding to membranal GRP78 on cells expressing the protein. Specifically, it is suggested that in sites of injury, characterized by a high level of cell apoptosis (such as apoptosis caused by hypoxia in sites of myocardial infract), a high level of GRP78 is expressed on the membrane of these cells thereby leading to a high level of binding to the peptide described herein. Thus, in disease states involving cell death and endothelial damage such as in myocardial ischemia, the peptides described herein will, upon local injection to the diseased site, penetrate to the sites of injured endothelium and apoptosis, bind to GRP78 on these endothelial and apoptotic cells and reduce the extent of damage by (i) inhibition of the apoptosis cascade in the injured cells (ii) enhance blood flow to site by promoting capillary formation and (iii) recruit EPCs to the site thus promoting the formation of a new, coherent endothelial lining. For example, in such cases where the blood supply to a specific organ is reduced due to blood vesicle narrowing (such as due to coronary atherosclerosis or stenosis in renal arteries) damage to the organ may be caused due to lack of crucial blood ingredients such as nutrition and most importantly, oxygen. Such conditions may lead to hypoxia- induced organ damage characterized by apoptosis (such as in the case in myocardial ischemia). These cells at the site of narrow blockage and/or apoptotic cells within the damaged organ will then express GRP78 to a higher extent. By applying the peptide described herein locally to the site of narrowness/blockage a reduction in the level of organ damage is achieved due to the peptide binding to GRP78 expressing apoptotic cells and to the damaged endothelial intima surrounding it and reduce the damage to the organ.

During percutaneous transluminal coronary balloon angioplasty (PTCA) a balloon is inserted through a catheter and inflated within a vascular organ such as an artery in order to widen the lumen of the organ (for example, during artery stenosis). The coating of such a balloon with the peptide described herein may be beneficial due to the peptide being distributed, upon the inflation of the balloon in the desired site, thus enabling the recruitment and formation of an EPC population in the site of stenosis and exerting cardioprotective activity (anti-apoptotic and pro-angiogenesis activity) at the site of myocardial ischemia.

In the treatment of a valvular disease, an artificial valve is implanted in or instead of the original valve or a valvuloplasty device is inserted. The coating of such a valve or device with the peptide described herein will enable the formation of endothelial cells covering on the valve or device surface thereby reducing the well known risk of thrombus formation due to interaction between blood factors and artificial surfaces.

Medical devices are also inserted temporarily or permanently to repair congenital cardiovascular malformations such as closure devices to close arterial or ventricular septal defects or balloon and stent to dilate aortic contraction.

During many medical procedures sutures are used for connecting blood vesicles one to the other. The coating of such sutures with the peptide described herein is beneficial since the surface of such sutures will comprise a population of EPCs via the chemoattraction ability of the peptides.

A list of cardiovascular diseases which are characterized by a site of cardiomyocyte cell death via an apoptosis mechanism and are treated using invasive procedures involving implantable medical devices and which are treatable by the method described herein include, but are not limited to angina pectoris; Myocardial Infraction, Congestive Heart Failure, myocarditis, myocardial hypertrophy, cardiomyopathy such as dilated cardiomyopathy and hypertrophic cardiomyopathy; arrhythmia, chronic coronary ischemia; during invasive procedures such as cardiac surgery, radiofrequency catheter ablation of arrhythmia foci, coronary thrombolysis, coronary angioplasty (with or without stent placement); and coronary artery bypass grafts or combinations of these.

The angiogenesis promoting activity of the peptides described herein may be utilized when angiogenesis is therapeutically beneficial. For example during the implantation of a stent in a site of narrowed blood vessel (such as in coronary arthrosclerosis) the injection of a solution of the peptide described herein and/or the distribution of the peptide in the site upon release from the medical device, may promote the formation of collateral blood vessels thus enabling superior blood flow to organs distal to the site of vascular narrowness. Similarly, other impaired blood vessels can be similarly treated.

In some embodiments, the peptide described herein is delivered to the site of cardiovascular disease during an invasive procedure already used for treating the disease. The bodily site and the peptide are brought into contact through the procedure used to treat the disease. Therefore, by depositing the peptide onto the medical device used during the procedure (such as the catheter, the stent, the PTCA balloon) or the local injection of a solution of the peptide during the procedure, utilizing the procedure technique, an improved therapeutic effect is achieved.

Thus, in some embodiments, the contacting of the bodily site with the peptide is effected by implanting the implantable medical device. In some embodiments, the implantable medical device is a transient insertable medical device, and contacting the bodily site with the peptide comprises delivering the peptide to the bodily site via the transient insertable device.

Implanting the medical device may be performed by any method known in the art. For example, for implantation of a stent, an angiogram is first performed to determine the appropriate positioning for stent therapy. An angiogram is typically accomplished by injecting a radiopaque contrasting agent through a catheter inserted into an artery or vein as an x-ray is taken. A guidewire is then advanced through the lesion or proposed site of treatment. Over the guidewire is passed a delivery catheter that allows a stent in its collapsed configuration to be inserted into the passageway. The delivery catheter is inserted either percutaneously or by surgery into the femoral artery, brachial artery, femoral vein, or brachial vein, and advanced into the appropriate blood vessel by steering the catheter through the vascular system under fluoroscopic guidance. A stent having attached thereto a peptide as described herein an optionally additional matrices and/or agents, as described herein may then be expanded at the desired area of treatment. A post-insertion angiogram may also be utilized to confirm appropriate positioning. Accordingly, there is provided a use of an implantable medical device as described herein, for treating or preventing a cardiovascular disease, as described herein.

Further provided is a use of at least one peptide, according to the embodiments of the invention, in the manufacture of an implantable medical device for treating or preventing a cardiovascular disease.

Further provided is a peptide as described herein, identified for use in a method of treating a cardiovascular disease in a subject undergoing an implantation of an implantable medical device configured for treating the cardiovascular disease. Accordingly, there is provided a peptide as described herein in the manufacture of a medicament for use in a method of treating a cardiovascular disease in a subject in need thereof via implantation of an implantable medical device 9permanent or transient). As discussed hereinabove, during the procedure for implantation of the medical device, the injection of a solution of the peptide described herein may be beneficial. For example, during the implantation of a stent (such as in cases of coronary atherosclerosis) using a delivery catheter, as described hereinabove, a solution of the peptide described herein may be injected via the same catheter. The solution of the peptide includes any solution suitable for injection in procedures as described herein. For example the viscosity of the solution should be such so as to allow the injection of the solution from the proximal side of the device to its distal site (located in proximity to the site of interest). Furthermore, the solution should comprise appropriate buffers and salts so as to allow the appropriate amount of the peptide to be dissolved within the liquid.

The peptides of the present invention can be utilized in any of the methods described herein, either per se or as a part of a pharmaceutical composition, which further comprises a pharmaceutically acceptable carrier.

According to a feature of the present invention, such a pharmaceutical composition is packaged in a packaging material and identified in print, in or on the packaging material, for use in a method of treating or preventing a cardiovascular disease, for example myocardial ischemia, as described hereinabove, whereby the methods involves an implantable medial device and the composition is for being administered via the device. As used herein a "pharmaceutical composition" refers to a preparation of one or more of the peptides of the present invention (as active ingredient), or physiologically acceptable salts or prodrugs thereof, with other chemical components including but not limited to physiologically suitable carriers, excipients, lubricants, buffering agents, antibacterial agents, bulking agents (e.g. mannitol), antioxidants (e.g., ascorbic acid or sodium bisulfite), anti-inflammatory agents, anti-viral agents, chemotherapeutic agents, anti-histamines and the like. The purpose of a pharmaceutical composition is to facilitate administration of a compound to a subject. The term "active ingredient" refers to a compound, which is accountable for a biological effect.

The terms "physiologically acceptable carrier" and "pharmaceutically acceptable carrier" which may be interchangeably used refer to a carrier or a diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered compound.

Herein the term "excipient" refers to an inert substance added to a pharmaceutical composition to further facilitate administration of a compound.

Examples, without limitation, of excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.

As stated above, techniques for formulation and administration of compounds as medicaments may be found in "Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, PA, latest edition, which is incorporated herein by reference.

Pharmaceutical compositions of the present invention may be manufactured by processes well known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.

Pharmaceutical compositions for use in accordance with the present invention thus may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active peptides described herein into preparations which, can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.

As the peptides of the present invention are injected through a procedure for the treatment of a cardiovascular disease the peptides are formulated in formulation suitable for injection. Therefore the peptides described herein may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution,

Ringer's solution, or physiological saline buffer.

The preparations described herein may be formulated for parenteral administration, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multidose containers with optionally, an added preservative. The compositions may be suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.

Pharmaceutical compositions for parenteral administration include aqueous solutions of the active preparation in water-soluble form. Aqueous injection suspensions may contain substances, which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.

Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water, before use.

Pharmaceutical compositions suitable for use in context of the present invention include compositions wherein the active ingredients are contained in an amount effective to achieve the intended purpose. More specifically, a therapeutically effective amount means an amount of active ingredient effective to prevent, alleviate or ameliorate a condition and/or symptoms thereof and/or effects thereof.

Determination of a therapeutically effective amount is within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.

For any preparation used in the methods of the invention, the therapeutically effective amount or dose can be estimated initially from cell culture assays. For example, a dose can be formulated in animal models to achieve a circulating concentration range that includes the IC^Q as determined in cell culture. Such information can be used to more accurately determine useful doses in humans.

Toxicity and therapeutic efficacy of the peptides described herein can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., by determining the IC^Q and the LD50 (lethal dose causing death in 50 % of the tested animals) for a subject compound. The data obtained from these cell culture assays and animal studies can be used in formulating a range of dosage for use in human. The dosage may vary depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. (See e.g., Fingl, et ai, 1975, in "The Pharmacological Basis of Therapeutics", Ch. 1 p.l). The amount of a composition to be administered will, of course, be dependent on the subject being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician, etc.

The present invention further provides a process for preparing an article of manufacturing which comprises an implantable medical device and at least one peptide capable of chemoattracting endothelial progenitor cells deposited on at least a portion of the surface of the medical device, the process comprising contacting the device with at least one peptide as described herein, thereby obtaining the article of manufacture.

As discussed hereinabove, the surface of the device may further have a biocompatible matrix deposited thereon, in which case the process is further affected by depositing the matrix onto the surface of the device.

The processes of depositing onto the device's surface the peptide and the biocompatible matrix may be performed sequentially or simultaneously.

The process may further comprise applying to the surface of the device or to the biocompatible matrix deposited in the device, simultaneously or sequentially, a therapeutically effective amount of a drug for the treatment of cardiovascular diseases, as described herein.

The process may further comprise applying to the surface of the device or to the biocompatible matrix deposited on the device, simultaneously or sequentially, a therapeutically effective amount of an agent that stimulates the endothelial progenitor cells to form an endothelium on the surface of the medical device, as described herein.

Any of the peptides, matrices and additional agents described herein can be applied onto the device's surface by methods known in the art. For example, the device can be incubated in a solution or suspension containing the peptide, additional agent and/or matrix, optionally in the presence of additional reagents that promote attachment/adherence of the peptide/agent and/or matrix to the surface.

An exemplary solution includes a solvent such as, but not limited to, phosphate buffered saline.

The devices may be coated in advance or, alternatively, immediately prior to the implantation procedure.

Alternatively, a chemical cross-linking with or without a spacer-molecule can be ensured between the surface/matrix and the peptide drug and/or agent. The specific architecture of this spacer arm allows control of the biodegradability of the cross-linking and as such the pharmacokinetics of the added peptide drug and/or agent.

Further according to embodiments of the invention, there is provided a method of preparing an enriched, isolated population of endothelial progenitor cells, the method being effected by implanting an implantable medical device according to embodiments of the invention, in a body, to thereby chemoattract endothelial progenitor cells to a surface of the device; retrieving the medical device from the body; and isolating the endothelial progenitor cells from the medical device, thereby obtaining an enriched, isolated population of endothelial progenitor cells. The term "population" describes a plurality of cells, herein EPCs, with characteristic proportions in particular stages of the cell cycle

The term "enriched, isolated population of EPCs", as used herein, relates to an isolated population of cells in which the percentage of EPCs is higher than their percentage in a blood of a subject. In some embodiments, the percentage of EPCs in the isolated population is higher than in the normal blood by 10%, 1 20%, 30%, 40% and even 50%.

The term "isolated" as used herein, describes a population of EPCs which have been separated from other cells in the blood and/or body.

As used herein the term "about" refers to ± 10%. The terms "comprises", "comprising", "includes", "including", "having" and their conjugates mean "including but not limited to".

The term "consisting of means "including and limited to".

The term "consisting essentially of means that the composition, method or structure may include additional ingredients, steps and/or parts, but only if the additional ingredients, steps and/or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.

As used herein, the singular form "a", "an" and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a compound" or "at least one compound" may include a plurality of compounds, including mixtures thereof.

Throughout this application, various embodiments of this invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

Whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range. The phrases "ranging/ranges between" a first indicate number and a second indicate number and "ranging/ranges from" a first indicate number "to" a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals therebetween. As used herein the term "method" refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts. As used herein, the term "treating" includes abrogating, substantially inhibiting, slowing or reversing the progression of a condition, substantially ameliorating clinical or aesthetical symptoms of a condition or substantially preventing the appearance of clinical or aesthetical symptoms of a condition.

It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements. Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples.

EXAMPLES

Reference is now made to the following examples, which together with the above descriptions illustrate some embodiments of the invention in a non limiting fashion.

EXAMPLE l

Peptides identified by Phage Display Peptide Library screening

Peptides that potentially bind endothelial cells were identified by positive affinity selection (Le., biopanning) of a random phage display peptide library using human umbilical vein endothelial cells [HUVECs, ECs (the two abbreviations are used interchangeably throughout the document)], followed by Enzyme-Linked Immunosorbent Assay (ELISA) of positive phage clones to ECs.

Materials and Experimental Methods

Phage Display Peptide Library: The Random Phage Display Peptide Library employed in this study was purchased from New England Biolabs (NEB), Inc. (Beverly, MA, USA). The phage display library is based on a combinatorial library of random peptide 12-mers fused to a minor coat protein (pill) of M13 phage. The displayed 12- mer peptides are expressed at the N-terminus of pill. The library consists of about 2.7xlO⁹ electroporated sequences amplified once to yield 20 copies of each sequence in 10 μl of phage suspension. The phage display peptide library was screened by five rounds of positive affinity selection (biopanning) using differentially-treated ECs: a) ECs without treatment (under normoxia), b) ECs following 3 hours of hypoxia treatment, and c) ECs following 24 hours of hypoxia treatment. Each positive selection was preceded by a negative selection using human Peripheral Blood Lymphocytes (PBLs). Each round of biopanning was effected by elution of the bound phage with 0.2 M glycine-HCl and incubation of the unbound phages on the second EC plate. This procedure was executed three times. Phages of the three elution steps were pooled for the second round of biopanning and so on. After the fifth round of biopanning, 40 individual clones from each group of cells screened were isolated so that, in all, 120 individual clones were obtained.

Human Umbilical Vein Endothelial Cells (HUVECs): HUVECs (ECs), were isolated by Collagenase digestion as previously described [Jaffe et al, J. Clin. Invest., 52(ll):2757-64, 1973]. ECs were cultured with M199 supplemented with 20% FCS, 10⁴ units of penicillin, 10 mg/ml of streptomycin sulfate, 10 mg/ml of neomycin sulfate (Biological Industries, Kibbutz Beit Haemek, Israel), 25 μg/ml of EC growth supplement (Biomedical Technologies, Inc., Stoughton, MA, USA), and 5 U/ml of Heparin (SIGMA, Rehovot, Israel). HUVECs were harvested with Trypsin (0.25%), EDTA (0.05%, Biological Industries, Kibbutz Beit Haemek, Israel) and incubated on 60 mm petri dishes coated with 1% gelatin for 24 hours. Following incubation, cells were washed and incubated with M199 supplemented with 10% FCS. ECs were subjected to four different treatments: a) no treatment, b) 3 hours of hypoxia, c) 6 hours of hypoxia, or d) 24 hours of hypoxia. Subsequently, monolayers were washed with PBS and dried overnight. Cells were rehydrated with PBS containing 5% FCS and 0.1% sodium azide and maintained at 4 ⁰C until biopanning.

Hypoxia treatment: ECs were subjected to hypoxia for 3, 6, or 24 hours in a gas mixture containing 94% N₂, 5% CO₂, and 1% O₂ in a hypoxia chamber (Billups- Rothenberg, Delmar, CA, USA).

Screening of positive clones by ELISA: The binding of positive clones from each group was re-evaluated by ELISA. For this purpose, ECs under normoxic conditions, ECs following 3, 6, or 24 hours of hypoxia, or human PBL (as controls) were plated at 20 x 10³ cells/well on 96 well plates. Plates were re-hydrated by overnight incubation at 4 ⁰C in PBS supplemented with 3% BSA, followed by three washes with PBS. Phages from each of the 120 clones isolated were dispersed on the ELISA plates at concentrations of 10¹⁰, 10⁹, or 10⁸ phage per well and incubated for two hours at room temperature. Plates were then washed three times with PBS containing 0.05% Tween, followed by three washes with double distilled water (DDW). After washing, an anti-M13 HRP antibody (Amersham Pharmacia Biotech UK Limited, Buckinghamshire, UK) at a 1:5,000 dilution was added and incubated for 2 hours at room temperature ⁰C, following which the plates were washed 5 times in the presence of PBS and 0.05% Tween-20. The HRP reaction was carried out using 100 μl of tetramethyl benzidine liquid substrate (DAKO TMB substrate chromogen, DAKO Corporation, Carpinteria, CA, USA) for a 15 minute-incubation following which the reaction was terminated by the addition of 50 μl of 1 M HCl. Plates were read at 450 nm in an ELISA reader (SLT 400ATC, SLT LAB Instruments, , Austria).

Statistical and graphical methods: Statistical analysis was effected by analysis of variance (ANOVA) with appropriate post-hoc tests, generally Dunnett's, for a comparison to a control or Tukey-Kramer HSD for multiple comparisons. Results were considered statistically significant at P < 0.05. Results

EC-binding peptides were selected using phage display peptide library: A phage display peptide library was subjected to five rounds of positive affinity selections (biopanning) using ECs under physiological conditions (i.e., normoxia) or following hypoxia. The second step of selection of peptide-presenting phage was effected by ELISA using ECs and lymphocyte-coated plates as controls. Fifteen different peptide- presenting phages at a concentration of 10⁹ (Figure IA) and 10¹⁰ (Figure IB) phages per well were screened by ELISA on four different EC preparations (ECs at normoxic conditions and ECs following 3, 6, and 24 hours of hypoxia). Figure IA and Table 1 hereinbelow, illustrate selected peptides which exhibited statistically significant differences (p < 0.05) between binding of NO phage (Le., unmodified M13 phage) and binding of certain peptide-presenting phages as determined using ANOVA analysis of 10⁹ selected phage indicated.

Table 1 Peptide-presenting phage

Peptide-presenting phage selected from EC (ECs at normoxic conditions), H3 (ECs following 3 hours of hypoxia), H6 (ECs following 6 hours of hypoxia), and H24 (ECs following 24 hours of hypoxia). P < 0.05.

Similarly, statistically significant peptides which were selected using ANOVA analysis performed on 10¹⁰ phages (p < 0.05) are shown in Figure IB.

Altogether, these findings demonstrate the identification of specific peptide- presenting phages which are capable of specifically binding ECs under either normoxia or hypoxia.

EXAMPLE 2 Chemical synthesis of EC and EPC binding proteins derived from the ADAM 15 protein

Materials and Experimental Methods Peptide synthesis: ADOPepl, ADOPep2 and ADOPep3 (SEQ ID NOs: 1, 16 and

17, respectively), were synthesized by SynPep (Dublin, CA, USA) according to the ADAM15 amino acid sequence (GenBank Accession No. Q13444). HPLC purity was greater than 97%. The peptides were dissolved in water in a concentration of 1 mg/ml.

Results A 4 amino-acid sequence, HWRR, was identified as a common motif present on

ADOPepl, 2 and 3 ADAM15 derived peptides, that binds to the GRP78 receptor on EC (Table 2, hereinbelow).

Table 2 Amino acid sequences of the ADOPeptides of the invention

The bolded text in each peptide amino acid sequence corresponds to the common 4- amino acid motif HWRR (SEQ ID NO:13). The common motif HWRR (SEQ ID NO: 13) of all AdoPeps was tested for its ability to bind and/or chemoattract endothelial cells. To test if the 5 amino acid motifs HWRRP (motif A; SEQ ID NO:2), HWRRA

(motif B; SEQ ID NO: 15) or AHLLP (motif C; SEQ ID NO: 18) can bind to the GRP78 on EC, synthetic peptide having amino acid sequences corresponding to SEQ ID NOs:2, 15 and 18 were used in a competitive assay for the binding of bio tiny lated ADOPepl to endothelial cells. As shown in Figure 2, while the peptides having amino acid sequence corresponding to Motif A and B exhibited a significant inhibition of the binding of ADOPepl^Blot to endothelial cells, the peptide corresponding to Motif C exhibited a moderate inhibition of binding.

In addition, endothelial cells were incubated under hypoxia conditions in the absence or presence of ADOPepl, Motif A or C and the Ql level of apoptosis was determined using FACS analysis. As shown in Figures 3A-D and 4 while ADOPepl and Motif A and B peptides were capable of inhibiting the hypoxia induced apoptosis, the motif C peptide exhibited no effect on hypoxia induced apoptosis.

To further test the biological activity of motifs A, B, C on endothelial cells, a migration assay was performed on EC under hypoxia. As shown in Figures 5A-B, while the ADOPepl, Motif A and B peptides induced endothelial cell migration at a concentration of about 10 ng/ml, motif C peptide exhibited no significant effect on endothelial cell migration.

Altogether, these results demonstrate the identification of minimal amino acid sequences from the ADOPeps which are capable of decreasing hypoxia induced apoptosis and inducing endothelial cell migration. These peptides (e.g., SEQ ID NOs:2 and 15) can be used to inhibit hypoxia induced apoptosis, induce angiogenesis and treat ischemic diseases.

EXAMPLE 3 ACTIVITY ASSAYS Exemplary peptides were selected for testing their ability to bind and chemoattract EPCs. These exemplary peptides, according to some embodiments of the present invention, included the ADAM15-derived 12 amino acid peptides, ADOPepl, ADOPep2, ADOPep3 as set forth by SEQ ID NOs: 1, 16, 17, a HWRR containing 5 amino acid peptide MotifA as set forth by SEQ ID NO:2 as well as one exemplary peptide, RoY, identified by Phage Display Peptide Library screening as set forth by SEQ ID NO: 3. Materials and Experimental Methods

Immune precipitation (IP) experiments of endothelial cells with biotinylated ADOPepl:

Preparation of HUVEC lysates: HUVEC were seeded in 90 mm Petri dishes plates for 24 hours with complete Endothelial Cell Growth Medium (PromoCell. Heildelberg, Germany). Cells were washed twice with PBS and buffer lysate (50 mM Tris Cl (pH 8, 150 mM Na Cl, 0.02% Na azide, 0.1% SDS, 100 μg/ml PMSF, 1 μg/ml protease inhibitors, 1% NP-40) was added for 20 minutes on ice. After incubation, cells were scraped with a rubber policeman and transferred to a chilled microfuge tube. After centrifugation of lysate at 12,000 g for 2 minutes at 4 ⁰C, supernatant was transferred to a fresh microfuge tube and stored at -70 ⁰C.

Immunoprecipitation (IP): Aliquots samples of lysates (500 μl) were incubated with 10 μg of biotinylated ADOPepl for one hour at 4 ⁰C by gently mixing. Strep tavidin Sepharose beads (Amersham Biosciences, Uppsla, Sweeden) (50 μl) were added to the sample for a second incubation one hour at 4 ⁰C by gently mixing. After then, sample was centrifuge for 20 seconds at 12,000 g and the pellet was saved and further washed 3 times. Finally the pellet was suspended in 100 μl of sample buffer for Western blot analysis. For ELISA, protein was eluted by 5 minutes incubation of the sample at room temperature in 1 ml of Tris-Glycin 0.2 M (pH 2.2), followed by titration with 1 M Tris (pH 9.1). Polyacrylamide Gel Electrophoresis (PAGE) analysis: The protein band containing GRP78 which was immunoprecipitated with the biotinylated ADOPepl (GRP78 immune-precipitated protein) (30 μl) was applied to minigel lane and run at standard conditions (60 mA for 2 minigels, 1.4 hours) in a 10% Tris Acrylamide gel. Gel was stained with Coomassie blue. Band was cut and sent to mass-spectroscopy for protein identification.

Western Blot analysis: Following PAGE analysis, proteins transfer was performed for 2 hours in wet conditions at 40 V in a nitrocellulose membrane, following which the gel was stained with Coomassie blue. The nitrocellulose membrane was blocked for 2 hours at room temperature by incubation with PBS containing 0.5% Tween-20 and 5% non-fat milk. Incubation of the membrane with biotinylated ADOPepl (5 μg/ml in PBS-Tween) was performed over night at 4 ⁰C by gently shaking. After then, membrane was washed 3 times (15 minutes each) in PBS-Tween. Incubation with the secondary antibody Peroxidase-conjugated Streptavidin (1 μg/ml) (JacksonlmmunoResearch, PA, CA USA) was performed for 45 minutes at room temperature followed by 3 washes (15 minutes each) in PBS-Tween. For GRP78 immunostaining, the membrane was blocked as described hereinabove and further incubated for 24 hours at 4 ⁰C with anti-GRP78 antibody (Santa Cruz Biotechnologies, CA, USA) at a concentration of 2 micrograms per ml, followed by 3 washes with PBS- Tween. Incubation with a secondary anti-goat FITC (Jackson ImmunoResearch Laboratories, PA, USA) at a dilution of 1:5000 was performed for 45 minutes at room temperature, followed by 3 washes in PBS-Tween. ECL was performed using SuperSignal West Pico Chemiluminescent Substrate (Pierce, IL, USA).

FACS analysis of and GRP78 binding to Endothelial Cells: HUVEC were harvested by trypsin and 100,000 cells per sample were suspended in PBS + 5% FCS + 0.1% Na azide. Goat polyclonal anti GRP78 (Santa Cruz Biotechnologies, CA, USA) at a concentration of 1 μg/100,000 cells was added for 40 minutes on ice. Cells were washed and stained with anti-goat Jr¹IlC (Jackson ImmunoResearch Laboratories, PA, USA). Samples were analyzed using a fluorescence activated cell sorter (FACScan Beckton Dickinson, CA, USA).

Competitive binding of ADAMl 5 derived peptides ADOPepl, ADOPepl and ADOPep3 to GRP78 receptor: Endothelial cells (20,000 per well) were seeded in 96 well plates for 24 hours in the presence of the complete medium. Plates were washed with PBS over night and rehydrated with PBS, 0.1% Na Azide and 5% FCS. ADOPepl, ADOPep2 and ADOPep3 were added to washed plates for 2 hours at room temperature at concentrations of 0.01, 0.1 and 1 microgram per ml. Anti-GRP78 antibody (goat polyclona IgG, Santa Cruz Biotechnology, CA, USA) was added to the plates for 1 hour at room temperature at a concentration of 2 micrograms/ml per well. After washing, bound anti-GRP78 antibody was detected by incubation with anti-goat IgG Peroxidase conjugated (Jackson Immuneresearch Laboratories, PA, USA). After 5 washes with PBS-0.1% Tween 20, 100 μl/well of TMB+ Substrate-Chromogen (DAKOCytomation, CA, USA) was added for a maximum of 30 minutes. Reaction was stopped with 1 N HCl. Color developed was determined by an ELISA reader at 450 nm. Peripheral Blood Mononuclear cells or Cord Blood Mononuclear cells isolation: The most commonly used method to obtain EPCs is cultures of unprocessed cord or peripheral mononuclear cells by adherence of the cells to fibronectin coated culture plate.

Peripheral blood mononuclear cells (PBMC) were isolated from 20 ml of peripheral blood anticoagulated with heparin from healthy volunteers (mean age 49.7±10 years), counted and incubated in 75 cm² tissue culture flasks coated with 0.1 mg/ml fibronectin (Biological Industries, Beit HaEmek, Israel) at a density of 1 x 10⁶/ml.

Cord Blood Mononuclear cells (CBMC) were isolated from 20 ml human cord blood by density gradient centrifugation over histopaque-10771 (1.077g/ml, Sigma) according to the manufacturer's protocol. After centrifugation for 30 minutes at 1600 rpm, cells were removed and transferred to a new conical tube.

All blood samples were obtained, processed, and analyzed individually in independent experiments. The residual Ficoll in the solution containing the PBMC was washed away with PBS, and the cells were then adjusted to a density of 10 x 10⁶ cells/ml in Endothelial Cell Growth Medium (PromoCell, Germany) without supplements. 1 x 10⁶ cells per sample in 100 microliter PBS were used for identification of endothelial progenitor cells markers CD34, CD 133, CD31 and KDR by flow cytometry. Endothelial Progenitor Cells (EPC) cultivation:

Cord Blood Mononuclear cells or peripheral blood mononuclear cells were incubated in endothelial cell growth media supplemented with Hydrocortisone at 1 microgram/ml, Gentamycine at 50 microgram/ml and 5% Fetal Calf Serum in a humidified 5% CO₂ incubator at 37 ⁰C in the presence of the following peptides: ADoPepl (SEQ ID NO: 1) at 10, 50 and 100 ng/ml, RoY (SEQ ID NO:3) at 50 ng/ml, or Motif A (SEQ ID NO:2) at 50 ng/ml for 7 days. After 3 days of culture, non-adherent cells were removed by washing with PBS, and adherent cells were maintained in new media with or without the presence of peptides for a total of 7 days followed by FACS analysis of EPC surface markers. FACS analysis of EPC:

EPC are a heterogeneous group of cells that can be characterized by the expression of surface markers, such as CD31, CD34, CD133 and VEGFR-2 (KDR or Hk-I).

Fluorescence-activated cell sorting (FACS) analysis was performed in order to detect the expression level of EPC surface markers, on adherent cells, after 7 days in culture on fibronectin coated plates. After gently detaching the cells with trypsin, cells were washed with PBS. Cells (2 x 10⁵) were incubated in FACS buffer (PBS with 5% FCS and 0.1% sodium azide) for 30 minutes at 4 ⁰C in the presence of anti-CD45- Fluorescein isothiocyanate (FITC) and anti-CD34-phycoerythrin (PE) (both from DAKO cytomation), anti-CD133-PE (Miltenyi Biotec, Auburn, CA) and anti-CD31- FITC and anti-KDR- FITC (both from Santa Cruz Biotechnologies, USA). A FITC conjugated IgG antibody or a PE conjugated IgG antibody served as isotype control. After staining, cells were washed with PBS for FACS analysis and quantitative fluorescence analysis was performed with a FACS-Calibur instrument and Cellquest Software (Becton-Dickinson). Three samples from different individuals were used in this test. The results are presented as mean±S.E. Cell Migration assay:

Cell migration was measured by using 24 well-modified Boyden chambers ThinCert (greiner bio-one, Germany) with 8 micrometerpore-size filters. Peptides ADoPepl, Motif A, RoY, were diluted at lOOng/ml in Endothelial Cell Growth Medium (PromoCell, Germany) without supplements and 300 microliters were placed in the lower compartment of a Boyden chamber. Cells were suspended in 200 microliter of medium at a density of 1 x 10⁶/ml and placed in the upper compartment in duplicates. After overnight incubation at 37 ⁰C in 5% CO₂ the cells that had not migrated were removed from the upper surface of the filters and those that migrated were collected for identification of EPC surface markers by FACS analysis. XTT determination of EPC bound to a peptide coated nitrocellulose sheet:

Peptides ADoPepl or Motif A, at 2 micrograms per ml, in Carbonate buffer pH 9.1, were incubated overnight in a 15 ml tube with a piece of 0.5 cm² nitrocellulose sheet. The nitrocellulose sheet was removed from the carbonate buffer and washed 3 times with PBS, followed by blocking with PBS-BSA 10 mg/ml for 2 hours at room temperature.

The nitrocellulose sheet was incubated with biotinylated ADoPepl peptide and the quantity of bound peptide to nitrocellulose was determined using the ELISA technique at 450 nm. The Nitrocellulose piece was washed with PBS and cut in 1 mm pieces.

The nitrocellulose pieces were inserted in 96 well plates. 1 x 10⁵ CBMC after Ficoll Hipaque density separation were added to the wells for 24 hours. After 24 hours the nitrocellulose pieces were removed and inserted into new wells of the 96 well-plate. XTT assay (Biological Industries, Beith Ha Emek, Israel) was performed following manufacturer instructions. Briefly, XTT solutions were added to the wells which previously contained the peptide coated nitrocellulose pieces (for assessment of unbound CMBCs) and to wells containing the peptide coated nitrocellulose pieces (for assessment of bound CMBCs) and the quantity of bound/unbound cells was determined by an ELISA reader at a wave length of 450nm.

Results

ADOPepl, ADOPep2, ADOPep3 and Motif A, bind the endothelial receptor GRP78: Immune precipitation (EP) of endothelial cells lysate with biotinylated ADOPepl was analyzed by PAGE. As can be seen in Figures 6A-B, following immunoprecipitation with ADOPepl a major single protein band is present at 78 kDa. In order to confirm that this band is the ADOPepl peptide binding receptor, the separated proteins were transferred to a nitrocellulose membrane which was further stained with biotinylated ADOPepl followed by Chemiluminescent Substrate. As can be seen in Figure 7, indeed the same band was stained by the labeled peptide in two different experiments (both lanes in the PAGE shown in Figure 7). The protein receptor band was cut from the gel and analyzed by mass-spectrometry.

The results of mass-spectrometry are presented in Table 3 hereinbelow. The receptor was identified as the glucose-regulated protein [Homo sapiens] GRP78 protein (GenBank Accession No. CAB71335; Gi: 6900104) with 22 peptides digested from the isolated band. Table 3

IP with ADOPepl

To further confirm that indeed the ADOPepl receptor on endothelial cells is the GRP78 protein, the IP experiment was repeated and Western blot analysis was performed using an anti GRP78 antibody. Results presented in Figure 8 demonstrate the positive staining with anti-GRP78. Competitive binding of ADAMlS derived peptides ADOPepl, ADOPep2 and ADOPep3 to GRP78 receptor:

To further test if the peptides of the invention bind to the GRP78 receptor on EC, a competitive binding assay was performed on EC which were incubated with the ADOPeps-1, 2 and 3, exemplary peptides according to embodiments of the present invention as set forth by SEO ID NOs: 1, 16 and 17, prior to binding with the anti-

GRP78 antibody. As demonstrated in Figure 9 (which represents a summary of 4 experiments), all 3 ADOPeps (i.e., ADOPepl, ADOPep2 and ADOPep3) show some degree of inhibition of binding of anti-GRP78 antibody to EC while scrambled peptide (sRoY) was not effective in the competitive binding to the receptor on EC.

AdoPepl and Motif A peptides compete on the binding to the same receptor on endothelial cells:

As shown in Figures 10 A-D, AdoPepl and Motif A, exemplary peptides according to embodiments of the present invention as set forth by SEQ ID NOs: 1 and 2, inhibited anti GRP78 binding to endothelial cells under hypoxic conditions. Ten micrograms of AdoPepl and Motif A inhibited anti GRP78 binding in approximately 80% and 60% respectively, while motif C, a control peptide which does not contain the conserved motif HWRR (having the amino acid sequence of AHLLP; SEQ ID NO: 18) did not inhibit anti GRP78 binding to endothelial cells. Ability of exemplary peptides Ro Y, AdoPepl and Motif A, to recruit EPCs:

The ability of RoY, AdoPepl and MotifA, to promote endothelial cell progenitor cells (EPCs) propagation and recruitment from cord and adult human peripheral blood was studied.

Increase in percent EPCs in cord blood mononuclear cells cultured with peptides: EPC are a heterogeneous group of cells that can be characterized by the expression of surface markers, such as CD31, CD34, CD133 and VEGFR-2 (KDR or FIk-I).

Expression of the marker CD34 is also found on a lower level on mature endothelial cells, and the search for more specific stem cell markers led to the discovery of CD133 (also termed AC133), which is highly expressed on immature stem cells (but whose expression is lost during the differentiation to mature endothelial cells.

In order to study the binding of peptides RoY, AdoPepl and MotifA to EPCs, the percent of EPCs in a population of cord blood mononuclear cells, cultured on fibronectin coated plates, in the presence/absence of the peptides was determined and compared. The EPC percent was quantified, after one week in culture, by detecting the percent of CD34, CD31 and CD133/CD31 positive cells using FACS analysis. As can be seen in Figure 11, RoY peptide induced a 51%, 67% and 39% increase in CD34, CD31 and CD133/CD31 positive cells, respectively, in the population of cord blood mononuclear cells.

Motif A induced an increase only in CD34 positive cells (45%) and not in CD133/CD31 positive cells, suggesting the propagation of a more mature progenitor cell subpopulation.

The effect of different concentrations of peptide ADoPepl on human cord blood EPC, after one week in culture, is presented in figures 12A and 12B. AdoPepl was added to cultures in increasing concentrations of from 10 to 100 ngrams per ml. At a concentration of 10 ngrams per ml, AdoPepl induced a 86% increase in CD34/KDR positive cells and 80% increase in the CD133 positive cells (see Figure 12A and 12B respectively).

The capacity of the peptides to induce EPC migration:

In order to study the level of chemoattraction of EPCs to RoY, AdoPepl and

MotifA, the ability of the peptides to induce EPCs migration through an 8 micrometer porous membrane was analyzed using Boyden chambers, in which cord or peripheral blood mononuclear cells were plated across an 8 micrometer membrane from the peptide and the migrating CD34 positive cells were collected and analyzed by FACS.

As shown in Figure 13, in the case of cord blood mononuclear cells, both RoY and AdoPep 1 induced the migration of CD34 positive cells, although their percent in the cell population remained similar to that before migration. The percent of CD34 positive cells migrating towards MotifA, was lower than in the control cell population.

As shown in Figures 14A and 14B, in the case of peripheral blood mononuclear cells, the percent of CD34 positive cells in the population of cells migrating towards AdoPepl was increased 4 folds from 0.2% to 0.81% (see, Figure 14A) and the percent of CD133/CD31 positive cells increased 9 folds from 1.4% to 13% (see, Figure 14B).

These data indicate that AdoPep 1 induces a selected and preferred migration and concentration of EPCs and might therefore be used as a specific EPCs chemoattractant. In contrast to AdoPep 1, the RoY and MotifA induced migration of human peripheral blood mononuclear cells was similar to the migration of control cells (incubated without any peptide) and the concentration of EPCs was similar to that of in the control cells population. Binding of peptides to solid matrix does not interfere with EPC chemoattraction:

In order to test whether coating of AdoPep 1 to a solid matrix might induce its EPC binding, a nitrocellulose sheet was coated with AdoPep 1 and Motif A and placed in a multiwell plate and the ability of mononuclear cells to bind the sheet was determined.

Figure 15 demonstrates that the coating of peptides onto a nitrocellulose membrane does not interfere with the ability to bind EPCs from cord blood mononuclear cells as determined by the significantly higher number of cells binding to the peptide coated membrane as compared to the extent of cell binding to control membranes which were not coated with any peptide.

The experimental data presented herein show that RoY, AdoPep 1 and MotifA induced an increase in percentage of EPCs in a population of cord mononuclear cell population, after one week incubation with the peptides. Induction of migration of EPCs from cord blood was obtained by both RoY and AdoPepl peptides (12 amino acid peptides). In addition, ADoPepl was also able to selectively recruit EPCs from adult human peripheral blood and thus lead to an EPC enrichment of the cell population. It has also been demonstrated that the ability of Motif A (a 5 amino acid peptide) and AdoPepl to bind EPCs, when attached to a solid matrix, was preserved..

The obtained data clearly indicates that percutaneous therapy of coronary and other vascular artery diseases by peptide bound-stent devices utilizing the peptides described herein can be used for prevention of early and late in-stent thrombosis, by promoting rapid endotheliazation at stent deployment sites. EXAMPLE 4 membrane GRP78 expression on smooth muscle cell and fibroblasts and Lack of migration of smooth muscle cells toward AdoPepl

Materials and Experimental Methods

Detection of membrane GRP8 on Human smooth muscle cells using FACS:

Human smooth muscle cells were isolated from human umbilical cords. Cells were incubated in DMEM medium supplemented with fetal bovine serum and antibiotics in 25cm² flasks. Cells were harvested with trypsin and samples of 100,000 cells were stained with polyclonal rabbit anti GRP78 for 30 minutes at 4 ⁰C followed by anti rabbit FITC for additional 30 minutes. Cells were analyzed by FACScalibur (Becton Dickinson).

Detection of membrane GRP8 on Human fibroblasts using FACS: Fibroblasts were obtained from human gingival tissue. Cells were incubated in DMEM medium supplemented with fetal bovine serum and antibiotics in 25 cm² flasks. For FACS analysis, cells were harvested with trypsin and samples of 100,000 cells were stained with polyclonal rabbit anti GRP78 for 30 minutes at 4 ⁰C followed by anti rabbit FITC for an additional 30 minutes. Cells were analyzed by FACScalibur (Becton Dickinson). Smooth muscle cells Migration assay:

Cell migration was measured by using 24 well-modified Boyden chambers ThinCert (greiner bio-one, Germany) with 8 micrometerpore-size filters. Cells (50,000/well) were placed in the upper compartment in serum-free DMEM medium. Medium containing ADoPepl peptide (100 ng/ml) was added to the lower compartment. Medium containing no peptide served as control. After 14 hours of incubation, the medium was removed, a Cells that did not migrate were removed from the upper surface of the filters and those that migrated were collected and transferred in duplicates to a 96 well plate. An XTT assay (Biological Industries, Beith Ha Emek, Israel) was performed following manufacturer instructions. Briefly, XTT solutions were added to the wells and the quantity of cells was determined by an ELISA reader at a wave length of 450 nm. Results

The results are presented in Figures 16-18 and show that while 30 % of smooth muscles cell population express GRP78 on their membrane (see, Figure 16) human fibroblasts do not (see, Figure 17). Incubation of the fibroblasts under hypoxic conditions did not induce an expression of membrane GRP78.

Although smooth muscle cells express GRP78, these cells do not migrate toward AdoPepl peptide, as could be deduced from the similar amount of muscle cells migrating toward AdoPepl and toward the control (without peptide; see, Figure 18)

EXAMPLE 5

Chemoattraction of EPCs by AdoPepl

Materials and Experimental Methods

Evaluation ofCD133 and GRP78 expression in EPCs:

Human peripheral blood mononuclear cells (PBMC) were obtained from four healthy donors (PMBCl, PMBC2, PMBC3 and PMBC4). To elucidate if EPCs from these cells express GRP78 on their surface, the PMBC from each of the four donors were double labeled with CD133 and GRP78 fluorescent antibodies and analyzed using

FACS (FACScalibur (Becton Dickinson)).

Isolation ofCD133+ and CD133^" subpopulation: The CD 133 positive cells were then isolatedfrom each of the four PMBQusing anti-CD133 magnetic beads in 90 % purity. The separated CD133⁺ cells were double stained with anti CD133 and anti GRP78 antibodies. Migration assay:

PBMC were separated to CD133 positive and CD133 negative cell subpopulations using anti CD133 magnetic beads. The two subpopulations were tested for migration in a Boyden chamber in the presence or absence Adopepl peptide as the chemoattractant. Cells that migrated were analyzed first by FACS to determine percent of GRP78 positive cells and secondly, by the XTT assay, to estimate the number of migrating cells. Results

Stem cells (CD133⁺) isolated from human peripheral blood express GRP78 on their surface:

As discussed hereinabove, CD 133 is highly expressed on immature stem cells such as EPCs, but is not expressed upon differentiation of stem cells to mature endothelial cells). Therefore, CD133⁺ expression was used as a marker for EPC type cells.

The level of GRP78 expression in cells expressing CD133 (i.e. EPCs) was assessed by double labeling_Human peripheral blood mononuclear cells (PMBC) from four healthy donors with anti-CD133 and anti-GRP78 antibodies. The results are presented in Figure 19 and demonstrate that 75 %, 43 %, 91 %, and 86 % of the CD133 positive cells from each of the four donors also expressed GRP78 (See, Figure 19A). Similar results were obtained after isolation to CD133 positive and CD133 negative cell subpopulation with 35-75 % of the CD133⁺ cells also expressing GRP78 on their surface (see, Figure 19B). A representative FACS analysis dot plot for the anti-GRP78 (bottom axis)/anti-CD133 (left axis) staining of PMBCl and PMBC2 groups is shown in Figure 19C, and demonstrate that 75 % and 35 % of the cells respectively express both CD133 and GRP78 proteins.

Chemoattraction of CDl 33* stem cell migration by AdoPepl: The ability of CD133⁺/GRP78⁺ cell subpopulation, as compared to CD133^"

/GRP78⁺ subpopulation, to migrate toward AdoPepl, was studied. The results are presented in Figures 20A-C and show that the percent of CD133⁺/GRP78⁺ migrating cells was 2.3 and 4.6 fold higher than the percent of CD133^"/GRP78⁺ with or without the peptide respectively (see, Figure 20A). A superior ability of AdoPepl in promoting the migration of immature endothelial cells (CD 133 positive cells), as compared to mature endothelial cells (CD133 negative cells), was determined (see, Figure 20B). A representative FACS analysis dot plot for the anti-GRP78-FITC (bottom axis)/anti- CD133 (left axis) staining of the CD133 positive and CD133 negative cell subpopulations that migrated toward the AdoPepl peptide or toward the control (no peptide) is shown in Figure 2OC.

These results suggest that AdoPepl is capable of chemoattracting EPCs (CD133 positive cells) to a greater extent as compared to ECs (CD133 negative cells). EXAMPLE 6 AdoPepl protective effect against hypoxia-induced apoptosis in cardiomyocytes The ability of AdoPepl to reduce apoptosis in cardiomyocytes exposed to hypoxic conditions was tested. The involvement of the protein GRP78 in the AdoPepl cardioprotective effect was also investigated.

Material and Methods Cardiomyocytes Cultures: Primary cardiomyocyte cultures were prepared from neonatal (1-3 days old)

C57BL mouse hearts as previously described [Sreejit P, Kumar S and Verma RS. 2008 In Vitro Cell.Dev.Biol. Animal;,44:45-50]. Briefly, the ventricles were digested with RDB proteolytic enzyme (Biological Institute, Ness-Ziona ,Israel). Cells were then resuspended in complete Dulbecco's Modified Eagle Medium (DMEM, Biological Industries, Beit Haemek, Israel) containing 10 % horse serum and antibiotics. To remove fibroblasts, isolated cells were first filtered through a 70μm cell strainer (Millipore, Billerica, MA, USA) and then pre-plated on uncoated dishes for 40 min. Myocardial cells in the supernatant were then centrifuged and plated on 1 % gelatin- Collagen coated Petri-dishes at a concentration of 5XlO⁵CeIIsZmI in complete medium. By this procedure, the cultures were composed of approximately 96 % cardiac muscle cells. Beating cells were observed starting at day 3 post plating of cells. Experiments under starvation conditions, were performed using medium without the 10 % horse serum.

Determination of Cardiomyocyte membrane GRP78 by FACS analysis: Cultures of cardiomyocytes were incubated under normoxic conditions in a humidified incubator with 95 % air and 5 % CO² at 37 ⁰C, or under hypoxic conditions in a hypoxia chamber (Billups-Rothenberg, Del Mar, CA, USA) exposed to a gas mix of 94 % Nitrogen, 5 % CCh and 1 % O² for 4 hours. Cells were incubated with or without the addition of 20ng/ml of ADoPepl 14 hours before hypoxia, during hypoxia or 14 hours post hypoxia. Cells were removed for fluorocytometric analysis with trypsin- EDTA (SIGMA, Rehovot, Israel) and stained with anti GRP78 polyclonal antibody (Santa Cruz, Biotechnologies, CA, USA) followed by anti-goat FITC. Isotype control was determined by anti-goat FITC for non specific binding. Percent GRP78 positive cardiomyocytes was analyzed by fluorescence cell sorter (FACScan Beckton Dickinson, San Jose, CA₅USA,). Results were expressed as mean of percent membrane positive cells ±SE in 5 different experiments. Determination of total GRP78 protein in cardiomyocytes by western blot analysis:

Cardiomyocytes were incubated with 20 ng/ml ADoPepl under starvation conditions for 14 hours and 4 hours hypoxia. For determination of total GRP78 protein, cells were lysed in lysis buffer [50 mM Tris HCl (pH 7.5), 120 mM NaCl, 1 % (v/v) Nonidet P-40, 25mM sodium fluoride, ImM sodium pyrophosphate, 0.1 mM sodium orthovanadate, ImM PMSF, ImM benzamidine, and leupeptin (20 μg/ml).)]. Proteins were separated by SDS-PAGE gel electrophoresis and transferred onto nitrocellulose membranes. Blots were incubated with anti GRP78 polyclonal antibody (Santa Cruz, Biotechnologies, CA, USA). To verify equal protein loading, blots were also stained with mouse monoclonal anti-Actin (MP Biomedicals, Illkirch, France). Protein bands were detected with SuperSignal Ultra Chemiluminescent Substrate (Pierce, Thermo Scientific, IL, USA) on X-ray film and densitometry ratio (GRP78/Actin) was determined in 3 different experiments.

Determination of Cardiomyocyte GRP78 gene expression by semi quantitative Reverse transcriptase (RT) PCR:

Cardiomyocytes were incubated with 20 ng/ml ADoPepl in starvation conditions for 14 hours followed by 4 hours of hypoxia. Total RNA was extracted from cultures with EZRNA reagent (Biological Industries, Beit Haemek, Israel). First strand cDNA was prepared from 1.5 μg total RNA with random primers using Superscript II First-Strand Synthesis System for RT-PCR. Semi quantative RT-PCR was performed using specific primers to target GRP78 gene: GRP78 Foward 5' aagcagatgcagcaagatcc, Reverse 5' tgtagatgctgccattgttcg. Each PCR reaction was performed in a 50μl reaction mixture containing 20ng of cDNA and lOpmol primers. The PCR mixture was amplified with the following profile: an initial denaturation at 94 ⁰C for 5 minutes, denaturating at 94⁰C for 45 seconds, annealing at 58⁰C for 1 minute and extension at 72 ⁰C for 1 minute. The RT-PCR products obtained by electrophoresis and stained by Ethidium bromide, were detected under UV light, and photographed. The amount of PCR product was quantitatively determined by measuring the density of the specific bands and normalizing with GAPDH as reference genes in 3 different experiments. Determination of hypoxia-induced apoptosis in cardiomyocytes: Cardiomyocytes in 5 different experiments were divided into 5 groups; Cultures were incubated under normoxic conditions for 14 hours in starvation conditions (group 1) or followed by 4 hours exposure to hypoxia (group 2). ADoPepl was added to cultures at 20ng/ml either at the 14 hours before hypoxia (group 3), during the 4 hours of hypoxia (group 4) or after hypoxia for 14 hours incubation (group 5). For the determination of apoptosis by FACS analysis, cardiomyocytes were harvested and stained with Annexin V-FITC kit (Mebcyto Apoptosis kit, MBL, Naka-Ku Nagoya, Japan) following the manufacturer's instructions. Cells were analyzed using a fluorescence activated cell sorter (FACScan Beckton Dickinson, San Jose, CA, USA). Cells were double stained with the fluorescent markers Annexin-FTTC and Propidium iodide (PI). The Annexin- V-positive and Pi-negative cells were scored as apoptotic cells.

Transfection of cardiomyocytes with Small Interfering RNA (siRNA) Oligomers:

RNA interference of GRP78 was induced with small interfering RNA (siRNA) directed against the mouse GRP78 mRNA. A pool of three different nucleotide siRNA specific primers were use to target human GRP78 mRNA sequence. The siRNAs started at position 1198 CUCGGGCCAAAUUUGAAGAtt (SEQ ID No: 47), 1829 GACACCUGAAGAAAUUGAAtt (SEQ ID No: 48) and 2163 GAAGAGGAUACAUCAGAAAtt (SEQ ID No: 49). A scrambled siRNA from position 1567 was used as negative control. All oligonucleotides were obtained from Santa Cruz Biotechnology (CA, USA). Cardiomiocytes were seeded at a density of 100,000 cells/cm² the day before transfection and were 60 % confluent when they were transfected with 5, 20 and 50 nmol/L positive or 50 nM scramble oligonucleotides in Opti-MEM 1 without serum and antibiotics and Oligofectamine Reagent (Invitogen, CA, USA). Before transfection, the cells were washed once with media without serum and antibiotics, transfected for 4 hours and serum was added. The reduction in GRP78 protein was estimated by Western blot analysis, 48 hours after transfection. Transfected cardiomyocytes with 50 nM GRP78 siRNA were preincubated with the peptide and exposed to 4 hours hypoxia for the determination of membrane GRP78, percent Annexin V positive cells (as described hereinabove) and Caspase 3/7 activity. Caspase enzymes participate in a series of reactions that are triggered in response to proapoptotic signals and result in the cleavage of protein substrates. Caspase enzymes specifically recognize a 4-5 amino acid sequence on the target substrate, which include an aspartic acid residue which is the target for cleavage. Caspase 3/7 activity was determined in transfected cells by FACS analysis using CaspaTag Caspase -3/7 in situ assay kit (Chemicon International, CA, USA) following manufacturer instructions. Statistical analysis: The results of quantitative analysis of western blot and flow cytometry were compared by one way factorial ANOVA followed by Tukey analysis. For reverse transcription-PCR results were compared only by factorial ANOVA. For all analyses, p<0.05 was accepted as statistically significant.

Results Hypoxia-induced membrane GRP78 expression in cardiomyocytes:

The percent of membrane GRP78 positive cardiomyocytes, as determined by fluorocytometry in normal conditions was doubled under hypoxic conditions. Figures 21A and 21B summarize 5 repeated experiments that determine the percent of membrane GRP78 positive cardiomyocytes. As can be seen in Figure 21A, exposure to 4 hours of hypoxia significantly increased the percent of GRP78 positive cells to 50 ± 5.2 as compared to 21.5 ± 4.7 under normoxic conditions (p<0.004). Incubation of cardiomyocytes with ADoPepl, before exposure to hypoxia, during hypoxia or after hypoxia decreased the percent of GRP78 positive cells compared to similar cultures without the peptide (p<0.053, p<0.005, p<0.01 respectively). Figure 21B depicts one representative fluorocytometry of cardiomyocytes membrane GRP78 staining and shows the ability of AdoPepl to reverse the hypoxia-dependent membranel GRP78 expression.

AdoPepl induces an increase in total GRP78 in cardyomyocites exposed to hypoxic conditions: In order to examine whether the hypoxia-induced membrane GRP78 expression increase is also accompanied by a total cell increase in GRP78 protein levels, the cardiomyocytes were lysed and evaluated by Western blotting using anti-GRP78 antibodies. The results of 3 repeated experiments are presented in Figure 22A. As can be seen in Figure 22A, hypoxia induced a 1.4 fold significant (p< 0.02) increase in the total GRP78 levels (from 4.9 ± 0.8 to 6.7 ± 0.33). Addition of AdoPepl peptide to cultures led to an additional dramatic increase in the total cell GRP78 level from 3.5 ± 0.33 to 8.7 ± 0.21 (p< 0.0002). Figure 22B demonstrates a representative Western blot membrane stained with anti-GRP78 and anti Actin from one of the three experiments conducted, and shows the ability of AdoPepl to enhance even more the hypoxia dependent increase in total cell GRP78 levels.

Hypoxia but not AdoPepl induced GRP78 gene expression in cardiomyocytes: Following the hypoxia and AdoPepl dependent increase in membrane and total

GRP78 in cardiomyocytes, the effect of hypoxia and AdoPep-1 on GRP78 gene expression was examined by RT-PCR and normalized with GAPDH as reference gene in 3 different experiments. As can be seen in Figure 23A, while hypoxia by itself significantly doubled gene expression (p<0.03), the addition of AdoPepl peptide to the cells cultures did not effect GRP78 gene expression (as deduced from the similar GRP78 expression levels obtained with or without addition of peptide). Figure 23B is a representative PCR analysis from one of the three experiments conducted, which shows the hypoxia dependent enhancement in GRP78 gene expression which was unaffected by incubation with AdoPepl. AdoPepl inhibits hypoxia-induced apoptosis in cardiomyocytes:

The involvement of membrane GRP78 in hypoxia induced apoptosis in cardiomyocytes was studied by FACS analysis using the fluorescent markers Annexin V and PI. The results of five repeated experiments are presented in Figure 24A. Four hours of hypoxia almost tripled the percent of apoptotic cells, as determined by Annexin V positive cells (increase from 19.61 ± 5.6 to 56.91 ± 4.9 percent; p< 0.0006). The percent of hypoxia induced apoptosis was however significantly inhibited when GRP78 binding ADoPepl peptide was added before or during hypoxia (from 56.91 ± 4.9 to 36.53 ± 3.37 or to 36.34 ± 4.8 respectively; p<0.04). The addition of the AdoPepl peptide post hypoxia reduced apoptosis to 41.12. ± 6.3 but was not significant. These results suggest that the hypoxia induced apoptosis in cardiomyocytes is triggered via a GRP78 mediated mechanism. Figure 24B is a representative FACS analysis from one experiment of the double labeling of cardiomyocytes with PI and Annexin V, which shows the hypoxia induced apoptosis in cardiomyocytes and the ability of AdoPepl to partially reverse this hypoxia dependent cell death.

Silencing GRP78 using siRNA abolishes AdoPepl peptide cardioprotection: In order to further explore the AdoPepl protective effect against hypoxia- induced apoptosis-and its mediation through GRP78, the effect of silencing the expression of GRP78 by siRNA on GRP78 expression was first assessed and then preservation of the AdoPepl protective ability in such cells was determined.

Therefore, cardiomyocytes, exposed to hypoxic condition with and without the

AdoPepl peptide, were transfected with small interfering RNA (siRNA) oligomers and the expression of membrane GRP78 was examined. As can be seen in Figure 25A, summarizing 3 repeated experiments, increasing concentrations of siRNA oligomers resulted in significant reduction (p<0.002) in total GRP78 in cell lysates reaching, at 50 nM of antisense oligodeoxynucleotide, a 3 fold reduction. Shown in Figure 25B is a representative gel from one of the three experiments conducted demonstrating the reduced total GRP78 bands with increasing concentrations of siRNA ranging from 5 nM to 50 nM. The effect of siRNA on the level of membrane GRP78 was also examined using anti-GRP78 antibody and FACS analysis. The results are presented in

Figure 25C and show that while 62.2 % of cells under hypoxic conditions with control scrambled SiRNA were GRP78 positive, only 13.3 % of cells with siRNA expressed membrane GRP78. Furthermore, addition of the AdoPepl peptide to these cells was unable to further reduce membrane GRP78 levels.

Secondly, the effect of GRP78 silencing on the level of hypoxia-induced apoptosis was assessed by fluorocytometry using AnnexinV, in three separate experiments. As can be seen in Figure 26A, silencing GRP78 did not prevent hypoxia- induced apoptosis as assessed from the similar level of apoptosis in cardiomyocytes transfected with siRNA groups as compared to cardiomyocytes transfected with control scrambled siRNA (60.24 ± 10.68 % and 65.2 ± 9.73 % respectively).

Addition of ADoPepl binding peptide to cardiomyocytes with silenced GRP78 ■ RNA also did not prevent hypoxia-induced apoptosis (62.24 ± 5.84 %) while in non- silenced cells addition of the peptide led to a significant inhibition of apoptosis (34.58 ± 9.98 %; p<0.01). The results suggest that AdoPepl peptide lost its protective effect when the GRP78 expression was silenced. Figure 26B is a representative FACS dot plot analysis of apoptosis of cardiomyocytes under hypoxia without and with peptide in cells with control scrambled RNA and in cells with the silenced RNA, which shows the ability of AdoPepl to reverse the hypoxia induced apoptosis only in cardiomyocytes expressing GRP78. In order to further establish that the AdoPepl protective effect is mediated through GRP78 the activity level of the Apoptosis related protein Caspase 3/7 was assessed following hypoxia with/without the peptide using fluorocytometry. As can be seen in Figure 26C, the percent of cells, when exposed to hypoxic conditions that bind Caspase 3/7 was reduced from 78.2 % percent to 43.4 % by AdoPepl. Silencing GRP78 expression using siRNA cancelled the effect of the peptide on caspase activity in hypoxic cardiomyocites as could be deduced from the similar level of Caspase 3/7 binding levels in cells incubated with without and with the peptide (72 % and 73.6% respectively).

These results strengthen the conclusion that the cardio protective effect of AdoPepl is via a GRP78 dependent mechanism.

EXAMPLE 7 Angiogenesis effect of RoY in the treatment of Myocardial Infraction

The angiogenesis effect of RoY, a peptide according to some embodiments of the present invention, was assessed in a mouse modal of Myocardial infraction.

Methods

10 microgram of RoY peptide were administered intravenously to mice suffering from Myocardial Infraction (MI) one day after the MI occurrence. Mice treated with saline served as control. 1, 7, 14, 30 and 60 days following the initiation of the myocardial infract, histological section were taken from the mice heart and stained with anti-CD31 for evaluation of the level of capillary density in the infract area. The results were statistically analyzed using the ANOVA and Tukey HSD tests.

Results

The intravenous administration of the RoY angiogenic peptide to mice post acute myocardial infarction induced increased capillary density in the border line of myocardial zones progressively and significantly from 15 to 60 days post infarction (see Figure 27). As shown in Figure 27 A and 27B the level of capillary density in mice that received RoY treatment was significantly higher than in control mice (p=0.03 in the ANOVA test and p=0.001 in the Tukey test). Presented in Figure 27C are representative histological sections from the myocardial infract zone of a mousethat received the RoY treatment , 60 days after the MI, (right pictures) as compared to a control mouse that received saline (left pictures) showing the enhancement in CD31 staining (as a measure of capillary density) in mice that received treatment.

These results suggest that the peptides of the present invention are capable of promoting angiogenesis after cardiac ischemic insults such as in Myocardial Infraction.

Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims.

All publications, patents and patent applications mentioned in this specification are herein incorporated in their entirety by reference into the specification, to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated herein by reference. In addition, citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention. To the extent that section headings are used, they should not be construed as necessarily limiting.

Claims

WHAT IS CLAIMED IS:

1. An article of manufacture comprising an implantable medical device and at least one peptide deposited on at least a portion of a surface of said medical device, said peptide being capable of chemoattracting endothelial progenitor cells and is being selected from the group consisting of:

(b) a peptide which comprises an amino acid sequence selected from the group consisting of HWRRAHLLPRLP (ADOPEPl; SEQ ID NO:1), HWRRP (MOTIFA; SEQ ID NO:2), and YPHIDSLGHWRR (RoY; SEQ ID NO:3);

(c) a peptide which comprises an amino acid sequence selected from the group consisting of amino acid sequences as set forth by SEQ ID NOs:4 (VPWMEPAYQRFL), 5 (LLADTTHHRPWT), 6 (QPWLEQAYYSTF), 7 (SAHGTSTGVPWP) , 3 (YPHIDSLGHWRR) and 9 (TLPWLEESYWRP);

(d) a peptide which comprises an amino acid sequence selected from the group consisting of amino acid sequences as set forth by SEQ ID NOs: 10, 11 and 12, the peptide being at least 6 and no more than 50 amino acid residues in length; and

(e) a peptide which comprises an amino acid sequence HWRR as set forth by SEQ ID NO: 13.

2. The article of manufacture of claim 1, wherein said peptide which comprises an amino acid sequence HWRR as set forth by SEQ ID NO: 13 consists of 4 or 5 amino acids.

3. The article of manufacture of claim 1, wherein said peptide has an amino acid sequence selected from the group consisting of the amino acid sequences as set forth by SEQ ID NOs: 4, 6, 7 and 9.

4. The article of manufacture of claim 1, wherein said peptide has an amino acid sequence selected from the group consisting of the amino acid sequences as set forth by SEQ ID NOs: 4, 6 and 9.

5. The article of manufacture of claim 2, wherein said amino acid sequence is HWRRP (SEQ ID NO:2) or HWRRA (SEQ ID NO: 15).

6. The article of manufacture of claim 1, wherein said peptide has an amino acid sequence as set forth by SEQ ID NO:1.

7. The article of manufacture of claim 1, wherein said peptide has an amino acid sequence as set forth by SEQ ID NO:3.

8. The article of manufacture of claim 1, wherein said peptide has an amino acid sequence selected from the group consisting of the amino acid sequences as set forth by SEQ ID NOs: 1, 16 and 17.

9. The article of manufacture of any of claims 1-8, wherein said peptide is a linear peptide.

10. The article of manufacture of any of claims 1-9, wherein said peptide consists of no more than 12 amino acids.

11. The article of manufacture of any of claims 1-10, wherein said peptide is capable of selectively chemoattracting endothelial progenitor cells, such that the amount of endothelial progenitor cells that migrate towards the peptide is at least twice the amount of non-progenitor endothelial cells.

12. The article of manufacture of any of claims 1-11, wherein said implantable medical device is a blood-contacting medical device.

13. The article of manufacture of any of claims 1-12, wherein said medical device is selected from the group consisting of a stent, a stent graft, a vascular graft, a synthetic vascular graft, a heart valve, a vascular prosthetic filter, a pacemaker, a pacemaker lead, a defibrillator, a patent foramen ovule septal closure device, a vascular clip, a vascular aneurysm occluder, a hemodialysis graft, an atrioventricular shunt, an aortic aneurysm graft device or components, a venous valve, a sensor, a suture, a vascular anastomosis clip, an indwelling venous catheter, an indwelling arterial catheter, a vascular occluder and a vascular sheath.

14. The article of manufacture of any of claims 1-12, wherein said medical device is a stent.

15. The article of manufacture of any of claims 1-14, wherein said medical device further comprises a therapeutically active agent for treating a vascular disease, said therapeutically active agent being deposited onto at least a portion of said surface of said medical device.

16. The article of manufacture of claim 15, wherein said therapeutically active agent is an anti-proliferative agent.

17. The article of manufacture of any of claims 1-16, further comprising an agent that stimulates a formation of an endothelium on the surface of the medical device by said endothelial progenitor cells, said agent being deposited on at least a portion of said surface of said medical device.

18. The article of manufacturing of any of claims 1-12, wherein said medical device is a transiently insertable medical device.

19. The article of manufacturing of claim 18, wherein said transiently insertable medical device is a balloon catheter.

20. The article of manufacture of any of claims 1-19, wherein said medical device further comprises a biocompatible matrix deposited on said surface.

21. The article of manufacture of claim 20, wherein said peptide is linked to said biocompatible matrix.

22. The article of manufacture of any of claims 1-19, wherein said peptide is deposited directly on said surface of said medical device directly.

23. The article of manufacture of any of claims 1-19, wherein said peptide is linked to said surface via a spacer.

24. The article of manufacture of any of claims 1-23, being identified for use in a method of treating a cardiovascular disease.

25. The article of manufacture of claim 24, being packaged in a packaging material and identified in print, in or on said packaging material, for use in a method of treating a cardiovascular disease in a subject in need thereof.

26. The article of manufacture of any of claims 24 and 25, wherein said method comprises implanting said medical device in a bodily site afflicted by said cardiovascular disease in said subject.

27. The article of manufacture of any of claims 24 to 26, wherein said method further comprises locally administering to said bodily site a solution of said peptide, said solution of said peptide being for reducing or preventing apoptosis in said bodily site and/or for inducing angiogenesis in said bodily site.

28. The article of manufacture of any of claims 24 to 27, wherein said bodily site is a cardiac lumen.

29. The article of manufacture of any of claims 26 to 27, wherein said bodily site is selected from the group consisting of a cardiac lumen, a renal artery, a renal vein, a coronary artery, a coronary vein, a cerebral artery, a cerebral vein, a cervical artery, a cervical vein , a stomach artery, a stomach vein, a hepatic artery, a portal vein, a mesenteric artery, a mesenteric vein, an arm artery, an arm vein, a hand artery, a hand vein, a leg artery, a leg vein, a foot artery, and a foot vein.

30. The article of manufacture of any of claims 27-29, wherein implanting the medical device and locally administering said solution of said peptide are effected via the same technique.

31. The article of manufacture of claim 30, wherein implanting said medical device and co-administering said solution of said peptide are both effected via catheterization.

32. A method of treating a vascular disease in a subject in need thereof, the method comprising:

(i) contacting a bodily site afflicted by the cardiovascular disease with an implantable medical device configured for treating the vascular disease; and

(ii) contacting said bodily site with a peptide as described in any of claims 1-11, by means of said implantable medical device.

33. The method of claim 32, wherein said implantable medical device has said peptide deposited on at least a portion of a surface thereof.

34. The method of claim 33, wherein contacting said bodily site with said peptide is effected by implanting said implantable medical device.

35. The method of claim 32, wherein said implantable medical device is a transient insertable medical device, and contacting said bodily site with said peptide comprises delivering said peptide to said bodily site via said transient insertable device.

36. The method of any of claims 32 to 35, wherein said bodily site is a cardiac lumen.

37. The method of any of claims 32 to 35, wherein said bodily site is selected from the group consisting of a cardiac lumen, a renal artery, a renal vein, a coronary artery, a coronary vein, a cerebral artery, a cerebral vein, a cervical artery, a cervical vein , a stomach artery, a stomach vein, a hepatic artery, a portal vein, a mesenteric artery, a mesenteric vein, an arm artery, an arm vein, a hand artery, a hand vein, a leg artery, a leg vein, a foot artery, and a foot vein.

38. A peptide as described in any of claims 1-11, identified for use in a method of treating a cardiovascular disease in a subject undergoing an implantation of an implantable medical device configured for treating the vascular disease.

39. A method of preparing an enriched, isolated population of endothelial progenitor cells, the method comprising: implanting an article comprising an implantable medical device as described in any of claims 1-28 in a body, to thereby chemoattract said cells to a surface of said device; retrieving said medical device from said body; and isolating said endothelial progenitor cells from said medical device, thereby obtaining an enriched, isolated population of endothelial progenitor cells.

40. A process for preparing an article of manufacturing which comprises an implantable medical device and at least one peptide capable of chemoattracting endothelial progenitor cells deposited on at least a portion of the surface of the medical device, the process comprising contacting the device with at least one peptide as described in any of claims 1-11, thereby obtaining the article of manufacture.

41. The process of claim 40, wherein the article of manufacture further comprises a biocompatible matrix coating a surface of said device, the process further comprising applying said matrix to the surface of the device.

42. The process of claim 41, wherein said contacting said device with said peptide and said applying said matrix are performed sequentially or simultaneously.