Use of α-v-integrin (CD51) inhibitors for treatment of cardiac fibrosis

Abstract

To provide a method of treating cardiac fibrosis in a patient.SOLUTION: The present invention relates to a method of treating cardiac fibrosis in a patient, comprising administering to the patient a therapeutically effective amount of an αV-integrin inhibitor. Pharmacological blockade of αV-containing integrins improved cardiac function and survival after MI by reducing infarct size and attenuating expansion of reactive cardiac fibrosis. Notably, after pharmacological blockade of αV-containing integrins, a total myocardial fibrogenesis area as well as interstitial fibrosis in the remote myocardial area are significantly reduced. These data identify a novel mechanism regulating cardiac fibrosis in response to an ischemic injury and suggest that pharmacological targeting of αV-integrins may provide clinical benefit in the treatment of cardiac fibrosis.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to the use of an αV-integrin (CD51) inhibitor for the treatment of myocardial fibrosis.

Background Art

[0002] Heart failure (HF) remains a major cause of death and hospitalization worldwide and represents a significant burden on healthcare costs. 1-6 Current drug therapies limit the peripheral effects of cardiac dysfunction, but the number of therapies that affect primary and deleterious cardiac remodeling at the myocardial level is limited. The etiology of HF is diverse, but HF is typically associated with various physiological and morphological changes that include myocardial fibrosis. 7-9 Indeed, myocardial fibrosis is characterized by the overproduction and deposition of extracellular matrix (ECM) proteins in the myocardium, causing disruption of normal tissue architecture, reduced tissue extensibility, mechanical and electrical dysfunction, and ultimately accelerating progression to HF. 10,11 However, since the mechanisms contributing to myocardial fibrosis are not fully understood, there is currently no effective antifibrotic treatment strategy that can complement current HF therapies. 9,11,12

[0003] Activated cardiac fibroblasts are essential for the production of ECM proteins that accumulate during myocardial fibrosis, but recent studies have demonstrated that cardiac fibroblasts are a typical example of a very heterogeneous cell population. 10-14 The exact nature of activated fibroblasts and the resulting causes of myocardial fibrosis have not yet been fully elucidated. 9,12 Proliferation and activation of resident fibroblasts 15 , transdifferentiation of endothelial cells and / or epicardial cells after injury by endothelial-mesenchymal transition and epithelial-mesenchymal transition, respectively 16,17 , migration of hematopoietic bone marrow-derived cells and perivascular cells 18Various mechanisms have been reported, including. Another model proposes that a population of progenitor cells resident in the tissue is activated in response to stress and becomes the major cellular source of organ fibrosis, including the heart. In our recent study, we identified a novel cardiac cell population that resides in the myocardium and exhibits fibrotic behavior in response to ischemic heart injury. 19 This population was identified based on the expression of Pw1 / Peg3 (hereinafter referred to as PW1), a pan-stem cell marker, using a transgenic Pw1-β-galactosidase reporter mouse model. 20,21 Utilizing the strong and productive expression of the β-gal reporter enzyme, we discovered that a significant proportion (~22%) of fibroblasts are derived from ischemic heart PW1-expressing cells, suggesting that cardiac PW1 + cells may contribute to myocardial fibrosis by directly generating fibroblasts and serving as an additional source of ECM.

[0004] Summary of the Invention The present invention relates to the use of an αV-integrin (CD51) inhibitor for the treatment of myocardial fibrosis. Specifically, the present invention is defined by the claims.

[0005] Detailed Description of the Invention Activated cardiac fibroblasts are essential for the production of extracellular matrix proteins that accumulate during myocardial fibrosis, and PW1 + cardiac adult stem cells have recently been proposed as a cellular source of fibroblasts in the ischemic heart. Here, the inventors have identified αV-integrin (or CD51) as an essential regulator of the fibrotic behavior of PW1 + cardiac adult stem cells. Using a combination of transcriptomics and proteomics approaches, the inventors identified the presence of αV-integrin in the plasma membrane of cardiac PW1 + cells. Expression analysis revealed that the expression of αV-integrin is sensitive and specific to cardiac PW1 + cells. More than 93% of the cardiac PW1 + cells sorted by FACS express CD51 and are mutually PW1+ More than 85% of the cells were CD51 isolated by FACS + It is recovered among heart cells. Inhibiting αV-integrin reduces the expression profile of fibrosis-promoting genes and the ability of heart PW1 + to differentiate into cells. Heart PW1 + Cells showed predominant expression of the αVβ1 complex, a putative key mediator of organ fibrosis via TGF-β activation. As a result, pharmacological blockade of αV-containing integrins improved cardiac function and survival after MI by reducing infarct size and attenuating the expansion of reactive myocardial fibrosis. In particular, after pharmacological blockade of αV-containing integrins, interstitial fibrosis decreased significantly in both the total myocardial fibrosis area and the remote myocardial area. These data identify a new mechanism for regulating myocardial fibrosis in response to ischemic injury and suggest that pharmacological targeting of αV-integrin may offer clinical benefits in the treatment of myocardial fibrosis.

[0006] Accordingly, a first object of the present invention relates to a method for treating myocardial fibrosis in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of an αV-integrin inhibitor.

[0007] As used herein, the term "myocardial fibrosis" has its ordinary meaning in the art and refers to a condition characterized by excessive production and deposition of extracellular matrix (ECM) proteins in the myocardium that causes disruption of normal tissue architecture, reduced tissue extensibility, and mechanical and electrical dysfunction. Myocardial fibrosis occurs in response to aging, exposure to certain drugs, or various heart diseases such as myocardial infarction and hypertension. Following acute myocardial infarction, the sudden loss of a large number of cardiomyocytes triggers an inflammatory response and ultimately the dead myocardium is replaced by collagen scar. Several other pathophysiological conditions induce more insidious interstitial and perivascular deposition of collagen in the absence of a complete infarct. Aging is associated with progressive fibrosis and may contribute to the development of dilated heart failure in the elderly. Pressure overload induced by hypertension or aortic stenosis results in extensive myocardial fibrosis that is initially associated with increased stiffness and diastolic dysfunction. Volume overload due to valvular regurgitant lesions can also cause myocardial fibrosis. Hypertrophic cardiomyopathy and dilated cardiomyopathy after viral infection are also often associated with the development of significant myocardial fibrosis. Furthermore, various toxic insults (such as alcohol and anthracyclines) and metabolic disorders (such as diabetes and obesity) induce myocardial fibrosis.

[0008] Specifically, the αV-integrin inhibitor of the present invention is particularly suitable for restricting the development of reactive interstitial fibrosis in viable myocardium. More specifically, the αV-integrin inhibitor of the present invention is suitable for improving the cardiac function of a subject suffering from myocardial fibrosis. As used herein, the term "cardiac function" refers to the function of the heart, including the overall and local functions of the heart. As used herein, the term "overall" cardiac function refers to the function of the entire heart. Such function can be measured, for example, by stroke volume, ejection fraction, cardiac output, myocardial contractility, etc. The term "local cardiac function" refers to the function of a part or region of the heart. Such local function can be measured, for example, by wall thickening, wall motion, myocardial mass, segmental shortening, ventricular remodeling, new myogenesis, the ratio of myocardial cell proliferation and programmed cell death, angiogenesis, and the size of fibrous and infarcted tissue. More specifically, the αV-integrin inhibitor of the present invention is suitable for improving the survival rate of a subject suffering from myocardial fibrosis.

[0009] As used herein, the terms "treatment" or "treating" refer to both prophylactic or preventive treatment, as well as curative or disease-modifying treatment, which include the treatment of patients at risk of developing a disease or suspected of having developed a disease, as well as patients diagnosed as being ill or suffering from a disease or medical disorder, and include the suppression of clinical recurrence. Treatment can be carried out on subjects having a medical disorder or who may ultimately be at risk of developing a disorder to prevent, cure, delay the onset of, reduce the severity of, or improve one or more symptoms of the disorder or a recurrence disorder, or to extend the lifespan of the subject beyond what would be expected in the absence of such treatment. "Treatment regimen" means a pattern of treatment of a disease, for example, a pattern of dosing used during treatment. A treatment regimen may include an induction regimen and a maintenance regimen. The term "induction regimen" or "induction period" refers to a treatment regimen (or a part of a treatment regimen) used for the initial treatment of a disease. A common goal of an induction regimen is to provide a high level of drug to the patient at the beginning of the treatment regimen. The induction regimen can employ (partially or wholly) a "loading regimen", which may include administering a larger amount of drug than the physician uses during the maintenance regimen, administering the drug more frequently than the physician administers the drug during the maintenance regimen, or both. The term "maintenance regimen" or "maintenance period" refers to a treatment regimen (or a part of a treatment regimen) used for the maintenance of a patient during the treatment of a disease, for example, to keep the patient in a remission state for a long period (months or years). The maintenance regimen can employ continuous treatment (for example, administering the drug regularly, for example, weekly, monthly, annually, etc.) or intermittent treatment (for example, interrupted treatment, intermittent treatment, treatment at recurrence, or treatment upon achievement of specific predetermined criteria [for example, signs of the disease, etc.]).

[0010] As used herein, the terms "αV-integrin" or "CD51" have their ordinary meaning in the art and refer to the protein encoded by the ITGAV gene in humans. An exemplary human amino acid sequence of CD51 is represented by SEQ ID NO: 1. Integrins are integral membrane proteins that are heterodimers composed of an α-chain and a β-chain. αV undergoes post-translational cleavage to generate a heavy chain and a light chain that are disulfide-bonded, and this binds to multiple integrin β-chains to form various integrins. Among the known related β-chains (β-chains 1, 3, 5, 6, and 8; "ITGB1", "ITGB3", "ITGB5", "ITGB6", and "ITGB8"), each can interact with extracellular matrix ligands.

[0011]

Chemical Structure

[0012] As used herein, the term "αV-integrin inhibitor" refers to any compound, whether natural or not, that can inhibit the activity or expression of αV-integrin. This term encompasses any antagonist currently known in the art or that may be identified in the future, and any chemical substance that, when administered to a patient, results in the inhibition or downregulation of a biological activity associated with αV-integrin. Thus, an inhibitor can inhibit the expression or activity of αV-integrin, modulate or block the signal transduction pathway of αV-integrin, and / or block the binding of αV-integrin to its binding partners. This term also encompasses inhibitors of expression.

[0013] A significant number of αV-integrin antagonists (Goodman et al, Trends in Pharmacological Sciences, 2012, 33, 405; Tucker, Gordon C. "Alpha v integrin inhibitors and cancer therapy." Current opinion in investigational drugs (London, England: 2000) 4.6 (2003): 722-731.; Hatley, Richard JD, et al. "An αv‐RGD Integrin Inhibitor Toolbox: Drug Discovery Insight, Challenges and Opportunities." Angewandte Chemie International Edition 57.13 (2018): 3298-3321.) that include inhibitory antibodies, peptides, and small molecules have been disclosed in the literature.

[0014] In some embodiments, the αV-integrin inhibitor of the present invention is an antibody, and more particularly, an antibody having specificity for αV-integrin.

[0015] Thus, as used herein, the term “antibody” is used to refer to any antibody-like molecule having an antigen-binding region, and this term includes Fab’, Fab, F(ab’)2, single domain antibodies (DABs), TandAb dimers, Fv, scFv (single-chain Fv), dsFv, ds-scFv, Fd, linear antibodies, minibodies, diabodies, bispecific antibody fragments, biobodies, tribodies (scFv-Fab fusion antibodies, bispecific or trispecific respectively); sc-diabodies; κ(λ)-bodies (scFv-CL fusion antibodies); BiTE (bispecific T-cell engager, scFv-scFv tandem that attracts T cells); DVD-Ig (dual variable domain antibody, bispecific format); SIP (small immunoprotein, a type of minibody); SMIP (“small modular immunopharmaceutical” scFv-Fc dimer); DART (ds stabilized diabody “dual affinity retargeting”); and antibody fragments containing antigen-binding domains such as small antibody mimics containing one or more CDRs. Techniques for preparing and using various antibody-based constructs and fragments are well known in the art (see, e.g., Kabat et al., 1991, specifically incorporated herein by reference). In particular, diabodies are further described in EP 404,097 and WO 93 / 11161; while linear antibodies are further described in Zapata et al. (1995). Antibodies can be fragmented using conventional methods. For example, treatment of an antibody with pepsin can generate F(ab’)2 fragments. The resulting F(ab’)2 fragments can be treated to reduce disulfide bridges to create Fab’ fragments. Papain digestion can lead to the formation of Fab fragments.Fabs, Fab’, and F(ab’)2, scFvs, Fvs, dsFvs, Fds, dAbs, TandAbs, ds-scFvs, dimers, minibodies, diabodies, bispecific antibody fragments, and other fragments can also be synthesized by recombinant techniques or chemically synthesized. Techniques for creating antibody fragments are well known and described in the art. For example, each of Beckman et al., 2006; Holliger & Hudson, 2005; Le Gall et al., 2004; Reff & Heard, 2001; Reiter et al., 1996; and Young et al., 1995 further describes and enables the creation of effective antibody fragments. In some embodiments, the antibodies of the invention are single-chain antibodies. As used herein, the term “single-domain antibody” has its general meaning in the art and refers to the single-chain variable domain of an antibody of the type found in camelid mammals that are naturally lacking a light chain. Such single-domain antibodies are also “nanobodies®”. For a general description of (single) domain antibodies, see the prior art cited above, as well as EP 0 368 684, Ward et al. (Nature 1989 Oct 12; 341 (6242): 544-6), Holt et al., Trends Biotechnol., 2003, 21(11):484-490; and WO 06 / 030220, WO 06 / 003388.

[0016] In some embodiments, the antibody is a humanized antibody. As used herein, “humanized” refers to an antibody in which some, most, or all of the amino acids outside the CDR regions have been replaced with the corresponding amino acids derived from a human immunoglobulin molecule. Methods of humanization include, but are not limited to, those described in U.S. Pat. Nos. 4,816,567, 5,225,539, 5,585,089, 5,693,761, 5,693,762, and 5,859,205, which are incorporated herein by reference.

[0017] In some embodiments, the antibody is a fully human antibody. Fully human monoclonal antibodies can also be prepared by immunizing transgenic mice that contain most of the human immunoglobulin heavy and light chain loci. See, e.g., U.S. Pat. Nos. 5,591,669, 5,598,369, 5,545,806, 5,545,807, 6,150,584, and the references cited therein (the contents of which are incorporated herein by reference).

[0018] In some embodiments, the antibody of the invention is a single-chain antibody. As used herein, the term "single-domain antibody" has its general meaning in the art and refers to the single heavy-chain variable domain of antibodies of the type found in camelid mammals that are naturally lacking a light chain. Such single-domain antibodies are also "nanobodies (registered trademark)".

[0019] In some embodiments, the antibody will contain a human heavy chain constant region sequence but will not induce antibody-dependent cell-mediated cytotoxicity (ADCC). In some embodiments, the antibody of the invention does not contain an Fc domain that can substantially bind to the FcgRIIIA (CD16) polypeptide. In some embodiments, the antibody of the invention lacks an Fc domain (e.g., lacks the CH2 and / or CH3 domains), or contains an Fc domain of the IgG2 or IgG4 isotype. In some embodiments, the antibody of the invention consists of or comprises a Fab, Fab', Fab'-SH, F(ab')2, Fv, diabody, single-chain antibody fragment, or multispecific antibody comprising a plurality of different antibody fragments. In some embodiments, the antibody of the invention is not linked to a toxic moiety. In some embodiments, one or more amino acids selected from amino acid residues can be replaced with different amino acid residues such that the antibody alters C2q binding and / or reduces or abrogates complement-dependent cytotoxicity (CDC). This approach is described in more detail in U.S. Pat. No. 6,194,551 by Idusogie et al.

[0020] Antibodies having specificity for αV-integrin are well known in the art and typically include integrimab and abciximab. Integrimab is a pan-αv human monoclonal antibody that recognizes αvβ1, αvβ3, αvβ5, and αvβ6 integrins. Integrimab competitively binds and blocks both αvβ3 and αvβ5 integrins. Abciximab is a humanized monoclonal IgG2 antibody that specifically targets all αv integrins (Mitjans F, et al. J Cell Sci 1995; 108:2825-38; Monnier Y, et al. Cancer Res 2008:68; 7323-31).

[0021] In some embodiments, the αV-integrin inhibitor is cilengitide. Cilengitide is a cyclic peptide antagonist that inhibits ανβ1, ανβ3 and ανβ5. The IUPAC name of cilengitide is 2-[(2S,5R,8S,11S)-5-benzyl-11-{3-[(diaminomethylidene)amino]propyl}-7-methyl-3,6,9,12,15-pentaoxo-8-(propan-2-yl)-1,4,7,10,13-pentaazacyclopentadecane-2-yl]acetic acid.

[0022] In some embodiments, the αV-integrin inhibitor is an inhibitor of αV-integrin expression, respectively. An "inhibitor of expression" refers to a natural or synthetic compound having a biological effect of inhibiting gene expression. In a preferred embodiment of the present invention, the inhibitor of gene expression is siRNA, an antisense oligonucleotide or a ribozyme. For example, antisense oligonucleotides, including antisense RNA molecules and antisense DNA molecules, act to directly block the translation of αV-integrin mRNA by binding to αV-integrin mRNA, preventing protein translation, or increasing mRNA degradation, and reducing the level and thus the activity of αV-integrin in the cell. For example, antisense oligonucleotides complementary to a unique region of the mRNA transcript sequence encoding αV-integrin, at least about 15 bases in length, can be synthesized, for example, by conventional phosphodiester methods. Methods of using the antisense method to specifically inhibit the gene expression of a gene with a known sequence are well known in the art (see, for example, U.S. Pat. Nos. 6,566,135; 6,566,131; 6,365,354; 6,410,323; 6,107,091; 6,046,321; and 5,981,732). Small interfering RNAs (siRNAs) can also function as inhibitors of expression for use in the present invention. The expression of the αV-integrin gene can be reduced by contacting a patient or cell with a small double-stranded RNA (dsRNA), or a vector or construct that causes the production of a small double-stranded RNA, such that the expression of the αV-integrin gene is specifically inhibited (i.e., RNA interference or RNAi). The antisense oligonucleotides, siRNAs, shRNAs and ribozymes of the present invention can be delivered in vivo alone or with a vector. In its broadest sense, a "vector" is any vehicle that can facilitate the transfer of an antisense oligonucleotide, siRNA, shRNA or ribozyme nucleic acid into a cell and typically into a cell that expresses αV-integrin.Typically, a vector transports nucleic acids into cells by reducing degradation compared to the extent of degradation that would occur in the absence of the vector. Generally, vectors useful in the present invention include, but are not limited to, plasmids, phagemids, viruses, and other vehicles (which have been engineered by the insertion or incorporation of antisense oligonucleotide, siRNA, shRNA, or ribozyme nucleic acid sequences) derived from viral or bacterial sources. Viral vectors are a preferred type of vector and include, but are not limited to, nucleic acid sequences from the following viruses: retroviruses such as Moloney murine leukemia virus, Harvey murine sarcoma virus, mouse mammary tumor virus, and Rous sarcoma virus; adenovirus, adeno-associated virus; SV40-type viruses; polyomavirus; Epstein-Barr virus; papillomavirus; herpes virus; vaccinia virus; poliovirus; and RNA viruses such as retroviruses. Other vectors known in the art but not named can also be readily used. In some embodiments, the inhibitor of expression is an endonuclease. The term "endonuclease" refers to an enzyme that cleaves phosphodiester bonds within a polynucleotide chain. Some, such as deoxyribonuclease I, cleave DNA relatively nonspecifically (regardless of the sequence), while many, typically called restriction endonucleases or restriction enzymes, cleave only at very specific nucleotide sequences. The mechanism underlying endonuclease-based genomic inactivation generally requires an initial step of single- or double-stranded DNA cleavage, which can then trigger two different cellular mechanisms for DNA repair that can be exploited for DNA inactivation: error-prone non-homologous end joining (NHEJ) and high-fidelity homologous recombination repair (HDR). In certain embodiments, the endonuclease is CRISPR-cas.As used herein, the term "CRISPR-cas" has its ordinary meaning in the art and refers to the related "clustered regularly interspaced short palindromic repeats", which are segments of prokaryotic DNA containing short repeats of base sequences. In some embodiments, the endonuclease is CRISPR-cas9 derived from Streptococcus pyogenes. The CRISPR / Cas9 system is described in US 8697359B1 and US 2014 / 0068797. In some embodiments, the endonuclease is CRISPR-Cpf1, which is a recently characterized CRISPR from Provotella and Francisella 1 (Cpf1) by Zetsche et al. ("Cpf1 is a Single RNA-guided Endonuclease of a Class 2 CRISPR-Cas System (2015); Cell; 163, 1-13).

[0023] "Therapeutically effective amount" means an amount of an active ingredient sufficient to treat or alleviate symptoms with a reasonable benefit / risk ratio applicable to any medical treatment. Of course, the total daily usage amount of the compounds and compositions of the present invention will be determined by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dosage level for any particular subject will depend on the disorder being treated and the severity of the disorder; the activity of the particular compound being used; the particular composition being used, the age, weight, general health, sex and diet of the subject; the time of administration, the route of administration, and the rate of excretion of the particular compound being used; the duration of treatment; drugs used in combination with the active ingredient; and various factors including similar factors well known in the medical arts. For example, it is within the skill of the art to start administration of the compound at a level lower than that required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. However, the daily dosage of the product can vary within a wide range of from 0.01 to 1,000 mg per adult per day. Typically, the composition contains 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250 and 500 mg of the active ingredient for adjustment of the dosage to the subject being treated. Pharmaceutical products typically contain from about 0.01 mg to about 500 mg of the active ingredient, typically from 1 mg to about 100 mg of the active ingredient. An effective amount of the drug is usually supplied at a dosage level of from 0.0002 mg / kg to about 20 mg / kg body weight per day, particularly from about 0.001 mg / kg to 7 mg / kg body weight per day.

[0024] Typically, the active ingredient of the present invention (e.g., an αV-integrin inhibitor) is combined with a pharmaceutically acceptable excipient and, optionally, a sustained-release matrix such as a biodegradable polymer to form a pharmaceutical composition. The term "pharmaceutically" or "pharmaceutically acceptable" refers to molecular components and compositions that do not cause side effects, allergic reactions or other adverse reactions when properly administered to mammals, particularly humans. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, capsule material, or any type of formulation aid. The carrier may also be a solvent or dispersion medium containing, for example, water, ethanol, polyols (such as glycerol, propylene glycol, and liquid polyethylene glycol), suitable mixtures thereof, and vegetable oils. Appropriate fluidity can be maintained, for example, by the use of coatings such as lecithin, by maintaining the required particle size in the case of dispersion media, and by the use of surfactants. Prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc. In many cases, it may be preferable to include isotonic agents, such as sugars or sodium chloride. Sustained absorption of the injection composition can be brought about by the use of absorption delaying agents in the composition, such as aluminum monostearate and gelatin. In the pharmaceutical composition of the present invention, the active ingredient of the present invention can be administered in unit dosage forms as a mixture with conventional pharmaceutical supports. Suitable unit dosage forms include oral dosage forms such as tablets, gel capsules, powders, granules and oral suspensions or solutions, sublingual and buccal dosage forms, aerosols, implants, subcutaneous, transdermal, topical, intraperitoneal, intramuscular, intravenous, subdermal, transdermal, intrathecal and intranasal dosage forms and rectal dosage forms.

[0025] The present invention will be further illustrated by the following drawings and examples. However, these examples and drawings should in no way be construed as limiting the scope of the present invention.

Brief Description of the Drawings

[0026]

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Example

[0027] Method: All procedures and animal care were approved by our institutional research committee and complied with the guidelines for animal care in Directive 2010 / 63 / EU of the European Parliament.

[0028] Myocardial infarction by LAD ligation Male 8-week-old C57BL / 6 or PW1 reporter (PW1 nLacZ ) mice were anesthetized in an induction chamber with 2% isoflurane mixed with 1.0 L / min 100% O2 and placed supine on a heating pad to maintain body temperature. The mice were intubated with an endotracheal tube and then connected to a rodent ventilator (180 breaths / min, tidal volume 200 μl). During the surgical procedure, anesthesia was maintained with 1.5 - 2% isoflurane containing O2. The chest was accessed through the intercostal space from the left side to incise the pericardium. The left anterior descending coronary artery (LAD) was exposed and surrounded with an 8.0 prolene suture at the apex of the left ventricle. The suture was looped for a short time to whiten the arterial area and confirm ligation. Mice were analyzed 7 days after permanent LAD ligation.

[0029] Male C57BL / 6J mice (Janvier labs) or PW1 reporter (PW1 nLacZ ) mice were intraperitoneally administered siramesine 10 mg / kg / day (Adooq bioscience, ref: A12372) or vehicle for 14 days. The LAD surgery was performed 7 days after the start of siramesine or vehicle administration. The mice were divided into 4 groups: MI-CIL (siramesine), MI-Veh (vehicle), Ctrl-CIL, and Ctrl-Veh. On day 14, the animals were euthanized, the hearts were carefully harvested, incubated in 4% formaldehyde solution (Ted Pella) at 4°C for 2 hours, then incubated in 30% sucrose / PBS solution for 24 hours, followed by OCT embedding and freezing in liquid nitrogen vapor. Echocardiography was performed on days 0 and 7 to evaluate cardiac function.

[0030] Cell isolation and fluorescence-activated cell sorting Before euthanasia, the animals were injected with heparin (100 UI / 25 g). The right atrium was carefully incised with surgical scissors, and the heart was perfused with 10 mL PBS (Gibco) through the right ventricle to remove blood. After harvesting the heart, the atria were excised, and the ventricles were sliced thinly (0.5 - 1 mm 3)。Cardiomyocytes were dissociated at 37°C for 30 minutes by enzymatic digestion with 480 U / mL collagenase II (Worthington) in DPBS (GIBCO). Digestion was stopped with DMEM (Life Technologies) supplemented with 10% FBS (Sigma) and 1% penicillin / streptomycin (P / S; Life Technologies), and the cell suspension was filtered through a 100 μm Falcon cell strainer and then centrifuged at 416 g for 10 minutes at 4°C. The cell pellet was resuspended in 500 μL of red blood cell lysing (RBCL) buffer to lyse red blood cells for 30 seconds at RT. DPBS was added and then centrifuged for 10 minutes under the same conditions as above. Next, the cell pellet was resuspended in 300 μL of HBSS (Gibco) containing 1% FBS (HBSS-1% FBS) and immunostained for 45 minutes on ice in the dark. The list of antibodies used is shown in Supplementary Table 1. After incubation, HBSS-1% FBS was added and the cells were centrifuged at 416 g for 10 minutes at 4°C to obtain PW 1+ Cell staining was performed. For this, 300 μL of HBSS-1% FBS containing 60 μM 5-dodecanoylaminofluorescein di-β-D-galactopyranoside (C 12 FDG, ThermoFisher Scientific), a substrate for β-gal detection by flow cytometry, was added to the cells and then incubated at 37°C for 1 hour with constant stirring in the dark. The reaction was stopped by the addition of HBSS-1% FBS. After cell centrifugation, the pellet was resuspended in 200 μL of HBSS-1% FBS, propidium iodide (Sigma), a vital dye, was added, and the sample was filtered through a 50 μm Filcon (BD bioscience) immediately before analysis.

[0031] The threshold for viability discrimination was determined by comparing the conditions with and without the vital dye. All immunostained populations were obtained by comparing the signals from unstained cells and the FMO (Fluorescence Minus One) condition. The threshold for B-Gal activity was obtained by comparing B-Gal negative and positive cells.

[0032] RNA-seq Using 300 ng of total RNA extracted from freshly isolated cells, library preparation was performed with the SureSelect Strand-Specific RNA kit (Agilent) according to the manufacturer's instructions. The quality of the resulting library was checked and quantified by peak integration with the Bioanalyzer High Sensitivity DNA LabChip (Agilent). Twelve purified libraries of equal amount were pooled, and each library was tagged with a different index. The miRNA pooled library was finally sequenced on an Illumina Hiseq 1500 machine using a Rapid flow cell. The pool was loaded onto 2 lanes of the flow cell. Paired-end sequencing of 2 × 100 bp was performed.

[0033] Read data that did not pass through the Illumina filter were discarded, and after trimming low-quality (q < 28) sequencing bases using the Cutadapt program 24 we restricted our downstream analysis to read data with a length exceeding 90 bp. The selected read data were from the RSEM package 25 and ENSEMBL from the whole mouse reference genome 26It was mapped to the mouse reference transcriptome created from the gtf transcript annotation file from . The RSEM program was used to align and estimate a large number of transcripts in each of the 12 processed samples. Transcripts with abundance counts exceeding 10 in more than two samples (N = 36,948) were considered expressed and retained for further analysis. The abundances of transcripts assigned to the same gene were combined to profile the expression of 16,403 genes. Next, principal component analysis was performed on the 12 expression profiles, and it was revealed that the first three principal components explained approximately 84% of the total variation in the gene expression profiling. The proportions of the variation explained by the first, second, and third components were 39.4%, 28.5%, and 15.7%, respectively. The analysis was performed in the R environment (version 3.2.2).

[0034] The Galaxy 15.10 instance was locally installed on the server machine. WolfPsort, TMHMM, and SignalP were obtained from the CBS prediction server collection (http: / / www.cbs.dtu.dk / services / ). Since NetNE was not implemented in Galaxy, NLStradamus and PredictNLS were used instead. Next, the RNA Seq of each population was processed through a pipeline designed to select sequences that contain a signal peptide, at least one transmembrane segment, no nuclear export signal, and are theoretically present in the plasma membrane. The putative membraneome was manually quality inspected for false positive sequences and compared to several databases (Qiagen's Ingenuity Pathway Analysis, Uniprot, Ensembl), so a deliberately loose filter was selected. Finally, all samples were compared, and PW1 + Sequences that are only present in cells but not sensitive to myocardial infarction were detected.

[0035] Proteomics PW1 +The cell sample sorted by FACS was prepared for MS analysis with an adaptation protocol because the cells recovered were too few to run a complete protocol. 300,000 PW1 + The sorted cells were centrifuged at 500 g for 10 minutes at 4°C. The pellet was lysed in 100 μL of lysis buffer (10 mM HEPES, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM DTT, 1 mM orthovanadic acid and protease inhibitor cocktail) and incubated on ice for 15 minutes. Next, mechanical cell disruption was performed by pipetting up and down 20 times with a Dounce Potter-type homogenizer. The sample was then centrifuged at 600 g for 10 minutes at 4°C. The supernatant was then centrifuged at 100,000 g for 25 minutes at 4°C. The pellet was washed with 150 μL of wash buffer and centrifuged under the same conditions as above. The final pellet was resuspended in 150 μL of 50 mM NH4HCO3 and homogenized. Membrane proteins were analyzed on an SDS-PAGE gel (4% stacking and 12% running gel) and stained with a silver nitrate staining protocol compliant with MS. All proteins were excised into one large strip and cut into 1 mm 3 cubes just prior to protein reduction, alkylation (DTT, iodoacetamide) and trypsin digestion (500 ng in 50 mM AMBIC, 5% ACN). Finally, peptides were extracted with 20 μL of 30% ACN 0.1% FA. Mass spectrometry was performed by injecting 4 μL into an online LC-MS consisting of a nanoHPLC Dionex Ultimate3000 with a 2-hour gradient and a Thermofisher QExactive +. All proteomics steps were carried out under the supervision of the Proteomics Facility of LaPitie-Salpetriere (P3S, UPMC), and LC-MS analysis was performed at the Proteomics Facility of the University of Paris-Descartes (3P5, Paris Descartes).

[0036] The software used was X!Tandem (Ver: 2015.04.01.1) using the X!Tandem pipeline (Ver: 3.4.3), and the reference proteome of Uniprot used was downloaded on December 12, 2016. Standard contaminants were removed. Applied filters: proteins with at least 1 peptide with an e-value less than 0.05; FDR peptide = 0.128; FDR protein = 0.95. Final protein score Log(e-value) < -2. In the final result, 1679 groups with 1831 subgroups were obtained.

[0037] The protein list obtained from LC-MS analysis was manually selected to retain only plasma membrane proteins. This observed PW1 + The membranome was matched against the putative PW1 + membranome. Only the proteins found in both lists were processed. Next, prospective candidates were selected by antibodies verified for their availability in the literature as well as flow cytometry and immunofluorescence staining.

[0038] Western blot analysis Proteins from mouse heart were extracted from frozen tissue with ice-cold RIPA buffer (50 mM Tris pH 7.4, 150 mM NaCl, 1% Igepal CA-630, 50 mM deoxycholic acid, and 0.1% SDS) containing antiprotease (Sigma-Aldrich), serine / threonine and tyrosine protein phosphatase inhibitors (Phosphatase Inhibitor Cocktails 2 and 3, Sigma-Aldrich) and 1 mM Na3VO4, or with urea-thiourea buffer (5 M urea, 2 M thiourea, 50 mM DTT, 0.1% [w / v] SDS (in PBS pH 7.4)) using a Dounce Potter-type homogenizer. After incubation at 4°C for 1 hour, the homogenate was centrifuged at 15,300 g for 15 minutes at 4°C and the supernatant containing the proteins was collected. The protein concentration of all samples was determined by a Bradford-based protein assay (Biorad).

[0039] After sorting, the hearts of 22 mice, PW1 + cells and the hearts of 16 mice, PW1 - The cells were pooled, centrifuged at 500 g for 15 minutes at 4°C, and dissolved in urea-thiourea buffer. Protein was extracted as described above.

[0040] Cardiomyocytes (CM) and non-cardiomyocytes (non-CM) cells were isolated from adult mouse hearts as previously reported (Ackers-Johnson et al, 2016) with minor modifications. After collecting the cells, CM was dissolved in RIPA and urea-thiourea buffer for protein extraction as described above, and non-CM was seeded in growth medium (DMEM 10% FBS 1% P / S). When the cells reached 80% confluence, protein was extracted with RIPA and urea-thiourea buffer.

[0041] Protein (10 - 30 μg) was mixed with NuPAGE™ LDS sample buffer, NuPAGE™ sample reducing agent, and extraction buffer, incubated at 70 °C for 10 minutes, and then loaded into the gel lanes of a NuPAGE™ Novex® 4 - 12% Bis-Tris gel (Life Technologies). After electrophoresis at 90 V for 3 hours with NuPAGE™ MES SDS running buffer (Life Technologies), the protein was transferred to a nitrocellulose membrane and blocked for 1 hour in Tris-buffered saline containing 0.1% Tween-20 (TBS-Tween) with 5% skim milk, with constant shaking. Next, the membrane was incubated overnight at 4 °C with primary antibodies specific to CD51 (Bioss), integrin β1 (Abcam), integrin β3 (Cell Signaling), integrin β5 (Cell Signaling), and integrin β8 (Sigma-Aldrich), diluted in 5% BSA / TBS-Tween, with constant shaking. Next, the membrane was washed with TBS-Tween and incubated for 1 hour at room temperature (RT) with a horseradish peroxidase-labeled secondary antibody diluted in 5% skim milk or BSA / TBS-Tween. Next, the membrane was washed again with TBS-Tween and then incubated with SuperSignal® West Pico PLUS chemiluminescent substrate (Life Tecnologies) for 5 minutes, followed by image processing using a Chemidoc® XRS+ camera (Biorad) and ImageLab™ software.

[0042] In vitro Pw1 + Pharmacological inhibition of cells PW1 sorted by FACS + Or PW1 - Cells were plated at 1 cm in culture dishes coated with adhesion factors (ThermoFisher Scientific). 2Seeded at a density of 25,000 cells per well. The cells were allowed to adhere overnight with 0.5 mL of growth medium (DMEM 10% FBS 1% P / S). The next day, the medium was changed. On day 5, the medium was changed and supplemented with 0.300 nM or 1 μM of sirencotide. On day 6, the medium was removed and the cells were washed twice with PBS. Next, the cells were lysed, RNA was extracted, and then RT-PCR was performed. Finally, Q-PCR was carried out with the selected fibrosis markers.

[0043] Statistical analysis The data of this manuscript were presented as mean ± SEM. When comparing more than two groups, one-way analysis of variance and pairwise comparison using Tukey's test for multiple comparisons were used to analyze the quantitative data. The Mann-Whitney U test was used to compare continuous variables between two groups. The log-rank (Mantel-Cox) test was used to compare the survival rates between groups. A P value < 0.05 was considered significant.

[0044] Results Bioinformatics analysis of cardiac PW1 + To define the putative membrainome of the cells First, we characterized the transcriptome profile of cardiac PW1 + by RNA sequencing. For this purpose, C 12 FDG, a fluorescent substrate for β-galactosidase activity, was used in our PW1 nLacZ reporter mouse model to FACS-sort cardiac PW1 + from fresh heart tissue. We obtained cardiac PW1 +were obtained from normal and also ischemic hearts (i.e., 7 days after MI). We then used the selected, aligned, and quality-controlled RNA-seq output files to predict the amino acid sequences of the corresponding genes, and then used a series of bioinformatics algorithms to identify putative membrane proteins (data not shown). Briefly, we expected the proteins to have a predicted N-terminal endoplasmic reticulum targeting signal peptide and a predicted transmembrane domain, but no subcellular localization signal (i.e., no ER retention signal motif, no mitochondrial targeting peptide, and no nuclear export signal). Thus, by progressive selection, we identified cardiac PW1 in both normal and ischemic conditions. + A list of 2040 candidates expressed in the plasma membrane was generated (data not shown). The properties of these 2040 candidates were further screened against available databases to identify cardiac PW1. + We identified 913 candidates that are likely expressed in the plasma membrane of cardiac cells (data not shown). We then implemented the same strategy to define the putative surface membraneome of other cell types (i.e., cardiomyocytes, cardiac fractions of non-myocytes, and embryonic stem cells) and performed a final screen to identify cardiac PW1 + We limited our list to 378 candidates that were only observed in the cell-derived dataset (data not shown). + Functional enrichment analysis of this short list of specific, condition-insensitive cell surface markers identified a significant number of transmembrane receptors, transporters, and to a lesser extent ion channels and enzymes (data not shown), however molecular functions were not explained for the majority of the identified candidates.

[0045] Proteomic analysis of cardiac PW1 + Cross-validate the expression of nine cell surface proteins in cells In parallel with the transcriptomics approach, we used mass spectrometry to characterize FACS-isolated cardiac PW1 +Proteomic analysis of the cells was also performed (data not shown). We identified the expression of 1,885 proteins and further analyzed these using available databases, restricting the dataset to 230 proteins located in the plasma membrane. We then directly compared the transcriptomic and proteomic datasets and confirmed nine candidate cross-identifications by both approaches (data not shown). Among these nine cell surface proteins, three transporters, four receptors, and two enzymes were found, and most of these candidates were involved in cell motility, adhesion to the matrix, and response to wounds (data not shown). Five of these candidates have already been reported in non-heart cells as plasma membrane clusters of differentiation (i.e., CD51-Itgav, CD140a-Pdgfra, CD172a-Sirpa, CD39-Entpd1, and CD163), and the remaining were further investigated in our study.

[0046] Itgav (CD51) is highly expressed in heart PW1 + cells. The expression of these five newly identified cell surface markers was analyzed by cytometry of PW1 nLacZ reporter mouse hearts isolated from C 12 FDG + cells. We observed that a total of 92.98 ± 1.01% of heart PW1 + cells expressed CD51 (data not shown). The other four new markers, as well as typical adult stem cell markers (i.e., CD44, CD34, CD166), were expressed at a lower percentage (i.e., approximately 50%) of heart PW1 + cells (data not shown), thus indicating that CD51 is a strong marker for identifying heart PW1 + cells. The selected CD45 - Ter119 - Further analysis in heart cells showed a high clustering of FDG and CD51 expression, thereby further confirming that resident heart PW1 + cells exhibit a high expression level of CD51 (data not shown). Conversely, the expression of CD51 was FDG+ CD45 + Ter119 - Observed in the majority of cardiac cells (data not shown).

[0047] Cardiac PW1 based on CD51 expression + To develop and validate a new cell sorting strategy, we first analyzed the expression of four other candidate markers in CD51-sorted cells from reporter mouse hearts, FDG nLacZ CD51 + CD51 + CD45 - Cells express exclusively CD140a (i.e., PDGF receptor alpha), while FDG + CD51 + CD45 + Cells were found to express CD172a (data not shown). Next, using our PW1 nLacZ reporter mice, to propose a new cell sorting strategy for cardiac PW1 + cells, we initiated cell selection using CD51, CD45, CD140a, and CD172. Briefly, the cardiac CD51 + cell population can be readily separated by the expression of CD45, CD140a, and CD172 (data not shown). Next, we evaluated β-galactosidase activity in each of these subpopulations, and with the new strategy, we were able to recover + CD45 - CD140a + CD172 - 84.57 ± 1.61% of PW1 + cells in the population, and + CD45 + CD140a - CD172 + 91.13 ± 0.81% in the population. These results indicate that CD51 is expressed in almost all cardiac PW1 + cells and is predominantly found in cells expressing PW1 in the myocardium + indicating that CD51 is expressed in almost all cardiac PW1

[0048] Cardiac PW1 +Cells express various αV-containing integrins CD51 (Itgav) belongs to the integrin family, which are transmembrane receptors that act as a bridge for cell-ECM binding and cell-cell interactions. Thus, first we analyzed CD51 expression in freshly isolated cardiomyocyte and non-myocyte fractions from normal mouse hearts. Huvec cells were used as a positive control. It was found that CD51 was expressed exclusively in the non-myocyte fraction (data not shown). Next, we sorted PW1 + and PW1 - cell fractions by FACS and found that CD51 expression was only observed in cardiac PW1 + cells (data not shown), which further supports the transcriptomics, proteomics, and cytometry data as a result. Western blot analysis further confirmed a significant increase in CD51 expression in ischemic hearts and more specifically in the infarct area (data not shown), which is consistent with the significant increase in the number of cardiac PW1 + cells that we previously showed to be predominantly located in the infarct area, as well as the predominant expression in cardiac PW1 + cells 19 .

[0049] The integrin αV subunit can bind to β subunits to form various integrin combinations that can be observed in even larger amounts in specific cell types. Thus, we searched for traces of integrins specific to cardiac PW1 + cells. First, we used our transcriptome dataset to evaluate the expression of Itgav and β subunits (including Itgb1, Itgb3, Itgb5, and Itgb8). Consistent with our Western blot data, Itgav was predicted to be abundant in cardiac PW1 + cells compared to other cell types studied. Next, we found that the mRNA expression level of Itgb1 was similar to that of other cell types studied in cardiac PW1 +It was found to be high in cells compared to other integrin β subunits (data not shown), which suggests a dominant αVβ1 complex in cardiac PW1 + cells. Western blotting of cardiac cell fractions (data not shown) confirmed the presence of ITGβ1, ITGβ3, ITGβ5, and ITGβ8 in non-muscle cells. The presence of not only ITGβ1 but also ITGβ3 was further confirmed in cardiac PW1 + cells, while ITGβ5 and ITGβ8 were not observed in these cells (data not shown). Consistently, ITGβ1 and ITGβ3 were overexpressed in the infarcted area of the heart after MI (data not shown). Overall, these results confirmed the dominant expression of CD51 (i.e., ITGAV) in PW1 + cardiomyocytes and suggested the important presence of αVβ1 and αVβ3 heterodimers in cardiac PW1 + cells.

[0050] PW1 + Targeting αV integrin reduces myocardial fibrosis after MI Recent evidence has shown that αV integrin is a central mediator of organ fibrosis via TGF-β activation 22,23 , indicating an important role of αVβ1 integrin in particular. Our results showed that CD51 can be used as a cell surface marker to identify PW1 + cells, but next we wanted to know whether CD51 could also be directly involved in the fibrotic behavior of cardiac PW1 19 as previously reported. + CD51 (αV integrin) recognizes and binds to the tripeptide sequence RGD (arginine, glycine, aspartic acid), an important motif for cell-cell and cell-ECM interactions. Based on this motif, a cyclic RGD pentapeptide (called cilengitide) was developed and shown to exhibit potent inhibitory activity against integrin αV, including αVβ1, αVβ3, and αVβ5.

[0051] To further evaluate the potential contribution of αV integrin seen in cardiac PW1+ cells, we examined the possibility of pharmacological blockade of αV integrin to prevent myocardial fibrosis in response to ischemic injury. Mice were pretreated with cilengitide or vehicle for 7 days and then subjected to MI by complete LAD ligation. Mice were further administered cilengitide or vehicle once daily for an additional 7 days, and then final evaluations of cardiac function and remodeling were performed (Figure 1a). Cilengitide administration was found to be associated with a significant improvement in survival 7 days after MI (Figure 1b) and correlated with a significant increase in cardiac function measured by left ventricular ejection fraction (Figure 1c). Next, collagen (Masson trichrome and picrosirius red) staining was performed on eight serial sections of the heart (data not shown). By quantification of digital images, a significant reduction in infarct size (38.8 ± 7.9% vs. 56.8 ± 14.9% for cilengitide vs. vehicle, p = 0.0008, Figure 1d) and a significant decrease in interstitial fibrosis measured in the remote myocardial region (15.9 ± 10.4% vs. 36.8 ± 13.1% for cilengitide vs. vehicle, p < 0.0001, Figure 1e) were demonstrated, while cilengitide administration was not associated with any changes in survival, cardiac function, or myocardial fibrosis in sham-operated animals (data not shown).

[0052] αV integrin blockade reduces fibrotic behavior or cardiac PW1 + cells Overall, these results suggest a primary blunting of the mechanism leading to the development of myocardial fibrosis in response to MI, which is a phenomenon in which cardiac PW1 + cells, which have previously been shown to exhibit fibrotic behavior after MI, may be involved, and here we have shown that those cells predominantly express the αV integrin that is the target of cilengitide. To test this hypothesis, we first determined whether pharmacological inhibition of CD51 affects newly FACS-isolated cardiac PW1 19 cells +We investigated whether it affects the expression of fibrosis-promoting genes in cells. After seeding these cells and culturing them for 4 days, they were treated with sirengeptide or vehicle for 48 hours while increasing the concentration. Sirengeptide treatment resulted in significant downregulation of aSma (or Acta2), mmp2, and tgfrb1 (Figure 2a), which suggests a decrease in TGF-β signaling and myofibroblast differentiation ability. However, the expression of Col1a1 was not affected by sirengeptide treatment, which supports the specific regulatory role for individual ECM genes.

[0053] Next, we nLacZ used a PW1 reporter mouse model to perform short-term lineage tracing of cardiac PW1 + cells. The stability (productivity) of the β-gal reporter enables the identification of cells derived from actual β-gal + cells. We examined in vivo β-gal activity and the expression of a typical fibroblast marker, namely vimentin, in cardiac sections of the heart after MI (7 days) and found that sirengeptide treatment was associated with a significant decrease in the proportion of cells co-expressing β-gal and vimentin (Figure 2b). Conversely, we found that the number of β-gal + cells that did not express vimentin increased significantly after sirengeptide treatment (Figure 2c). Overall, these results suggest that αV integrin blockade by sirengeptide affected the ability of cardiac PW1 + cells to differentiate into fibroblasts and contribute to fibrotic scarring.

[0054] Discussion: To our knowledge, here PW1 +Provide the first description of proteins expressed on the cell surface of cardiac adult stem cells. Identification of cell surface marker footprints is crucial for the isolation, characterization, and understanding of resident adult stem cells (as these cells typically only make up a very small fraction of the live cells within an organ). Furthermore, while the proposed markers are diverse, there is significant overlap between markers and the discriminatory power is limited. For example, here we show that typical stem cell markers (e.g., CD140a, CD44, CD166) are actually found on the surface of cardiac PW1 + cells, but are only seen in approximately 50% of these cells. To date, identification of PW1 + cells has been made possible using a transgenic PW1 reporter mouse model, which has proven to be very promising for demonstrating PW1 as a pan-tissue stem cell marker 19,20,27-29 . Here we used this model to specifically isolate cardiac PW1 + cells and then performed an unbiased multi-omics approach to further elucidate the cell surface membranome of these cells. Transcriptomic predictions helped us select a substantial number of membrane proteins identified by mass spectrometry, thus increasing the likelihood of identifying more specific cell surface markers from the limited number of remaining candidates. A similar approach was recently proposed to define the secretome of bone marrow stem cells 30 .

[0055] Next, we found that CD51 is expressed in the majority of cardiac PW1 + cells, a result that was not previously reported or predicted. Conversely, CD51 is predominantly seen in cells expressing PW1 in the myocardium, thus highlighting the discriminatory value of this new marker that may be useful in the future for the selection of PW1 + cells. However, it has not yet been determined whether CD51 specifically recognizes PW1 + cells in non-cardiac organs + .

[0056] The identification of this new cell surface marker next provided important information regarding the pathophysiological role of cardiac PW1 + cells. CD51 is a subunit of the αV-integrin, i.e., a subset of integrin family cell adhesion receptors, which have recently been suggested to be central mediators of organ fibrosis via TGF-β activation 22,23 . Surprisingly, we recently reported that cardiac PW1 + cells exhibit fibrogenic behavior in response to ischemic injury, particularly by directly giving rise to fibroblasts 19 . Our new results indicate that CD51 (or ITGAV) plays a direct role in controlling the fibrogenic fate of cardiac PW1 + cells. Indeed, we found that blockade of αV-integrin restricted the expression of fibrotic genes in isolated cardiac PW1 + cells and decreased the differentiation ability of cardiac PW1 + cells into fibroblasts in vivo in the MI mouse model. Pharmacological blockade of αV-integrin by cilengitide in this model significantly reduced fibrotic remodeling after MI, along with a significant decrease in infarct size and interstitial fibrosis. As a result, the survival rate and cardiac function of cilengitide-treated animals were improved. Since some integrins are expressed in endothelial cells, cilengitide has been suggested to have some anti-angiogenic effects 31 . However, the anti-angiogenic effect should worsen cardiac function after MI, so this mechanism could not explain our latest observations. Similarly, although ITGβ3 can be expressed by endothelial cells, we found a significant increase in ITGβ3 expression in the infarct area, i.e., the zone where blood vessels are usually severely reduced but PW1 + cells are abundant. Our data rather suggest that αV-integrin is within cardiac cells of PW1 +It has been shown to be predominantly expressed in cells, thus suggesting that the advantages observed in cardiac fibrotic remodeling are mainly achieved by reducing the fibrotic behavior of these cells. However, the contribution of circulating cells (such as inflammatory cells in response to myocardial necrosis) cannot be excluded.

[0057] The recognition of the role of integrins in non - cardiac organ fibrosis (i.e., lung and liver) is increasing, and genetic deletion or pharmacological inhibition of integrins has been associated with a reduction in fibrotic remodeling. 23,32,33 Interestingly, these recent studies have shown a very important role of αVβ1 in organ fibrosis. 23,32 The role of the αVβ1 heterodimer and mesenchymal - like cell αV integrins in the development of muscle and myocardial fibrosis has only recently been recognized. 23,34 Our current data further provide evidence for the role of cardiac PW1 + adult stem cells in the development of myocardial fibrosis via αV integrin. Our results are consistent with the hypothesis that cardiac PW1 + cells correspond to the cellular source of fibrosis. However, cardiac PW1 + cells may also contribute indirectly to fibrosis by integrating a more favorable microenvironment for the activation of resident fibroblasts or the remodeling of the extracellular matrix. Our data suggest some specific changes in individual ECM genes (especially those related to the TGF - β pathway) after αV - integrin blockade, i.e., observations worthy of further experimentation.

[0058] Finally, our study has shown that a new antifibrotic therapy can be developed by targeting αV - integrin expressed in fibrotic cardiac PW1 + cells. So far, heart failure treatment has been used to reduce the cardiac workload and indirectly reduce cardiac remodeling, but there is currently no effective antifibrotic treatment strategy that can complement the current HF treatment methods. 9,11,12To achieve antifibrotic therapy, a better understanding of the mechanisms contributing to myocardial fibrosis is needed, and our data report a new cell source for myocardial fibrosis. Importantly, in our study, pharmacological blockade of αV-integrin was associated with a favorable outcome in a mouse MI model. Antifibrotic strategies in MI, by restoring areas lacking viable cardiomyocytes to prevent cardiac rupture and limiting the development of replacement fibrosis, are often exposed to the risk of cardiac rupture 35 For example, strategies that limit the recruitment of inflammatory cells to the infarct site have recently been shown to delay the removal of dead cardiomyocytes and their replacement by scar tissue, thus increasing susceptibility to cardiac rupture 36 We did not observe such adverse outcomes; rather, we found a significant improvement in survival under sirengitide treatment. Coupled with a significant reduction in interstitial fibrosis in sirengitide-treated animals, this suggests that blockade of αV-integrin does not directly affect replacement fibrosis remodeling, but rather limits the development of reactive interstitial fibrosis in viable myocardium 12 。

[0059] Overall, these data identify cardiac PW1 + cells as a cause of myocardial fibrosis in response to ischemic injury via αV-integrin and suggest that pharmacological targeting of αV-integrin may offer clinical benefit in the treatment of myocardial ischemia.

[0060] References: Throughout this application, various references describe the state of the art to which the present invention pertains. The disclosures of these references are incorporated herein by reference for illustrative purposes.

[0061]

Table 1

Claims

1. A method of treating myocardial fibrosis in a patient in need thereof comprising administering to the patient a therapeutically effective amount of an αV-integrin inhibitor.

2. 2. The method of claim 1, wherein myocardial fibrosis occurs from aging, exposure to certain drugs, or in response to various cardiac diseases such as myocardial infarction or hypertension.

3. The method of claim 1, wherein the αV-integrin inhibitor is suitable for limiting the development of reactive interstitial fibrosis in viable myocardium.

4. The method of claim 1, wherein the αV-integrin inhibitor is suitable for improving cardiac function.

5. The method of claim 1, wherein the αV-integrin inhibitor is an antibody, more particularly an antibody having specificity for αV-integrin.

6. The method of claim 1, wherein the αV-integrin inhibitor is cilengitide.

7. The method of claim 1, wherein the V-integrin inhibitor is an inhibitor of αV-integrin.

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