STEM CELL DERIVED FACTORS FOR TREATING PATHOLOGIC CONDITIONS

FIELD OF THE INVENTION

The invention relates to cardiac disorders.

BACKGROUND OF THE INVENTION

Patient mortality and morbidity is increased by cell/tissue damage or death resulting from acute and chronic injury or disease of the heart muscle, such as myocardial infarction, cardiac failure, stroke, degenerative neurological disease, spinal injury, musculoskeletal diseases, hypertension, and diabetes.

SUMMARY OF THE INVENTION The invention is based upon the surprising discovery that paracrine factors secreted from mesenchymal stem cells (MSC) confer a therapeutic benefit to bodily tissues. Thus, stem cells serve as a factory of biologic products that are purified and administered to subjects.

The paracrine factors are useful in cellular and tissue protection, repair, and regeneration. Mesenchymal stem cells or progenitor cells that secrete cytoprotective paracrine factors preferably comprise an Akt gene (Akt-MSC). One or more secreted compounds (e.g., and isolated compound or a mixture of secreted compounds such as a MSC culture supernatant) confers a clinical benefit to a variety of injured, compromised, or disease tissues.

A method of reducing cell death or enhancing tissue repair is carried out by contacting an injured or diseased tissue with a composition comprising a paracrine factor of a mesenchymal stein cell (MSC). The composition is administered to healthy tissue that is determined to be at high risk of injury or to injured tissue following the occurrence of an injury. Preferably, the factor is a Secreted frizzled related protein (Sfrp). Optionally, the composition contains one or more paracrine factors, e.g., two, three, five, ten or more factors. The factors provide cell reparative benefits in a synergistic manner. For example, the composition contains one or more Sfrp, e.g., Sfrp-1, Sfrp-2, and Sfrp-3. In one embodiment, Sfrp-1 comprises an amino acid sequence of SEQ ID NO:5, a mature processed form of SEQ ID NO:5, or a fragment thereof; in another embodiment, Sfrp-2 comprises an amino acid sequence of SEQ ID NO:7, a mature processed form of SEQ ED NO:7, or a fragment thereof; and in yet another embodiment, Sfrp-3 comprises an amino acid sequence of SEQ ID NO:9, a mature processed form of SEQ ID NO:9,

or a fragment thereof. The amount of apoptotic cell death is reduced in the presence of a paracrine factor such as an Sfrp compared to in its absence.

Cytoprotective and cell reparative effects are conferred to many types of bodily tissues such as cardiac tissue. For example, in the case of a myocardial infarction, cardiac infarct size is reduced following contact of myocardial tissue with the paracrine factor.

Factors derived from Akt-MSCs, which have been genetically altered to contain a recombinant Akt gene sequence, confer a therapeutic benefit at each stage of a hypoxic cardiac event (early, middle, and late stage). Early on, factors confer a cell protective effect, followed by inotropy, angiogenesis, and cardiac remodeling. The invention also features methods of inhibiting cell damage, inducing or enhancing cell repair or regeneration or inhibiting an ischemic or reperfusion related injury in a subject. Cell damage or injury is inhibited by administering to the subject or contacting a cell with a composition containing a purified cytoprotective compound such as a substantially pure polypeptide, or a mixture of substantially pure polypeptides such as the Sfrp proteins described above. Other purified proteins, e.g., hi, h5, h8, hl2, and hl3 are also useful to prevent or reduce cell damage. Accordinglhy, a method of reducing cell death is carried out by contacting an injured or diseased tissue with a composition comprising a purified paracrine factor of a mesenchymal stem cell selected from the group consisting of hi, h5, h8, hl2 and hl3 or fragment thereof. For example, hi 2 comprises a fragment of SEQ ID NO: 17.

Similarly, cell repair or regeneration is induced by administering to the subject or contacting a cell with a composition containing a purified cytoprotective compound. Polypeptides or other compounds described herein are said to be "substantially pure" when they are within preparations that are at least 60% by weight (dry weight) the compound of interest. Preferably, the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight the compound of interest. Purity is measured by any appropriate standard method, for example, by column chromatography, polyacrylaminde gel electrophoresis, or HPLC analysis. The polypeptide is purified from MSC culture media or recombinantly produced. Cell or tissue damage is defined by a loss or diminution of cell function. Such loss or decrease in function leads to eventual cell death. The cell is a cardiac cell such as a cardiomyocyte, a kidney cell, a liver cell, a neurological (e.g., brain, spinal cord) cell, or a pancreatic cell. For example, a loss of cardiomyocyte function results in the loss of the

contractile function of the cell. Cardiomyocytes that have lost their ability to contract form round cells rather that rod shaped cells when cultured. Ischemia causes irreversible cellular/tissue damage and cell death. Reperfusion exacerbates ischemic damage by activating inflammatory response and oxidative stress. Oxidative stress modifies membrane lipids, proteins and nucleic acids resulting in cellular/tissue damage or death, and depression of cardiac, endothelial and kidney function.

Also included in the invention are methods of regenerating an injured myocai'dial tissue by administered to the tissue a composition containing a cytoprotective compound. The cardiac muscle has been damaged by disease, such as a myocardial infarction. By regenerating an injured myocardial tissue is meant restoring ventricular function and/or decreasing infarct size. Ventricular function is measured by methods known in the art such as radionuclide angiography.

A cytoprotective compound is a compound, which is capable of inhibiting cell damage such as oxidative-stress induced cell death or apoptosis. In addition to Sfrps, cytoprotective compounds include for example adipsin, adrenomedullin, chemokine (C-C motif) ligand 2, cysteine rich protein 61 , lysyl oxidase-like 2, or serine proteinase inhibitor.

The composition is administered to the subject prior to, at the time of, or shortly after (1 , 5, 10, 15, 30, 60 minutes; 1.5, 2, 4, 6, 12, 18, 24, 48 hours) identification of cell damage or identification of a symptom of ischemia or reperfusion injury. For example the composition is administered to a subject prior to a cardiac event or ischemic-reperfusion injury. Such a subject is a risk candidate for an ischemic event or condition. Symptoms of a cardiac event include for example, chest pain, aπn pain, fatigue and shortness of breath. For example, the composition is administered at the onset of symptoms, e.g., chest pain, associated with a cardiac event such as a myocardial infarction. The composition is administered systemically or locally. For example, the composition is administered directly, i.e., by myocardial injection to the cardiac tissue, or systemically, e.g., interperitoneally, orally, intravenously. In another example, administration of the composition is carried out by infusion into a coronary artery. Slow-release formulations, e.g., a dermal patch, in which diffusion of the composition from an excipient such as a polymeric carrier mediates drug delivery are also within the invention. Optionally, the subject is further administered VEGF or thyrosin beta 4. The composition is administered at a dose sufficient to inhibit apoptotic death or oxidative stress-induced cell death. To determine whether the composition inhibits oxidative- stress induced cell death, the composition is tested by incubating the composition with a primary or immortalized cell such as a cardiomyocyte. A state of oxidative stress of the cells is induced

(e.g., by incubating cells with H ₂ O ₂ ), and cell viability is measured using standard methods. As a control, the cells are incubated in the absence of the composition and then a state of oxidative stress is induced. A decrease in cell death (or an increase in the number of viable cells) in the compound treated sample indicates that the composition inhibits oxidative-stress induced cell death. Alternatively, an increase in cell death (or an decrease in the number of viable cells) in the compound treated sample indicates that the composition does not inhibit oxidative-stress induced cell death. The test is repeated using different doses of the composition to determine the dose range in which the composition functions to inhibit oxidative-stress induced cell death. A subject to be treated is suffering from or at risk of developing a condition characterized by aberrant cell damage such as oxidative-stress induced cell death (e.g., apoptotic cell death) or an ischemic or reperfusion related injury. A subject suffering from or at risk of developing such a condition is identified by the detection of a known risk factor, e.g., gender, age, high blood pressure, obesity, diabetes, prior history of smoking, stress, genetic or familial predisposition, attributed to the particular disorder, or previous cardiac event such as myocardial infarction or stroke.

Conditions characterized by aberrant cell damage or death include cardiac disorders (acute or chronic) such as stroke, myocardial infarction, chronic coronary ischemia, arteriosclerosis, congestive heart failure, dilated cardiomyopathy, restenosis, coronary artery disease, heart failure, arrhythmia, angina, atherosclerosis, hypertension, renal failure, kidney ischemia, ischemic hepatitis, hepatic vein thrombosis, cirrhosis, portal vein thrombosis, pancreatitis, ischemic colitis, or myocardial hypertrophy. Cardiac repair or regeneration is evaluated by detecting an improvement of symptoms such as chest pain or shortness of breath as well as by evaluation of heart function by standard methods such as cardiac magnetic resonance, echocardiography, and/or ventricular angiography. Also within the invention is a cell culture or preservation media containing purified

Sfrp2 and a method of maintaining inhibiting stem cell differentiation, e.g., inhibiting myogenesis, by contacting a population of isolated stem cells with purified Sfrp2. Isolated stem cells are selected from the group consisting of embryonic stem cells, mesenchymal stem cells, and hematopoetic stem cells. Stem cells are isolated from the tissue of origin by fractionation by cell surface markers or other distinguishing characteristics. Preferably, a population of isolated cells is at least 85% stem cells. More preferably, the population is 90, 95, 98, 99, 100% stem cells.

This factor is involved in the maintenance and self renewal of tissue specific and embryonic stem cells. For example, differentiation of stem cells, e.g., embryonic stem cells, is inhibited by Sfrp2. Myogenesis is inhibited by contacting stem cells with Sfrp2. In another example, bone marrow-derived hematopoetic stem are maintained in a stem cell state by contacting the cells with purified Sfrp2. Preservation of stem cells in this manner is useful in transport and storage of stem cells prior to transplantation into a subject for therapeutic purposes.

Unless othei ^{^} wise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. AU publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In the case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

Other features and advantages of the invention will be apparent from the following detailed description and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

Figs. IA and IB are bar graphs showing that Sfrps are expressed in mesenchymal stem cells. Fig.s IA shows levels of Sfrpl, Sfrp2 and Sfrp3 expression as estimated by microarray analysis and shows a nearly 10 fold upregulation of Sfrp2 in Akt-MSC compared to GFP-MSC. Fig. IB shows a quantitative real-time RT-PCR validation of mRNA expression levels that demonstrates a 100 fold upregulation of Sfrp2 gene expression in Akt-MSC compared to GFP- MSC. Fig. 2A is a photograph of results of a Western Blotting assay for Sfrp2. The data demonstrates presence of Sfrp2 protein in conditioned medium collected from AKT or GFP MSCs and inhibition of its accumulation in the medium in the presence of Pi3K inhibitor

Fig. 2B is a bar graph showing relative reduction in mRNA levels of Sfrp2 in Akt-MSC following knockdown of Sfrp2 with siRNA. Fig. 2C is a bar graph showing the effect of conditioned medium on apoptosis in

ARVCs. Caspase activity of ARVCs after 24 hours of hypoxia under different culture conditions (control conditioned medium, Ctr CM; Akt conditioned medium, Akt CM; Akt

conditioned medium following Sfrp2 knockdown, Akt CM minus Sfrp2) demonstrates reduction of caspase activity following Akt-CM treatment and attenuation of this effect following treatment with Akt CM minus Sfrp2. These data demonstrate that paracrine factors from Akt- MSCs mediate the survival signaling on cardiomycytes. Fig. 3A is a bar graph showing the effect of Sfrp2 on caspase activity. Cleaved-caspase

3 activity as measured by a fluorometric assay demonstrated decreased caspase activity in hypoxic cardiomyocytes following Sfrp2 treatment in a dose dependent manner. The activity was calculated as fold changes with the same control.

Fig. 3B is a bar graph showing the number of round shaped cardiomyocytes that were counted in 6 random high power fields (40X) following 24 hour hypoxic exposure with/without Sfrp2 treatment. Data is expressed as a percentage of total number of cells present.

Fig. 3 C is a series of representative high power field photographs demonstrating decreased number of round shaped cardiomyocytes following treatment with Sfrp2. Collectively, these data demonstrate that Sfrp2 decreases caspase 3 activity Fig. 4 is a bar graph showing that Sfrp2 decreased cardiac infarct size. Above each bar, is a photograph of TTC staining showing bi- ventricular sections of similar thickness perpendicular to the long axis of the heart. The staining data deomonstrates decreased infarct size with Akt-CM and Sfrp2 and attenuation of reduction in infarct size with Akt-Sfrp2. Infarct size is expressed as a percentage of the total ventricular area. Rat hearts were treated with PBS as control, Akt-MSCs CM (Akt), CM form Akt-MSCs that did express reduced levels of sfrp2 due to siRNA treatment (Akt-Sfrp2).

Fig. 5A is a photograph of an electrophoretic gel showing that Wnt3a mRNA expression as detected by RT-PCR is increased in hypoxic cardiomyocytes while expression of Wnt5 remains unchanged. The data indicate that hypoxic cardiomyocytes upregulate Wnt3a expression and that Sfrp2 blocks pro-apototic effects of Wnt3a.

Fig. 5B is a bar graph showing that Wnt3a (3nM) increases caspase activity of cardiomyocytes undergoing lrypoxia/reoxygenation injury; Sfrp2 at a similar concentration significantly attenuates Wnt3a induced caspase activity ( vs. normoxia, p<.05; vs. wnt+hypoxia/reoxygenation, p<.05, n=6/group). Fig. 6A is a bar graph showing genes upregulated by Sfrp2 under hypoxia. Microarray analysis demonstrates Sfrp2 mediated upregulation of Birclb gene expression in hypoxic cardiomyocytes.

Fig. 6B is a photograph of an electrophoretic gel showing the effect of Sfrp2 on mRNA levels on Birclb. RT-PCR confirmed increased Birclb expression in hypoxic cardiomyocytes following Sfrp2 treatment.

Fig. 6C is a photograph of results of a Western Blot showing that beta-catenin levels are increased by Sfrp2. Western blotting for nuclear and total βcatenin expression in ARVCs demonstrates a reduction of βcatenin following hypoxia and upregulation following treatment with Sfrp2.

Fig. 7 is a bar graph showing the effect of cytoprotective factor hl2 compared to IGF-I on myocyte apoptosis. Fig. 8 is a line graph showing caspase inhibition in cardiomyocytes by hl2.

Fig. 9 is a series of photographs electrophoretic gels showing that hl2 phosphorylates/activates AKT in cardiomyocytes

Fig. 10 is a photograph showing inhibition of cytochrome C release by hl2.

Fig. 11 is a photograph of an electrophoretic gel showing mitochondrial Bcl-2 protein stabilization by h 12.

DETAILED DESCRIPTION

The present invention is based upon the unexpected discovery of that MSC-secreted products confer a therapeutic benefit to injured or compromised tissues. Disclosed herein is a Akt-MSC mediated paracrine mechanism of organ protection and repair. More particularly, the invention provides purified polypeptides such as Srfps isolated from Akt-MSCs or recombinantly or synthetically produced and methods of using these polypeptides to prevent or reduce myocardial damage and ventricular dysfunction.

Akt genes

Akt-MSCs are produced by introducing (e.g., by retrovirus-mediated transduction) into mesenchymal stem cells isolated from the bone marrow an Akt coding sequence or fragment, e.g., Akt-1, Ak-2 or Akt-3. The Akt nucleic acid is human, mouse, or rat.

Exemplary human Akt-1 polypeptides include GenBank Accession numbers NP_005154 and AAH00479. Exemplary human Alct-2 polypeptides includes for example GenBank Accession numbers P31751 and NP_001617. Exemplary human Akt-3 polypeptides includes for example GenBank Accession numbers Q9Y243 and NP_005456. Exemplary nucleic acids encoding Akt include human Akt-1 available at GENBANK™ Accession No. NM_005163 (SEQ ID NO: 1), human Alct-2 available at GENBANK™ Accession No. NMJ)01626 (SEQ ID NO:2) and human Akt-3 available at GENBANK™ Accession No. AJ245709 (SEQ ID NO:3) (all of which are hereby incorporated by reference)or nucleic acids encoding the human Akt polypeptides described above. mRNA sequences and the corresponding coding region for human Akt are shown below.

AkM mRNA (SEQ ID NO:1)

1 atcctgggac agggcacagg gccatctgtc accaggggct tagggaaggc cgagccagcc

61 tgggtcaaag aagtcaaagg ggctgcctgg aggaggcagc ctgtcagctg gtgcatcaga

121 ggctgtggcc aggccagctg ggctcgggga gcgccagcct gagaggagcg cgtgagcgtc

181 gcgggagcct cgggcaccat gagcgacgtg gctattgtga aggagggttg gctgcacaaa

241 cgaggggagt acatcaagac ctggcggcca cgctacttcc tcctcaagaa tgatggcacc

301 ttcattggct acaaggagcg gccgcaggat gtggaccaac gtgaggctcc c ^' ctcaacaac

361 ttctctgtgg cgcagtgcca gctgatgaag acggagcggc cccggcccaa caccttcatc

421 atccgctgcc tgcagtggac cactgtcatc gaacgcacct tccatgtgga gactcctgag

481 gagcgggagg agtggacaac cgccatccag actgtggctg acggcctcaa gaagcaggag

541 gaggaggaga tggacttccg gtcgggctca cccagtgaca actcaggggc tgaagagatg

601 gaggtgtccc tggccaagcc caagcaccgc gtgaccatga acgagtttga gtacctgaag

661 ctgctgggca agggcacttt cggcaaggtg atcctggtga aggagaaggc cacaggccgc

721 tactacgcca tgaagatcct caagaaggaa gtcatcgtgg ccaaggacga ggtggcccac

781 acactcaccg agaaccgcgt cctgcagaac tccaggcacc ccttcctcac agccctgaag

841 tactctttcc agacccacga ccgcctctgc tttgtcatgg agtacgccaa cgggggcgag

901 ctgttcttcc acctgtcccg ggaacgtgtg ttctccgagg accgggcccg cttctatggc

961 gctgagattg tgtcagccct ggactacctg cactcggaga agaacgtggt gtaccgggac

1021 ctcaagctgg agaacσtcat gctggacaag gacgggcaca ttaagatcac agacttcggg

1081 ctgtgcaagg aggggatcaa ggacggtgcc accatgaaga ccttttgcgg cacacctgag

1141 tacctggccc ccgaggtgct ggaggacaat gactacggcc gtgcagtgga ctggtggggg

1201 ctgggcgtgg tcatgtacga gatgatgtgc ggtcgcctgc ccttctacaa ccaggaccat

1261 gagaagcttt ttgagctcat cctcatggag gagatccgct tcccgcgcac gcttggtccc

1321 gaggccaagt ccttgctttc agggctgctc aagaaggacc ccaagcagag gcttggcggg

1381 ggctccgagg acgccaagga gatcatgcag catcgcttct ttgccggtat cgtgtggcag

1441 cacgtgtacg agaagaagct cagcccaccc ttcaagcccc aggtcacgtc ggagactgac

1501 accaggtatt ttgatgagga gttcacggcc cagatgatca ccatcacacc acctgaccaa

1561 gatgacagca tggagtgtgt ggacagcgag cgcaggcccc acttccccca gttctcctac

1621 tcggccagφa gcacggcctg aggcggcggt ggactgcgct ggacgatagc ttggagggat

1681 ggagaggcgg cctcgtgcca tgatctgtat ttaatggttt ttatttctcg ggtgcatttg

1741 agagaagcca cgctgtcctc tcgagcccag atggaaagac gtttttgtgc tgtgggcagc

1801 accctccccc gcagcggggt agggaagaaa actatcctgc gggttttaat ttatttcatc

1861 cagtttgttc tccgggtgtg gcctcagccc tcagaacaat ccgattcacg tagggaaatg

1921 ttaaggactt ctacagctat gcgcaatgtg gcattggggg gccgggcagg tcctgcccat

1981 gtgtcccctc actctgtcag ccagccgccc tgggctgtct gtcaccagct atctgtcatc

2041 tctctggggc cctgggcctc agttcaacct ggtggcacca gatgcaacct cactatggta

2101 tgctggccag caccctctcc tgggggtggc aggcacacag cagcccccca gcactaaggc

2161 cgtgtctctg aggacgtcat cggaggctgg gcccctggga tgggaccagg gatgggggat

2221 gggccagggt ttacccagtg ggacagagga gcaaggttta aatttgttat tgtgtattat

2281 gttgttcaaa tgcattttgg gggtttttaa tctttgtgac aggaaagccc tcccccttcc

2341 ccttctgtgt cacagttctt ggtgactgtc ccaccggagc ctccccctca gatgatctct

2401 ccacggtagc acttgacctt ttcgacgctt aacctttccg ctgtcgcccc aggccctccc

2461 tgactccctg tgggggtggc catccctggg cccctccacg cctcctggcc agacgctgcc

2521 gctgccgctg caccacggcg tttttttaca acattcaact ttagtatttt tactattata 2581 atataatatg gaaccttccc tccaaattct Coding sequence = nucleotide 199-1641.

Akt-2mRNA (SEQ IDNO:2)

1 gaattccagc ggcggcgccg ttgccgctgc cgggaaacac aaggaaaggg aaccagcgca

61 gcgtggcgat gggcgggggt agagccccgc cggagaggct gggcggctgc cggtgacaga

121 ctgtgccctg tccacggtgc ctcctgcatg tcctgctgcc ctgagctgtc ccgagctagg

181 tgacagcgta ccacgctgcc accatgaatg aggtgtctgt catcaaagaa ggctggctcc

241 acaagcgtgg tgaatacatc aagacctgga ggccacggta cttcctgctg aagagcgacg

301 gctccttcat tgggtacaag gagaggcccg aggcccctga tcagactcta ccccccttaa

361 acaacttctc cgtagcagaa tgccagctga tgaagaccga gaggccgcga cccaacacct

421 ttgtcatacg ctgcctgcag tggaccacag tcatcgagag gaccttccac gtggattctc

481 cagacgagag ggaggagtgg atgcgggcca tccagatggt cgccaacagc ctcaagcagc

541 gggccccagg cgaggacccc atggactaca agtgtggctc ccccagtgac tcctccacga

601 ctgaggagat ggaagtggcg gtcagcaagg cacgggctaa agtgaccatg aatgacttcg

661 actatctcaa actccttggc aagggaacct ttggcaaagt catcctggtg cgggagaagg

721 ccactggccg ctactacgcc atgaagatcc tgcgaaagga agtcatcatt gccaaggatg

781 aagtcgctca cacagtcacc gagagccggg tcctccagaa caccaggcac ccgttcctca

841 ctgcgctgaa gtatgccttc cagacccacg accgcctgtg ctttgtgatg gagtatgcca

901 acgggggtga gctgttcttc cacctgtccc gggagcgtgt cttcacagag gagcgggσcc

961 ggttttatgg tgcagagatt gtctcggctc ttgagtactt gcactcgcgg gacgtggtat

1021 accgcgacat caagctggaa aacctcatgc tggacaaaga tggccacatc aagatcactg

1081 actttggcct ctgcaaagag ggcatcagtg acggggccac catgaaaacc ttctgtggga

1141 ccccggagta cctggcgcct gaggtgctgg aggacaatga ctatggccgg gccgtggact

1201 ggtgggggct gggtgtggtc atgtacgaga tgatgtgcgg ccgcctgccc ttctacaacc

1261 aggaccacga gcgcctcttc gagctcatcc tcatggaaga gatccgcttc ccgcgcacgc

1321 tcagccccga ggccaagtcc ctgcttgctg ggctgcttaa gaaggacccc aagcagaggc

1381 ttggtggggg gcccagcgat gccaaggagg tcatggagca caggttcttc ctcagcatca

1441 actggcagga cgtggtccag aagaagctcc tgccaccctt caaacctcag gtcacgtccg

1501 aggtcgacac aaggtacttc gatgatgaat ttaccgccca gtccatcaca atcacacccc

1561 ctgaccgcta tgacagcctg ggcttactgg agctggacca gcggacccac ttcccccagt

1621 tctcctactc ggccagcatc cgcgagtgag cagtctgccc acgcagagga cgcacgctcg

1681 ctgccatcac cgctgggtgg ttttttaccc ctgcc Coding sequence = nucleotide

204-1649.

Akt-3 mRNA (SEQIDNO:3)

1 gggagtcatc atgagcgatg ttaccattgt gaaagaaggt tgggttcaga agaggggaga

61 atatataaaa aactggaggc caagatactt ccttttgaag acagatggct cattcatagg

121 atataaagag aaacctcaag atgtggattt accttatccc ctcaacaact tttcagtggc

181 aaaatgccag ttaatgaaaa cagaacgacc aaagccaaac acatttataa tcagatgtct

241 ccagtggact actgttatag agagaacatt tcatgtagat actccagagg aaagggaaga

301 atggacagaa gctatccagg ctgtagcaga cagactgcag aggcaagaag aggagagaat

361 gaattgtagt ccaacttcac aaattgataa tataggagag gaagagatgg atgcctctac

421 aacccatcat aaaagaaaga caatgaatga ttttgactat ttgaaactac taggtaaagg

481 cacttttggg aaagttattt tggttcgaga gaaggcaagt ggaaaatact atgctatgaa

541 gattctgaag aaagaagtca ttattgcaaa ggatgaagtg gcacacactc taactgaaag

601 σagagtatta aagaacacta gacatccctt tttaacatcc ttgaaatatt ccttccagac

661 aaaagaccgt ttgtgttttg tgatggaata tgttaatggg ggcgagctgt ttttccattt

721 gtcgagagag cgggtgttct ctgaggaccg cacacgtttc tatggtgcag aaattgtctc

781 tgccttggac tatctacatt ccggaaagat tgtgtaccgt gatctcaagt tggagaatct

841 aatgctggac aaagatggcc acataaaaat tacagatttt ggactttgca aagaagggat

901 cacagatgca gccaccatga agacattctg tggcactcca gaatatctgg caccagaggt

961 gttagaagat aatgactatg gccgagcagt agactggtgg ggcctagggg ttgtcatgta

1021 tgaaatgatg tgtgggaggt tacctttcta caaccaggac catgagaaac tttttgaatt

1081 aatattaatg gaagacatta aatttcctcq aacactctct tcagatgcaa aatcattgct

1141 ttcagggctc ttgataaagg atccaaataa acgccttggt ggaggaccag atgatgcaaa

1201 agaaattatg agacacagtt tcttctctgg agtaaactgg caagatgtat aCgataaaaa

1261 gcttgtacct ccttttaaac ctcaagtaac atctgagaca gatactagat attttgatga

1321 agaatttaca gctcagacta ttacaataac accacctgaa aaatatgatg aggatggtat

1381 ggactgcatg gacaatgaga ggcggccgca tttccctcaa ttttcctact ctgcaagtgg

1441 acgagaataa gtctctttca ttctgctact tcactgtcat cttcaattta ttactgaaaa

1501 tgattcctgg acatcaccag tcctagctct tacacatagc aggggca

Coding sequence = nucleotide 11-1450

Intramyocardial transplantation of adult stem cells lias been proposed as a therapy to repair and regenerate damaged myocardium and to restore cardiac function after acute myocardial infarction (MI). Given their multipotency, low immunogenicity, amenability to ex vivo expansion and genetic modification, autologous bone marrow derived MSCs are suitable for this purpose. Injection of MSCs reduces post-infarction ventricular remodeling and in some cases improves left ventricular function. However prior to the invention, mechanism(s) underlying these therapeutic effects have not been clearly defined. In situ differentiation of the transplanted MSCs into cardiomyocytes and other constituent cardiac cell types has been suggested. Intramyocardial transplantation of MSCs transduced with a retroviral vector overexpressing the survival gene Akt markedly improves the therapeutic efficacy of MSCs in preventing ventricular remodeling and restoring cardiac function.

The data described herein shows that therapeutic effects seen with the administration of cells occur in less than 72 hours after infarction., These early dramatic effects cannot be readily attributed to myocardial regeneration or neoangiogenesis, but rather indicate that Akt-MSCs release biologically active factors that exert paracrine actions on the ischemic cardiomyocytes. Under hypoxic stimulation, genetically-modified bone marrow derived MSCs overexpressing the Akt gene release paracrine factors that exert cytoprotective effects on isolated cardiomyocytes. Intramyocardial injection of these substances reduces infarct size, prevents left ventricular dysfunction, and decreases in the number of apoptotic cardiomyocytes in vivo. In addition, no increase in microvessel density was observed in is the treated groups compared to controls 72 hours after the injection of the conditioned medium Thus, a significant portion of the salutary effects of Akt-MSCs transplantation is attributable to protection and functional recovery of ischemic myocardium, instead of, or in addition to, de novo cardiac repair and regeneration. The ability of bone marrow derived MSCs, especially Akt-MSCs, to produce factor(s) capable of protecting cardiomyocytes from cell death has not been previously demonstrated.

Secreted Frizzled-Related Proteins

The GENBANK™ Accession numbers of human Sfrps include BCO36503 (Sftpl), BC008666 (Sfrp2), and NM001463 (Sfrp3), hereby incorporated by reference. The amino acid sequence of exemplary Sfrp polypeptides and nucleotides encoding the polypeptides (coding sequences) are described below. The Sfrp polypeptides, mature processed forms, and/or fragments thereof are used in the cardioprotective and repair methods described herein.

Human SFRPl mRNA sequence (SEO ID NO:4)

1 cctgcagcct ccggagtcag tgccgcgcgc ccgccgcccc gcgccttcct gctcgccgca 61 cctccgggag ccggggcgca cccagcccgc agcgccgcct ccccgcccgc gccgcctccg 121 accgcaggcc gagggccgcc actggccggg gggaccgggc agcagcttgc ggccgcggag

181 ccgggcaacg ctggggactg cgccttttgt ccccggaggt ccctggaagt ttgcggcagg 241 acgcgcgcgg ggaggcggcg gaggcagccc cgacgtcgcg gagaacaggg cgcagagccg 301 gcatgggcat cgggcgcagc gaggggggcc gccgcggggc agccctgggc gtgctgctgg 361 cgctgggcgc ggcgcrtctg gccgtgggct cggccagcga gtacgactac gtgagcttcc 421 agtcggacat cggcccgtac cagagcgggc gcttctacac caagccacct cagtgcgtgg

481 acatccccgc ggacctgcgg ctgtgccaca acgtgggcta caagaagatg gtgctgccca 541 acctgctgga gcacgagacc atggcggagg tgaagcagca ggccagcagc tgggtgcccc 601 tgctcaacaa gaactgccac gccggcaccc aggtcttcct ctgctcgctc ttcgcgcccg 661 tctgcctgga ccggcccatc tacccgtgtc gctggctctg cgaggccgtg cgcgactcgt 721 gcgagccggt catgcagttc ttcggcttct actggcccga gatgcttaag tgtgacaagt

781 tccccgaggg ggacgtctgc atcgccatga cgccgcccaa tgccaccgaa gcctccaagc 841 cccaaggcac aacggtgtgt cctccctgtg acaacgagtt gaaatctgag gccatcattg 901 aacatctctg tgccagcgag tttgcactga ggatgaaaat aaaagaagtg aaaaaagaaa 961 atggcgacaa gaagattgtc cccaagaaga agaagcccct gaagttgggg cccatcaaga 1021 agaaggacct gaagaagctt gtgctgtacc tgaagaatgg ggctgactgt ccctgccacc

1081 agctggacaa cctcagccac cacttcctca tcatgggccg caaggtgaag agccagtact 1141 tgctgacggc catccacaag tgggacaaga aaaacaagga gttcaaaaac ttcatgaaga 1201 aaatgaaaaa ccatgagtgc cccacctttc agtccgtgtt taagtgattc tcccgggggc 1261 agggtgggga gggagcctcg ggtggggtgg gagcgggggg gacagtgccc cgggaacccg 1321 gtgggtcaca cacacgcact gcgcctgtca gtagtggaca ttgtaatcca gtcggcttgt

1381 tcttgcagca ttcccgctcc cttccctcca tagccacgct ccaaacccca gggtagccat 1441 ggccgggtaa agcaagggcc atttagatta ggaaggtttt taagatccgc aatgtggagc 1501 agcagccact gcacaggagg aggtgacaaa ccatttccaa cagcaacaca gccactaaaa 1561 cacaaaaagg gggattgggc ggaaagtgag agccagcagc aaaaactaca ttttgcaact 1621 tgttggtgtg gatctattgg ctgatctatg cctttcaact agaaaattct aatgattggc

1681 aagtcacgtt gttttcaggt ccagagtagt ttctttctgt ctgctttaaa tggaaacaga 1741 ctcataccac acttacaatt aaggtcaagc ccagaaagtg ataagtgcag ggaggaaaag 1801 tgcaagtcca ttatgtaata gtgacagcaa agggaccagg ggagaggcat tgccttctct 1861 gcccacagtc tttccgtgtg attgtctttg aatctgaatc agccagtctc agatgcccca 1921 aagtttcggt tcctatgagc ccggggcatg atctgatccc caagacatgt ggaggggcag

1981 cctgtgcctg cctttgtgtc agaaaaagga aaccacagtg agcctgagag agacggcgat 2041 tttcgggctg agaaggcagt agttttcaaa acacatagtt aaaaaagaaa caaatgaaaa 2101 aaattttaga acagtccagc aaattgctag tcagggtgaa ttgtgaaatt gggtgaagag 2161 cttaggattc taatctcatg ttttttcctt ttcacatttt taaaagaaca atgacaaaca 2221 cccacttatt tttcaaggtt ttaaaacagt ctacattgag catttgaaag gtgtgctaga

2281 acaaggtctc ctgatccgtc cgaggctgct tcccagagga gcagctctcc ccaggcattt

2341 gccaagggag gcggatttcc ctggtagtgt agctgtgtgg ctttccttcc tgaagagtcc 2401 gtggttgccc tagaacctaa caccccctag caaaactcac agagctttcc glttttttct 2461 ttcctgtaaa gaaacatttc ctttgaactt gattgcctat ggatcaaaga aattcagaac 2521 agcctgcctg tccccccgca ctttttacat atatttgttt catttctgca gatggaaagt 2581 tgacatgggt ggggtgtccc catccagcga gagagtttca aaagcaaaac atctctgcag

2641 tttttcccaa gtaccctgag atacttccca aagcccltat gtttaatcag cgatgtatat 2701 aagccagttc acttagacaa ctttaccctt cttgtccaat gtacaggaag tagttctaaa 2761 aaaaatgcat attaatttct tcccccaaag ccggattctt aattctctgc aacactttga 2821 ggacatttat gattgtccct ctgggccaat gcttataccc agtgaggatg ctgcagtgag 2881 gctgtaaagt ggccccctgc ggccctagcc tgacccggag gaaaggatgg tagattctgt

2941 taactcttga agactccagt atgaaaatca gcatgcccgc ctagttacct accggagagt 3001 tatcctgata aattaacctc tcacagttag tgatcctgtc cttttaacac cttttttgtg 3061 gggttctctc tgacctttca tcgtaaagtg ctggggacct taagtgattt gcctgtaatt 3121 ttggatgatt aaaaaatgtg tatatatatt agctaattag aaatattcta cttctctgtt 3181 gtcaaactga aattcagagc aagttcctga gtgcgtggat ctgggtctta gttctggttg

3241 attcactcaa gagttcagtg ctcatacgta tctgctcatt ttgacaaagt gcctcatgca 3301 accgggccct ctctctgcgg cagagtcctt agtggagggg tttacctgga acattagtag 3361 ttaccacaga atacggaaga gcaggtgact gtgctgtgca gctctctaaa tgggaattct 3421 caggtaggaa gcaacagctt cagaaagagc tcaaaataaa ttggaaatgt gaatcgcagc 3481 tgtgggtttt accaccgtct gtctcagagt cccaggacct tgagtgtcat tagttacttt

3541 attgaaggtt ttagacccat agcagctttg tctctgtcac atcagcaatt tcagaaccaa 3601 aagggaggct ctctgtaggc acagagctgc actatcacga gcctttgttt ttctccacaa 3661 agtatctaac aaaaccaatg tgcagactga ttggcctggt cattggtctc cgagagagga 3721 ggtttgcctg tgatttccta attatcgcta gggccaaggt gggatttgta aagctttaca 3781 ataatcattc tggatagagt cctgggaggt ccttggcaga actcagttaa atctttgaag

3841 aatatttgta gttatcttag aagatagcat gggaggtgag gattccaaaa acattttatt 3901 tttaaaatat cctgtgtaac acttggctct tggtacctgt gggttagcat caagttctcc 3961 ccagggtaga attcaatcag agctccagtt tgcatttgga tgtgtaaatt acagtaatcc 4021 catttcccaa acctaaaatc tgtttttctc atcagactct gagtaactgg ttgctgtgtc 4081 ataacttcat agatgcagga ggctcaggtg atctgtttga ggagagcacc ctaggcagcc

4141 tgcagggaat aacatactgg ccgttctgac ctgttgccag cagatacaca ggacatggat 4201 gaaattcccg tttcctctag tttcttcctg tagtactcct cttttagatc ctaagtctct 4261 tacaaaagct ttgaatactg tgaaaatgtt ttacattcca tttcatttgt gttgtttttt 4321 taactgcatt ttaccagatg ttttgatgtt atcgcttatg ttaatagtaa ttcccgtacg 4381 tgttcatttt attttcatgc tttttcagcc atgtatcaat attcacttga ctaaaatcac

4441 tcaattaatc aatgaaaaaa aaaaa

Human SFRPl protein sequence CSEO ID NO:5)

MGIGRSEGGRRGAALGVLLALGAALLAVGSASEYDYVSFQSDIG PYQSGRFYTKPPQCVDIPADLRLCHNVGYKKMVLPNLLEHETMAEVKQQASSWVPLL N

KNCHAGTQVFLCSLF APVCLDRPIYPCRWLCEA VPVDSCEPVMQFFGFYWPEMLKCDKF PEGDVCIAMTPPNATEASKPQGTTVCPPCDNELKSEAIIEHLCASEFALRMKIKEVKK ENGDiαCIVPKKKXPLKLGPIKXKDLKKLVLYLKNGADCPCHQLDNLSHHFLIMGRKVK SQYLLTAIHKWDKKNKEFKNFMKKMKNHECPTFQSVFK

Human SFRP2 mRNA sequence (SEO ID NO: 6)

1 caacggctca ttctgctccc ccgggtcgga gccccccgga gctgcgcgcg ggcttgcagc 61 gcctcgcccg cgctgtcctc ccggtgtccc gcttctccgc gccccagccg ccggctgcca 121 gcttttcggg gccccgagtc gcacccagcg aagagagcgg gcccgggaca agctcgaact

181 ccggccgcct cgcccttccc cggctccgct ccctctgccc cctcggggtc gcgcgcccac 241 gatgctgcag ggccctggct cgctgctgct gctcttcctc gcctcgcact gctgcctggg 301 ctcggcgcgc gggctcttcc tctttggcca gcccgacttc tcctacaagc gcagcaattg 361 caagcccatc cctgccaacc tgcagctgtg ccacggcatc gaataccaga acatgcggct 421 gcccaacctg ctgggccacg agaccatgaa ggaggtgctg gagcaggccg gcgcttggat

481 cccgctggtc atgaagcagt gccacccgga caccaagaag ttcctgtgct cgctcttcgc 541 ccccgtctgc ctcgatgacc tagacgagac catccagcca tgccactcgc tctgcgtgca 601 ggtgaaggac cgctgcgccc cggtcatgtc cgccttcggc ttcccctggc ccgacatgct 661 tgagtgcgac cgtttccccc aggacaacga cctttgcatc cccctcgcta gcagcgacca 721 cctcctgcca gccaccgagg aagctccaaa ggtatgtgaa gcctgcaaaa ataaaaatga

781 tgatgacaac gacataatgg aaacgctttg taaaaatgat tttgcactga aaataaaagt 841 gaaggagata acctacatca accgagatac caaaatcatc ctggagacca agagcaagac 901 catttacaag ctgaacggtg tgtccgaaag ggacctgaag aaatcggtgc tgtggctcaa 961 agacagcttg cagtgcacct gtgaggagat gaacgacatc aacgcgccct atctggtcat 1021 gggacagaaa cagggtgggg agctggtgat cacctcggtg aagcggtggc agaaggggca

1081 gagagagttc aagcgcatct cccgcagcat ccgcaagctg cagtgctagt cccggcatcc 1141 tgatggctcc gacaggcctg ctccagagca cggctgacca tttctgctcc gggatctcag 1201 ctcccgttcc ccaagcacac tcctagctgc tccagtctca gcctgggcag cttccccctg 1261 ccttttgcac gtttgcatcc ccagcatttc ctgagttata aggccacagg agtggatagc 1321 tgttttcacc taaaggaaaa gcccacccga atcttgtaga aatattcaaa ctaataaaat

1381 catgaatatt tttatgaagt ttaaaaatag ctcactttaa agctagtttt gaataggtgc 1441 aactgtgact tgggtctggt tggttgttgt ttgttgtttt gagtcagctg attttcactt 1501 cccactgagg ttgtcataac atgcaaattg cttcaatttt ctctgtggcc caaacttgtg 1561 ggtcacaaac cctgttgaga taaagctggc tgttatctca acatcttcat cagctccaga 1621 ctgagactca gtgtctaagt cttacaacaa ttcatcattt tataccttca atgggaactt

1681 aaactgttac atgtatcaca ttccagctac aatacttcca tttattagaa gcacattaac 1741 catttctata gcatgatttc ttcaagtaaa aggcaaaaga tataaatttt ataattgact 1801 tgagtacttt aagccttgtt taaaacattt cttacttaac ttttgcaaat taaacccatt 1861 gtagcttacc tgtaatatac atagtagttt acctttaaaa gttgtaaaaa tattgcttta 1921 accaacactg taaatatttc agataaacat tatattcttg tatataaact ttacatcctg

1981 ttttacctat aaaaaaaaaa aaaaa

Human SFRP2 protein sequence fSEO ID NO:7)

MLQGPGSLLLLFLASHCCLGSARGLFLFGQPDFSYKRSNCKPIF

ANLQLCHGIEYQNMRLPNLLGHETMKEVLEQAGAWIPLVMKQCHPDTKKFLCSLFAP V CLDDLDETIQPCHSLCVQVKDRCAPVMSAFGFPWPDMLECDRFPQDNDLCIPLASSDH LLPATEEAPKVCEACKNKNDDDNDIMETLCIWDF ALKIKVKEITYINRDTKIILETKS KTIYKLNGVSERDLKKSVLWLKDSLQCTCEEMNDINAPYLVMGQKQGGELVITSVKR W QKGQREFKRISRSIRKLQC

Human SFRP3 mRNA sequence (SEO ID N0:8)

1 gttgggaaag agcagcctgg gcggcagggg cggtggctgg agctcggtaa agctcgtggg 61 accccattgg gggaatttga tccaaggaag cggtgattgc cgggggagga gaagctccca 121 gatccttgtg tccacttgca gcgggggagg cggagacggc ggagcgggcc ttttggcgtc 181 cactgcgcgg ctgcaccctg ccccatcctg ccgggatcat ggtctgcggc agcccgggag

241 ggatgctgct gctgcgggcc gggctgcttg ccctggctgc tctctgcctg ctccgggtgc 301 ccggggctcg ggctgcagcc tgtgagcccg tccgcatccc cctgtgcaag tccctgccct 361 ggaacatgac taagatgccc aaccacctgc accacagcac tcaggccaac gccatcctgg 421 ccatcgagca gttcgaaggt ctgctgggca cccactgcag ccccgatctg ctcttcttcc 481 tctgtgccat gtacgcgccc atctgcacca ttgacttcca gcacgagccc atcaagccct

541 gtaagtctgt gtgcgagcgg gcccggcagg gctgtgagcc catactcatc aagtaccgcc 601 actcgtggcc ggagaacctg gcctgcgagg agctgccagt gtacgacagg ggcgtgtgca 661 tctctcccga ggccatcgtt actgcggacg gagctgattt tcctatggat tctagtaacg 721 gaaactgtag aggggcaagc agtgaacgct gtaaatgtaa gcctattaga gctacacaga 781 agacctattt ccggaacaat tacaactatg tcattcgggc taaagttaaa gagataaaga

841 ctaagtgcca tgatgtgact gcagtagtgg aggtgaagga gattctaaag tcctctctgg 901 taaacattcc acgggacact gtcaacctct ataccagctc tggctgcctc tgccctccac 961 ttaatgttaa tgaggaatat atcatcatgg gctatgaaga tgaggaacgt tccagattac 1021 tcttggtgga aggctctata gctgagaagt ggaaggatcg actcggtaaa aaagttaagc 1081 gctgggatat gaagcttcgt catcttggac tcagtaaaag tgattctagc aatagtgatt

1141 ccactcagag tcagaagtct ggcaggaact cgaacccccg gcaagcacgc aactaaatcc 1201 cgaaatacaa aaagtaacac agtggacttc ctattaagac ttacttgcat tgctggacta 1261 gcaaaggaaa attgcactat tgcacatcat attctattgt ttactataaa aatcatgtga 1321 taactgatta ttacttctgt ttctcttttg gtttctgctt ctctcttctc tcaacccctt 1381 tgtaatggtt tgggggcaga ctcttaagta tattgtgagt tttctatttc actaatcatg

1441 agaaaaactg ttcttttgca ataataataa attaaacatg ctgttaccag agcctctttg 1501 ctggagtctc cagatgttaa tttactttct gcaccccaat tgggaatgca atattggatg 1561 aaaagagagg tttctggtat tcacagaaag ctagatatgc cttaaaacat actctgccga 1621 tctaattaca gccttatttt tgtatgcctt ttgggcattc tcctcatgct tagaaagttc 1681 caaatgttta taaaggtaaa atggcagttt gaagtcaaat gtcacatagg caaagcaatc

1741 aagcaccagg aagtgtttat gaggaaacaa cacccaagat gaattatttt tgagactgtc 1801 aggaagtaaa ataaatagga gcttaagaaa gaacattttg cctgattgag aagcacaact 1861 gaaaccagta gccgctgggg tgttaatggt agcattcttc ttttggcaat acatttgatt 1921 tgttcatgaa tatattaatc agcattagag aaatgaatta taactagaca tctgctgtta 1981 tcaccatagt tttgtttaat ttgcttcctt ttaaataaac ccattggtga aagtcccaaa

2041 aaaaaaaaaa aaaaaaaa

Human SFRP3 protein sequence (SEO ID NO:9)

MVCGSPGGMLLLRAGLLALAALCLLRVPGARAAACEPVRIPLCK SLPWNMTI ¹ CMPNI-ILHHSTQANAILAIEQFEGLLGTHCSPDLLFFLCAMYAPICTIDFQH EPIKPCKSVCERARQGCEPILIKYRHSWPENLACEELPVYDRGVCISPEAIVTADGAD FPMDSSNGNCRGASSERCKCICPIRATQKTYFRNNYNYVIRAKVKEIKTKCHDVTAWE VKEILKSSLVNIPRDTVNLYTSSGCLCPPLNVNEEYIIMGYEDEERSRLLLVEGSIAE KWKDRLGiαCVKRWDMKLRHLGLSKSDSSNSDSTQSQKSGRNSNPRQARN

Coronary Disorders Many patients are either at risk for or have suffered from various types of heart failure, including myocardial infarction, symptomatic or imsymptomatic left ventricular dysfunction, or congestive heart failure (CHF). An estimated 4.9 million Americans are now diagnosed with CHF, with 400,000 new cases added annually. This year over 300,000 Americans will die from congestive heart failure. Without therapeutic invention, cardiac muscle does not normally have reparative potential. The ability to augment weakened cardiac muscle as described herein is a major advance in the treatment of cardiomyopathy and heart failure. Despite advances in the medical therapy of heart failure, the mortality due to this disorder remains high, where most patients die within one to five years after diagnosis.

Coronary disorders are categorized into at least two groups. Acute coronary disorders include myocardial infarction, and chronic coronary disorders include chronic coronary ischemia, arteriosclerosis, congestive heart failure, angina, atherosclerosis, and myocardial hypertrophy. Other coronary disorders include stroke, myocardial infarction, dilated cardiomyopathy, restenosis, coronary artery disease, heart failure, arrhythmia, angina, or hypertension. Acute coronary disorders result in a sudden blockage of the blood supply to the heart which deprives the heart tissue of oxygen and nutrients, resulting in damage and death of the cardiac tissue. In contrast, chronic coronary disorders are characterized by a gradual decrease of oxygen and blood supply to the heart tissue overtime causing progressive damage and the eventual death of cardiac tissue. Cvtoprotective Compounds

A cvtoprotective (i.e., cell protective or regenerative) compound is a compound that that is capable of inhibiting cell damage such as apoptosis induced or oxidative-stress induced cell death. Cytoprotective compounds also include compounds that induce cell repair and

regeneration. A cytoprotective compound is a polypeptide or nucleic acid encoding the polypeptide, the expression of which is increased in MSC-Akt cells under hypoxic conditions as compared to normoxic condition. For example, a cytoprotective polypeptide includes Sfrp-1, 2, and/or 3, adipsin, adrenomedullin, chemoldne (C-C motif) ligand 2, cysteine rich protein 61, lysyl oxidase-like 2, serine proteinase inhibitor or vascular endothelial growth factor or fragment thereof. Other proteins/polypeptides with cytoprotective and regenerative properties include hi, 5, 8, 12, and 13. In some aspects the compound is a nucleic acid that increases expression of a nucleic acid that encodes a polypeptide or an agonist of a cytoprotective polypeptide. Therapeutic Methods The invention provides methods of inhibiting cell or tissue damage and ischemic or reperfusion related injuries. Also included are methods of regenerating injured myocardial tissue. The therapeutic methods include administering to a subject, or contacting a cell or tissue with a composition containing a cytoprotective compound.

Cell/tissue damage is characterized by a loss of one or more cellular functions characteristic of the cell type which can lead to eventual cell death. For example, cell damage to a cardiomyocyte results in the loss contractile function of the cell resulting in a loss of ventricular function of the heart tissue. An ischemic or reperfusion related injury results in tissue necrosis and scar formation.

Injured myocardial tissue is defined for example by necrosis, scarring or yellow softening of the myocardial tissue. Injured myocardial tissue leads to one or more of several mechanical complications of the heart, such as ventricular dysfunction, decrease forward cardiac output, as well as inflammation of the lining around the heart ( i.e., pericarditis). Accordingly, regenerating injured myocardial tissue results in histological and functional restoration of the tissue. The cell is any cell subject to apoptotic or oxidative stress induced cell death. For example, the cell is a cardiac cell such as a cardiomyocyte, a liver cell or a kidney cell. Tissues to be treated include a cardiac tissue, a pulmonary tissue, or a hepatic tissue. For example, the tissue is an muscle tissue such as heart muscle. The tissue has been damaged by disease or deprivation of oxygen. Cells or tissues are directly contacted with a cytoprotective compound, e.g. by direct injection into the myocardium. Alternatively, the cytoprotective compound is administered systemically. The cytoprotective compounds are administered in an amount sufficient to decrease (e.g., inhibit) apoptosis induced or oxidative stress induced cell death as compared to

untreated cells or tissues. Cells undergoing apoptosis are identified by detecting cell shrinkage, membrane blebbing, caspase activation, chromatin condensation and fragmentation as is well know in the art. Cell undergoing oxidative stress are identified by detecting an increase production of reactive oxygen species (ROS). A decrease in cell death (i.e., an increase in cell viability) is measured by using standard cell viability measurements such as BrdU incorporation assay and trypan blue exclusion.

The methods are useful to alleviate the symptoms of a variety disorders, such as disorders associated with aberrant cell damage, ischemic disorders, and reperfusion related disorders. For example, the methods are useful in alleviating a symptom of stroke, myocardial infarction, chronic coronary ischemia, arteriosclerosis, congestive heart failure, dilated cardiomyopathy, restenosis, coronary artery disease, heart failure, arrhythmia, angina, atherosclerosis, hypertension, renal failure, kidney ischemia or myocardial hypertrophy. The disorders are diagnosed and or monitored, typically by a physician using standard methodologies. Alleviation of one or more symptoms of the disorder indicates that the compound confers a clinical benefit, such as a reduction in one or more of the following symptoms: shortness of breath, fluid retention, headaches, dizzy spells, chest pain, left shoulder or arm pain, and ventricular dysfunction Therapeutic Administration

The invention includes administering to a subject a composition comprising a cytoprotective compound (also referred to herein as a "therapeutic compound").

An effective amount of a therapeutic compound administered systemically in the range of about 0.1 mg/kg to about 150 mg/kg. Proteins or peptides are administered directly into the heart by injection at a dose of 1-1000 μg. For example, 10, 20, 30, 40, 50, 60, 75, 100 μg are administered by myocardial injection. Effective doses vary, as recognized by those skilled in the art, depending on route of administration, excipient usage, and coadministration with other therapeutic treatments including use of other anti-apoptotic agents or therapeutic agents for treating, preventing or alleviating a symptom of a particular cardiac disorder. A therapeutic regimen is carried out by identifying a mammal, e.g., a human patient suffering from (or at risk of developing) an cardiac disorder, using standard methods. The pharmaceutical compound is administered to such an individual using methods known in the art. Preferably, the compound is administered orally, nasally, topically or parenterally, e.g., subcutaneously, intraperitoneally, intramuscularly, and intravenously. The compound is administered prophylactically, or after the detection of an cardiac event such as a

heart attack. The compound is optionally formulated as a component of a cocktail of therapeutic drugs to treat cardiac disorders. Examples of formulations suitable for parenteral administration include aqueous solutions of the active agent in an isotonic saline solution, a 5% glucose solution, or another standard pharmaceutically acceptable excipient. Standard solubilizing agents such as PVP or cyclodextrins are also utilized as pharmaceutical excipients for delivery of the therapeutic compounds.

The therapeutic compounds described herein are formulated into compositions for administration utilizing conventional methods. For example, cytoprotective compounds are formulated in a capsule or a tablet for oral administration. Capsules may contain any standard pharmaceutically acceptable materials such as gelatin or cellulose. Tablets are formulated in accordance with conventional procedures by compressing mixtures of a therapeutic compound with a solid carrier and a lubricant. Examples of solid carriers include starch and sugar bentonite. The compound is administered in the form of a hard shell tablet or a capsule containing a binder, e.g., lactose or mannitol, a conventional filler, and a tableting agent. Other formulations include an ointment, suppository, paste, spray, patch, cream, gel, resorbable sponge, or foam. Such formulations are produced using methods well known in the art.

Cytoprotective compounds are effective upon direct contact of the compound with the affected tissue, e.g. heart muscle. Alternatively, cytoprotective compounds are administered systemically. Additionally, compounds are administered by implanting (either directly into an organ such as the heart or subcutaneonsly) a solid or resorbable matrix which slowly releases the compound into adjacent and surrounding tissues of the subject. For example, the compound is delivered to the cardiac tissue (i.e., myocardium, pericardium, or endocardium) by direct intracoronary injection through the chest wall or using standard percutaneous catheter based methods under fluoroscopic guidance for direct injection into tissue such as the myocardium or infusion of an inhibitor from a stent or catheter which is inserted into a bodily lumen. Any variety of coronary catheter, or a perfusion catheter, is used to administer the compound. Alternatively, the compound is coated or impregnated on a stent that is placed in a coronary vessel.

The present invention is further illustrated, but not limited, by the following examples. Example 1: The family of Secreted Frizzled related proteins mediate Akt-MSC cardiac protection and repair through paracrine mechanisms

Loss of myocardial tissue due to ischemia and infarction usually leads to inflammation, scarring and cardiac myocyte hypertrophy. However, since the myocardium has limited

endogenous repair and regenerative capacity, these compensatory pathophysiological responses to myocardial damage are frequently inefficient to sustain cardiac function, resulting eventually in cardiac dilation and failure.

Cellular cardiomyoplasty has been proposed as a potential approach for reconstitution of infarcted myocardium and recuperation of cardiac function. Several cell-based strategies have evolved using a variety of alternatives, such as skeletal muscle myoblasts, embryonic stem cells, fetal cardiomyocytes, myocardial stem cells and marrow-derived mesenchymal stem cells (MSC). Among these methods, the use of MSCs has shown much promise for clinical applications. Protection may result from differentiation of donor cells into cardiomyocytes, fusion of donor cells with host cardiomyocytes, or through enhanced myocardial perfusion. A significant mechanism by which cardiomyocyte survival/function is mediated by stem cells is through paracrine effects.

Intramyocardial transplantation of MSCs overexpressing the survival gene Akt (Akt- MSCs) resulted in reduced infarct size and volume, ventricular remodeling and cardiac dysfunction, 2 weeks after infarction, when compared to hearts transplanted with control MSCs alone. Moreover, conditioned medium from Akt-MSCs provided cytoprotection of cardiac myocytes exposed to hypoxia in vitro and once injected into infarcted hearts dramatically limited the infarct size and prevented ventricular dysfunction within 72 hours. Since this early effect cannot be readily explained by significant regeneration of cardiac myocytes from the donor cells or enhancement of angiogenesis, these data indicate that the observed effect is due to paracrine factors released by the Akt-MSCs that prevent myocyte loss.

Although it has been reported that native MSCs can secrete angiogenic factors and cytokines, the ability of bone marrow derived MSCs, especially Akt-MSCs, to produce factor(s) capable of acutely protecting the cardiomyocytes from cell death has not been previously documented. Given that apoptosis is the principal cause of myocytes loss in the acute phase of MI, therapeutic methods that prevent or reduce apoptotic cell death are effective in reducing the severity and extent of myocardial infarction. Paracrine factor(s) secreted by MSCs were identified, and biological evidence of their therapeutic potential is described below. A strategy was developed that allows large-scale identification and functional screening of secreted factors that are responsible for the enhanced cytoprotective effect of the Akt-MSCs. First, microarray analysis of Akt-MSC and control MSC under normoxia and 6 h of hypoxia was performed. Approximately 62 transcripts that were differentially regulated between the Akt-

MSC and control MSC under normoxia or hypoxia encode for known secreted proteins based on their annotation. Included in this list were three members of the secreted Frizzled-related protein (Sfrp) family, SM, Sfrp2 and Sfrp3. Sfrps bind to Wnt ligands or their frizzled receptors and modulate Wnt signaling. All three factors are associated with regulation of cell fate, differentiation, and cell death and cell growth. The data described herein provide evidence that Akt-MSCs exert an early protection to the injured heart by secreting Sfrps, which then modulate the X pathway in cardiac myocytes to prevent cell death.

The following material and methods were used to generate the data described below. Purification of mesenchymal stem cells and retroviral transduction. Bone marrow cells from 8-10 week-old wild type male C57BL/6J mice (Jackson

Laboratory), were collected in a-modified minimum essential media supplemented with 17% fetal bovine serum; 100 units/ml penicillin, 100 mg/ml streptomycin; amphotericin B 0.25 mg/ml. Mononuclear cells were then isolated from aspirates by Ficoll-Paque (Amersham Biosciences) gradient centrifugation. For the retroviral transduction, murine Aktl cDNA tagged with a c-myc epitope by PCR-ampliiϊcation was cloned into pMSCV-IRES-GFP vector. To overexpress Akt/GFP (Akt-MSC) or GFP alone (GFP-MSC), MSCs were infected with high- titer VSV-G pseudotyped retrovirus.

Gene expression profiling and RNA validation

Eight micrograms of total RNA from mouse GFP-MSCs and Akt-MSCs (n=3 per group) under normoxia or hypoxia (6 hours) were used for microarray analysis. Affymetrix GeneChips of Mouse Genome 430 2.0 Arrays (Affymetrix. CA), which allows analysis of -45000 transcrips, was performed in triplicate, and analyzed with Affymetrix Microarray Suite (MAS 5.0). For further analysis various Dhip was used. All possible comparisons (Akt-MSC normoxia vs. GFP-MSC normoxia, Akt-MSC hypoxia vs. GFP-MSC hypoxia, GFP-MSC hypoxia vs. GFP-MSC normoxia and Akt-MSC hypoxia vs. Akt-MSC normoxia) were tested. The transcripts were then annotated using various databases to compile a list of potent secreted candidates.

Gene expression profiling was determined by quantitative real-time RT-PCR (QPCR) for selected genes with appropriate primer mixtures ( TaqMan© Gene Expression Assays, No. 4331182) from Applied Biosystems (Sfrpl, Mm00489161; Sfrp2, Mm00485986; Sfrp3(Frzb), Mm00441378; Gapdh, Mm99999915). Conditioned media collection and concentration

Passage 3 to 5 GFP-MSCs and Akt-MSCs reached to 90% confluence in 10 cm dishes. The cells were then left either into a standard incubator or the hypoxic chamber in the medium (αMEM+ 0.2% FBS +ITS) for 6 hours. Plates with medium only were also left at the same conditions as control-conditioned medium. The medium was concentrated up to 50X using a Millipore system with membrane (Aniicon Ultra-15). Western Blotting

Proteins from conditioned medium from MSCs were separated by SDS page gel (Invitrogen) and transferred to nitrocellulose membranes (Bio-Rad). The blots were incubated with Sfrp2 primary antibody (Santa Cruz Biotechnology, Inc.) and then with appropriate secondary antibody conjugated with horseradish peroxidase (Amersham Biosciences). Complexes were detected by chemiluminescence (LumiGLO, Cell Signaling). Suppression of secreted factor effect by siRNA

GFP-MSCs or Akt-MSCs were incubated overnight with OptiMEM medium containing lμM siRNA for Sfrps (Sfrpl, sense (5'→3'): CGGAUUGUAAAGAACUGCATT (SEQ ID NO: 10), antisens (5'→3'): UGCAGUUCUUUAC AAUCCGTT (SEQ ID NO: 11); Sfrp2, sense (5'→3'): GGACGACAACGACAUCAUGTT (SEQ ED NO: 12), antisense (5'→3'): CAUGAUGUCGUUGUCGUCCTC (SEQ ID NO: 13); Sfrp3, sense (5'→3'):

CCGUCAAUCUUUAUACCACTT (SEQ ID NO:14), antisense (5'->3'):

GUGGUAUAAAGAUUGACGGTG (SEQ ID NO: 15); Ambion). Rhodamine- labeled GFP siRNA (Qiagen) was used to assess the efficiency of transfection. Cells were incubated in normal medium for 48 hours, then exposed to a serum free medium (αMEM+ 0.2% FBS +ITS) at normoxia or hypoxia as described above. The medium was concentrated for further analysis. The efficiency of the siRNA-mediated reduction of Sfrps was assessed by QPCR using 18S as a control. Adult rat ventricular myocyte isolation and quantification of apoptotic cardiomyocytes

Adult rat ventricular myocytes (ARVMs) were isolated by enzymatic dissociation. 1 x 10 ⁶ cells were incubated in 10cm dishes (Becton Dickinson) overnight with full 199 medium (0.2% albumin, 2mM carnitine, 5mM creatine, 5mM taurine and lμg/ml of recombinant human insulin, Sigma). On the following day, the medium was replaced with optimal medium according to different assays. Hypoxic condition was created by incubating the cells at 37 ⁰ C into a hypoxia chamber with an atmosphere of 5% CO ₂ /95% N ₂ . Oxygen level into the chamber was controlled to 0.5%. Apoptosis was determined by measuring the activity of cleaved-caspase 3 using a caspase-specific fluorogenic substrate according to the protocol for Caspase 3 assay kit (Sigma, St. Louis, MO). ARVMs were lysed after treatment with SFRPs for 24 hours under hypoxia. 5ul of cell extract was incubated in reaction buffer at room temperature for 1 hour. The enzyme- catalyzed release of 7-amino-4-methyl coumarin (AMC) was measured by a fluorescence microplate reader. Fluorescent units were converted to pmole AMC/μl sample/min/μg protein, using a standard curve of AMC. Quantitation of morphologic changes of ARVC following hypoxic exposure

Isolated cardiomyocytes were seeded in multi-well plates (Becton Dickinson, Franklin Lakes, NJ, USA) precoated with laminin (lμg/cm ² ) and left overnight in standard growth medium (Ml 99). One day later, the medium was replaced by serum-free medium with different doses of Sfrp2. The ARVCs were then placed in the hypoxia chamber. The viability of the ARVCs was evaluated on the basis of their morphology using a phase contrast microscope, and rod-shaped cardiomyocytes were considered viable. The number of round shaped cardiomyocytes was counted in 6 random high power fields and expressed as a percentage of total number of cells.

Myocardial infarction model and determination of infarct size

Ligation of the left anterior descending coronary artery was performed on 170 to 200 grams female Sprague Dawley rats (Harlan World Headquarters, Indianapolis, Indiana). A left

thoracotomy was performed under anesthesia, and the vessel was ligated with a silk suture at midway between the left atrium and the apex of the heart. The infarction was then assessed by the change of color and kinesis of the apex and the anterior-lateral wall. Thirty minutes later 250 μl conditioned media was injected in 5 different sites around the border zone. An equivalent amount of PBS was injected in the control group. Then the wound was sutured immediately.

Infarct size was analyzed by staining the tissue 5 min at 37 ⁰ C with planar morphometry in triphenyl tetrazolium chloride (TTC, Sigma Chemicals) followed by fixation of 12 hours in 10% phosphate-buffered formalin, and expressed as a percentage of the total ventricular area.

Akt regulated Expression of Sfrps in MSCs

Since the secreted frizzled-related sequence, protein 2 (Sfrp2) appeared to be expressed highly only in Akt-MSCs, and two other members of the same family (Sfrpl and Sfrp3) were also upregulated in these cells, efforts were focused on these molecules. First, MSCs-Akt and control Gfp-MSCs were cultured under normoxia or 6 hours of hypoxia and the RNA was collected and used to confirm the microarray data by quantitative PCR (Q-PCR). The expression pattern of all genes Sfrpl, Sfrp2 and Sfrp3 was consistent with the microarray results. Although both Sfrpl and Sfrp3 exhibited a consistent trend (P<0.1) of being expressed higher in Akt-MSCs, the most dramatic and significant differences were shown in the Sfrp2 levels (almost undetectable in control cells as opposed to high levels in Akt-MSCs). No significant changes were observed in the levels of all three genes in regard to hypoxia treatment in either control MSC or Akt-MSCs.

To further validate the observations at the protein level and to evaluate the effect of Akt on Sfrp2 expression, control mouse MSCs and Akt-MSCs were cultured under nomioxic or hypoxic conditions for 6 hours with PI3K inhibitor ( LY294002 50 mM) or vehicle. The conditioned medium was then collected and concentrated for protein analysis. Sfrp2 was highly expressed in the conditioned medium from the Akt-MSC cells at both normoxia and hypoxia. The levels of Sfrp2 were low or undetectable in the supernatant from GFP-MSCs under normoxia or hypoxia. Furthermore, the expression of Sfrp2 in the Akt-MSC cells was dependent to the PI3K pathway since inhibition of the PI3K, also abolished 8frp2 expression from the medium. Sfrpl and Sfrp3 showed similar patterns of protein expression.

Akt-MSCs promote cardiac myocyte cell survival after injury through Sfrp mediated paracrine effects

To determine whether Sfrps are a key mediator of the early cytoprotective effect of the conditioned medium in vitro, the effects of conditioned medium from cultured Akt-MSCs (Akt CM) and Akt-MSCs that did not express Sfrpl, Sfrp2 or Sfrp3 due to siRNA treatment (Akt- Sfφ2 CM) on the viability of adult rat cardiac myocytes (ARVCs) subjected to hypoxia were assessed. The conditioned media (CM) from Akt-MSCs of Gfp-MSCs was collected and concentrated after 6 hours of exposure to either normoxia or hypoxia. The CM was then added to ARVCs that were exposed to 24h of hypoxia. The experimental conditions included ARVCs that were incubated with either control conditioned medium (Ctr CM), conditioned medium from Akt-MSCs or Gfp-MSCs (Akt CM and Gfp CM) and conditioned medium Akt-MSCs or Gfp-MSCs that did not express Sfrp2 due to siRNA treatment (Akt minus Sfrp2 CM and Gfp minus Sfrp2 CM respectively). The data showed that ARVCs maintained under normoxic conditions for 24 hours were viable and exhibited their typical rod-shaped appearance. Exposure of ARVCs to 24 hours of hypoxia in control conditioned medium (Ctr CM) resulted in a 200% increase in cell death as indicated by caspase activity assay. Moreover, as expected the addition of Gfp CM had no effect whereas addition of Akt CM resulted in a reduction of caspase activity (64% as compared to Ctr CM) to levels similar to normoxic conditions. However, exposure of hypoxic myocytes to Akt minus Sfrp2 CM resulted in increased caspase activity compared to Ctr CM indicating higher cell death levels. Finally, reduction of Sfrp2 expression in the Gfp CM did not have did not have any significant impact on its effect on hypoxic cardiac myocytes. To examine the direct effect of Sfrps on ARVCs we also performed gain of function experiments in vitro. ARVCs were maintained in standard growth medium at normoxia or at 24h hypoxia. Sfrpl, Sfrp2, Sfrp3 or vehicle was then added at various concentrations and apoptosis levels were assessed as before by measuring caspase activity. Treatment with as low as 0.1 ng/ml of Sfrpl or Sfrp2 resulted in significant reduction in caspase activity (36% and 33% respectively). However, higher concentrations of Sfrpl showed reduced protection. On the contrary, Sfrp2 mediated reduction of cell death was positive correlated to higher concentrations of the molecule and seemed to plateau around the concentration of 10 ng/ml (55% reduction in caspase activity). Sfrp3 treatment reduced caspase activity only in concentrations higher than 10 ng/ml and overall was less potent that the other molecules (54% reduction at 500 ng/ml). Finally, to corroborate the results from the apoptosis assays, the relative number of healthy ARVCs after 24 hours of hypoxia was assessed based on their ATP synthesis levels. For this, again the cells were grown in normoxia or hypoxia with PBS or Akt CM, Akt-Sfrp2 CM, lOng/ml Sfrp2, or 500ng/ml Sfrp3 for 24 h. Exposure of ARVCs to 24hplus Akt CM increased

cell viability by 35% whereas medium from Akt cells that did not express Sfrp2 increased cell viability only by 9%. Treatment with Sfrp2 and Sfrp3 resulted in 24% and 17% increase in viability respectively.

Sfrp treatment protects the heart from myocardial injury To elucidate the therapeutic potential of the Sfrps, we studied the direct effects of Sfrps on infarct size by intramyocardial injection of Sfrp 1, Sfrp2 or Sfrp3 peptide. For this, lμg of Sfrpl, Sfrp2 or Sfrp3 were injected into 5 different sites in the heart at the infarct border zone 30 minutes after coronary artery occlusion. Additional groups included hearts injected with PBS as negative control, hearts injected with Akt CM as positive control and hearts injected with Akt minusSfrp2 CM to provide further evidence of Sfrp2 role in the protective Akt -MSC CM mediated cardiac protection in vivo. Hearts were isolated 72 hours later and infarct size was estimated by TTC staining. Injection of Sfrp2 had an effect of 69% reduction of infarct size, while injection of the same concentration of Sfrpl resulted in 50% reduction in infarct size and the same dose of Sfrp3 did not have any effect on infarct size.. Since Sfrp2have been shown to have the most potent effect from all the three Sfrps tested, its physiological significance in Akt- MSC mediated myocardial protection in vivo was also evaluated. Injection of Akt CM in infarcted hearts resulted in 71 % reduction in the infarct size after MI within 72 hours, whereas injection Akt minus Sfrp2 CM did not show any significant protection. These results indicate that Sfrps secreted from Akt-MSCs protects the myocardium from injury. Sfrps mediate cardioprotection

Despite vigorous efforts and the great potential of cell-based therapies for cardiac disease, the mechanisms underlying their therapeutic effect are still under debate. The data described herein indicates that MSCs exert an early protective effect in the injured myocardium by preventing myocyte cell death. This effect is enhanced by the overexpression of Akt and includes paracrine factors that regulate the Wnt signaling pathway in cardiac myocytes. Sfrp modulators of Wnt pathway protect from hypoxia cell death in vitro and result in reduction of infarct size in vivo.

Members of the Secreted frizzled-related protein (Sfrp) family act as modulators of the Wnt signaling pathway thereby influencing a range of biological processes, such as cell fate, cell adhesion, differentiation and survival. Sfrps are inhibitors of the Wnt signaling pathway. They act through binding of Wnts and altering their ability to bind to their frizzled receptors or by forming non functional complexes with the frizzled receptors themselves. However, some studies suggest that Sfrpl at low concentrations may actually promote Wnt signaling.

Furthermore, it has been reported that similar concentrations of Sfrpl and Sfrp2 result in different cellular responses. For instance, Sfrpl has been shown to sensitize cells to TNF induced apoptosis whereas Sfrp2 conferred resistance. A proposed explanation for these observations is that the Sfrp specific effects are closely dependent on the range of their Wnt partners present, the relative affinities of different Sfrps for Wnt or Frizzled receptors, tissues specific responses or biphasic responses to different concentrations of Sfrp. The present data support this mechanis, since the three different Sfrps tested confered variable degrees of protection to cardiac myocytes and these effect was dependent on their concentration levels. Prior to the invention, little was known about the role of Sfrps in cardiac tissue. Sfrpl has been associated with heart morphogeneiss, whereas Sfpr3 and Sfrp4 were found to be upregulated in volume overload induced hypertrophy. Evidence suggests that they are play a role during cardiac ischemia but again their role is diverse and not fully understood. For instance, overexpression of Sfrpl seemed to protect the heart from injury in a model of coronary ligation but has been reported to alleviate it and reverse the benefit of preconditioning in a model of ischemia /reperfusion. Similarly few studies have been conducted in regard to the role of Wnt singaling in cardiac myocyte survival. The present data provides evidence that Sfrp activates/ inhibits Wnt signaling.

The data do not exclude additional paracrine effects from other proteins secreted by the Akt-MSCs. Indeed, other secreted molecules are also expressed and are involved in different aspects of cardiac repair such as immunological responses, angiogenesis, recruitment/expansion of cardiac stem cells, regeneration and/or remodeling. For example, administration of vascular endothelial growth factor A (a growth factor with higher levels in Akt-MSCs) resulted in repaired myocardium by promoting angiogenesis and vascularization. Moreover, paracrine factors exert not only individual effects, but in some examples, one factor enhances the effect of another, i.e, a synergistic relationship is present between the different secreted factors expressed by the MSCs. In other examples, the presence of one factor inhibits the effects of one or more others.

Paracrine factors, e.g., Sfrps, contained in conditioned medium from Akt-MSCs are useful in therapeutic methods to prevent or reduce cell death, e.g., apoptotic cell death, of cardiac cells. The data indicates that simple administration of Sfrp2 alone or in combination with other molecules achieve cardioprotective results similar and in some cases better than those seen with stem cell based therapy. Methods that employ these paracrine factors have numerous advantages over cell based therapies. For example, many of the difficulties of stem cell based

therapy such as availability of cells, laborious maintenance of cell lines, limited alternative administration methods as well as difficulties in successful delivery and survival of the cells can be avoided. Administration of a peptide or a cocktail of peptides to the injured myocardium is a simpler, delivery methods, and dosages are more easily modified to achieve higher efficiency with lower toxicity or side effects and does not involve any of the ethical concerns associated with cell therapy.

Example 2: Secreted Frizzled Related Protein 2 is the Key Stem Cell Paracrine Factor Mediating Myocardial Survival and Repair

Using a comprehensive functional genomic strategy, Sfrp2 was shown to be a key stem cell paracrine factor that mediates myocardial survival and repair following ischemic injury. Sfrp2 modulates Wnt signaling, and cardiomyocytes treated with secreted frizzled related protein increase cellular β-catenin and up-regulate expression of anti-apoptotic genes. These findings demonstrate the key role played by Sfrp2 in mediating the paracrine effects of Akt mesenchymal stem cells on tissue repair and identify modulation of Wnt signaling as a strategy for treating heart disease.

Microarray data confirmed by Western blot analysis demonstrated that one of the most prominently expressed and secreted protein by Akt-MSC compared to native MSC is the Sfrp2. Quantitative PCR showed 100 fold up regulation of Sfrp2 mRNA in Akt-MSC compared to control MSC. Sfrps are secreted glycoprotein molecules that structurally resemble cell surface frizzled receptors but lack the transmembrane domain. They have been increasingly recognized as potent regulators of cellular Wnt signaling and have been implicated in diverse cellular processes such as regulation of cell fate, differentiation, proliferation and cell death.

Sfrp2 was found to play a major role in mediating the survival signal of Akt-MSC on the ischemic myocardium. The data shows that Sfrp2 exerted survival effects on ischemic cardiomyocytes and that the pro-survival effects of Akt-MSC were markedly attenuated upon knockdown of Sfrp2 using siRNA. Sfrp2 increased total cellular and nuclear β-catenin in cardiomyocytes in vitro. Stabilization of β-catenin has been demonstrated to protect neonatal rat cardiomyocytes against hypoxia/re-oxygenation induced apoptosis. The canonical Wnt, Wnt3a, was found to be up-regulated in ischemic cardiomyocytes in vitro, and Wnt3a induced apoptosis of cardiomyocytes. Sfrp2 blocked the pro-apoptotic effect of Wnt3a. The data indicate that Sfrp2 is a major paracrine mediator of Akt-MSC myocardial survival and reparative effects and indicate that modulators of Wnt signaling such as Sfrp2 are useful as therapeutic agents in the management of myocardial injury.

Experiments were carried out as described above. Further profiling of secreted factors to identify cytoprolective proteins is described below. Profiling of Secreted factors Expressed in MSCs

To identify potential Akt-MSC secreted candidate paracrine factors mediating myocardial cell survival following ischemic injury, Affymetrix GeneChip® Mouse Genome 430 2.0 Arrays, which allows analysis of approximately 45,000 transcripts was used. Expression levels and quality analysis were carried out with the Affymetrix MAS 5.0 software. Further analysis was performed using the dChip software based on the following filtering criteria: a) Transcripts expressed (P call) in at least one of the sample compared, b) Fold change: at least 1.2X, (90% lower bound confidence). Approximately 650 transcripts were differentially regulated between the Akt-MSC and the GFP-MSC. Included in this list were 169 transcripts with unassigned function. The set of 650 transcripts was queried for transcripts coding for secreted proteins. This analysis revealed 62 transcripts encoding for 51 unique genes that contribute to the paracrine effects of the MSC cells (Table 1). TabIe 1.

Probe set ^• Gene Title Gene Symbol Fold change Fold change Akt Akt vs. Gf p at vs. Gfp at normoxϊa hypoxia

1426858_at inhibin beta-B lnhbb -2.27 ^~ -4.34

1423635_at bone morphogenetic protein Bmp2 -3.82 -3.19 2

1456404_at a disintegrin-like and Adamtsδ -1.22 -3.08 metalloprotease (reprolysin type) with thrombospondin type 1 motif, 5 (aggrecanase-2)

1426152_a_c kit ligand/ stem cell factor Kit! -1.64 -2.78

1427760_s_at Proliferin PIf -3.15 -2.61

1431056_a_at lipoprotein lipase LpI -2 -2.58

1450658_at a disintegrin-like and Adamtsδ -1.71 -2.21 metalloprotease (reprolysin type) with thrombospondin type 1 motif, 5 (aggrecanase-2)

1449528_at c-fos induced growth factor Figf -2.27 -2.14

1438953_at c-fos induced growth factor Figf -3.02 -2.09

1415904_at lipoprotein lipase LpI -1.55 -2.08

Probe set Gene Title Gene Symbol Fold change Fold change Akt Akt vs. Gf p at vs. Gfp at normoxia hypoxia

1418450_at immunoglobulin superfamily IsIr -1.55 -2.06 containing leucine-rich repeat

1426951_at cysteine-rich motor neuron ^' Crimi -2.41 -2 1

1437218_at fibronectin 1 Fn 1 -1.89 -1.97

1438954_x_at c-fos induced growth factor Figf -3.03 -1.96

1435603_at secreted protein SST3 SST3 -1.12 -1.93

1422561_at a disintegrin-like and Adamtsδ -1.14 -1.91 metalloprotease (reprolysin type) with thrombospondin type 1 motif, 5 (aggrecanase-2)

1418061_at latent transforming growth Ltbp2 -2.66 -1.87 factor beta binding protein 2

1451243_at arginyl aminopeptidase Rnpep -1.34 -1.86 (aminopeptidase B)

1460302_at thrombospondin 1 Thbsi 1.03 -1.84

1417234_at matrix metalloproteinase 11 Mmp11 -1.59 -1.82

1438936_s_at Angiogenin Ang 1.18 -1.82

1447862_x_at thrombospondin 2 Thbs2 -1.33 -1.8

1425985_s_at mannan-binding lectin Maspi -1.72 -1.79 serine protease 1

1448117_at kit ligand Kitl -1.23 -1.79

1438937_x_at Angiogenin Ang -1.22 -1.76

1416164_at fibulin 5 Fbln5 -1.35 -1.72

1448823_at chemokine (C-X-C motif) Cxcl12 -1.1 -1.62 ligand 12

1415949_at carboxypeptidase E Cpe -1.33 -1.6

1416953_at connective tissue growth Ctgf -6.01 -1.57 factor

1449187_at platelet derived growth Pdgfa -2.33 -1.55 factor, alpha

1423396_at Angiotensinogen Agt -2.48 -1.51

1421228_at chemokine (C-C motif) Ccl7 -3.4 -1.25 ligand 7

Probe set Gene Title Gene Symbol Fold change Fold change Akt Akt vs. Gfp at vs. Gfp at normoxia hypoxia

1438133_a_at cysteine rich protein 61 Cyr61 -3.93 -1.18

1419662_at Osteoglycin Og n 2.19 -1.07

1420380_at chemokine (C-C motif) Ccl2 -6.73 1.01 ligand 2

1416039_x_at cysteine rich protein 61 Cyr61 -4.61 1.04

1417130_s_at angiopoietin-like 4 Angptl4 -1.04 1.02

1421991_a_at insulin-like growth factor Igfbp4 2.32 1.19 binding protein 4

1416371_at apolipoprotein D Apod 1.88 1.34

1423294_at mesoderm specific Mest 2.21 1.34 transcript

1416594_at secreted frizzled-related Sfrpi 2.23 1.42 sequence protein 1

1450325_at angiopoietin 4 Agpt4 2.43 1.6

1417634_at superoxide dismutase 3, Sod3 4.31 1.61 extracellular

1417256_at matrix metalloproteinase 13 Mmp13 2.21 1.74

1417633_at superoxide dismutase 3, Sod3 3.23 1.78 extracellular

1429348_at sema domain, Sema3c 2.61 1.92 immunoglobulin domain (Ig), short basic domain, secreted, (semaphorin) 3C

1451866_a_at hepatocyte growth factor Hgf 2.32 2.26

1429679_at leucine rich repeat Lrrc17 2.36 2.35 containing 17

1452436_at lysyl oxidase-like 2 Loxl2 1.8 2.62

1431591_s_at interferon, alpha-inducible G1 p2 4.75 2.71 protein

1448424_at secreted frizzled-related Sfrp3 3.15 3.14 sequence protein 3

1419043_a_at interferon-inducible GTPase ligpi 3.97 3.15 1

1419042_at interferon-inducible GTPase Iigpi 4.61 3.55 1

1451959_a_at vascular endothelial growth Vegfa -1.07 3.64 factor A

Probe set Gene Title Gene Symbol Fold change Fold change Akt Akt vs. Gf p at vs. Gfp at normoxia hypoxia

1447839_x_at Adrenomedullin Adm -3.72 4.03

1417867_at Adipsin Adn 3.5 4.15

1448254_at Pleiotrophin Ptn 5.21 4.48

1416211_a_at Pleiotrophin Ptn 5.68 4.79

1416077_at Adrenomedullin Adm -2.78 8.36

1419149_at serine (or cysteine) Serpinei -6.34 10.35 proteinase inhibitor, clade E, member 1

1448201_at secreted frizzled-related Sfrp2 10.04 11.66 sequence protein 2

Among these upregulated genes, Sfrp2 was the most dramatically upregulated. Other cytokines such as Vegf, Hgf and FGF were not differentially expressed between Akt-MSC and FP-MSC under normoxic conditions. The expression of Sfrp2 was Akt pathway dependent. The expression of the other Sfrp family members were minimally altered in Akt-MSC (Fig. IA). Akt regulated Expression of Sfrps in MSCs

The results of microarray analysis was confirmed by quantitative PCR (Q-PCR). RNA was collected from cultured Akt-MSC and GFP-MSC that were cultured in vitro. As shown in Fig. IB, the expression pattern of Sfrp 1, Sfrp2 and Sfrp3 was consistent with the microarray results. Neither Sfrp land Sfrp3 was significantly upregulated in Akt-MSC vs GFP-MSC, whereas Sfrp2 expression was 100 fold higher in Akt-MSC.

To further validate our observations at the protein level and to evaluate the effect of Akt on Sfrp2 expression, control mouse MSCs and Akt-MSCs were cultured for 6 hours with PBKinase inhibitor (LY294002 50 niM) or vehicle. The conditioned medium was then collected and concentrated for Western blot protein analysis. As shown in Fig. 2A, Sfrp2 was secreted at high levels into the conditioned medium from the Akt-MSC cells(lanes 1). The levels of Sfrp2 were low or undetectable in the conditioned medium of GFP-MSCs (lanes 2 ). Furthermore, the expression/release of Sfrp2 in the Akt-MSC cells was dependent on the PI3K pathway since inhibition of the PI3K, abolished Sfrp2 accumulation in the medium (lanes 3). Akt-MSCs promote cardiomyocyte cell survival through paracrine mechanisms mediated by Sfrp

To prove whether Sfrp2 is a key paracrine mediator of the survival signaling of Akt- MSC, the apoptotic response (caspase activity) of adult rat ventricular cardiomyocytes (ARVC) exposed to conditioned medium collected from Akt-MSC treated with siRNA (Akt-MSC minus Sfrp2 CM) against Sfrp2 was evaluated. ARVC were subjected to hypoxia for 24 hours in the presence of Akt-MSC CM, Akt-MSC minus Sfrp2 CM or standard growth medium (GM). (Figs. 2B,C). ARVCs maintained in standard growth medium under normoxic conditions for 24 hours were viable and exhibited their typical rod-shaped appearance while ARVC grown in the same medium and subjected to 24 hour hypoxia exhibited a 82% increase in caspase activity (Fig. 2C). Compared to hypoxic ARVC maintained in standard growth medium, hypoxic ARVC exposed to Akt-MSC CM exhibited a 40% reduction in caspase activity (Fig. 3B). Moreover, exposure of hypoxic cardiomyocytes to Akt minus Sfrp2 CM resulted in a significant increase in caspase activity compared to hypoxic ARVC treated with Akt CM. A 33% increase in caspase activity was observed in hypoxic ARVC following knockdown of Sfrp2 expression in Akt-MSC. These observations demonstrate the key role played by Sfrp2 in mediating survival effects of Akt-MSC CM on cardiomyocytes.

To examine the direct effect of Sfrp2 on ARVCs, gain of function experiments were carried out. ARVCs were maintained in standard growth medium at normoxia or subjected to 24h hypoxia. Sfrp2 or vehicle was then added at various concentrations and apoptosis levels were assessed by measuring caspase activity. Treatment with Sfrp2 resulted in significant reduction in caspase activity, and a dose dependent cytoprotective response was observed with increasing Sfrp2 concentrations up to 15nM (Fig. 3A).

The cytoprotective effects of Sfψ2 on cardiomyocytes was confirmed by observing changes in cardiomyocyte cell morphology following exposure to hypoxia. ARVC following exposure to 24 hour hypoxia, lose their typical rod shaped morphology, become round in shape, subsequently detach and die. Hypoxia alone increased the number of round shaped cardiomyocytes by approximately 36% (Fig3B, C). However when ARVC were treated with Sfrp2 (3nM), the number of round shaped cardiomyocytes was decreased by approximately 31% compared to untreated controls (Fig3B, C). The data strongly indicate that Sfrp2 promotes cardiomyocyte survival and protects cardiomyocytes from hypoxic injury. Suppression of Sfrp2 expression in Akt-MSCs reduces the paracrine protection of myocardial injury in vivo.

Experiments were carried out to evaluate the physiological significance of Sfrp2 in Akt- MSC mediated paracrine myocardial protection in vivo. To demonstrate the importance of Sfrp2

as a key paracrine factor mediating prosurvival effects of injected Akt-MSC, //; vivo effects of conditioned medium collected from Akt-MSC treated with siRNA against Sfrp2 were compared with those of untreated Akt-MSC CM. Akt-MSC treated with siRNA against Sfrp2 had a 60% decrease in Sfrp2 mRNA expression following 48 hours of exposure to siRNA (Fig. 2B). The conditioned medium either from untreated or siRNA treated cells was collected, concentrated and then injected into 5 different sites at the infarct border zone 30 minutes after coronary artery ligation (a standard model for MI). Hearts were then isolated 72 hours later and infarct size was estimated by TTC staining. The results were analyzed by an investigator blinded to the treatment groups. As shown (Figs 4A, B) injection of Alct CM in infarcted hearts resulted in 71% reduction in the infarct size after MI within 72 hours, whereas injection of conditioned medium from siRNA treated Akt-MSC did not show any significant protection. Collectively, these results indicate that Sfrp2 possesses cell survival signaling properties and mediates myocardial protective effects following myocardial infarction. Sfrp2 leads to upregulation of βcatenin in hypoxic cardiomyocytes Sfrp2 is an antagonist of Wnt signaling. Unlike Sfrpl which can potentiate Wnt signaling under certain conditions, Sfrp2 has not been known to activate Wnt signaling. However, evidence described herein indicates mat Sfrp2 increases total cellular as well as nuclear β catenin mimicking canonical Wnt signaling. Using Western blotting, Sfrp2 was found to induce a dose dependent increase in nuclear as well as total cellular β catenin levels in cardiomyocytes exposed to hypoxia (Fig. 5C). Increased β catenin within cardiomyocytes is associated with increased cellular protection against ischemic injury in vitro. These data indicate that Sfrp2 promotes the survival of cardiomyocytes against hypoxia induced apoptosis via potentiation of canonical signaling. Experiments were then carried out to determine if Wnts are up-regulated in cardiomyocytes exposed to 24 hour hypoxia. The data indicated that Wnt3a was expressed at very low levels in normoxic cells but increased in hypoxic cells (Fig. 5A). Cardiomyocytes were incubated both under normoxia and hypoxia/reoxygenation with Wnt3a alone and in combination with Sfrp2. The data demonstrated that under normoxic conditions, as compared to control cardiomyocytes Wnt3a treatment resulted in a modest increase in caspase 3 activity which was attenuated by Sfrp2 treatment. Furthermore, under hypoxia/reoxygenation conditions, Wnt3a treatment resulted in a significant increase in caspase activity which was inhibited by the addition of Sfrp2 (Fig.5B). Sfrp2 upregulates expression of anti-apoptotic gene Birclb in hypoxic cardiomyocvtes

To further investigate the molecular mechanism by which Sfrp2 protects cardiomyocytes from cell death, RNA from hypoxic cardiomyocytes following Sfrp2 treatment (IOng/mL) was collected and expression of multiple genes involved in cell survival/death pathways was determined using microarray analysis. Using an oligo GE Array for rat signal transduction pathways, gene expression of 95 marker genes associated with 18 different signaling pathways was analyzed. In this analysis, 43 genes showed differential expression between the Sfrp2 treated and the control cardiomyocytes. Sfrp2 upregulated the expression of Birclb, an anti- apoptotic gene belonging to the neuronal apoptosis inhibitoiy protein (NAIP) family. Expression of other cytoprotective genes such as Bcl2 were only minimally increased in hypoxic cardiomyocytes in the presence of Sfrp2 (Figs. 6 A, B). Sfrp-based therapy for cardiac disorders

Sfrp2 was identified as an Akt-MSC secreted protein exerting prosurvival effects on the myocardium. Several lines of evidence support the role of Sfrp2 as a principal mediator of anti apoptotic effects exerted on the myocardium by Akt-MSC. First, Sfrp2 expression is dramatically upregulated (100X) in Akt-MSC compared to GFP-MSC and its expression/secretion is dependent on the PB kinase/Akt pathway. Secondly, Sfrp2 conferred prosurvival effects on hypoxic cardiomyocytes. Moreover, knockdown of Sfrp2 expression resulted in the attenuation of the prosurvival action of Akt-MSC conditioned medium both in vitro and in vivo. Sfrp2 is a secreted glycoprotein molecule that structurally resembles cell surface Frizzled receptors but lacks the latter 's transmembrane domains. Sfrps compete with the frizzled receptor for Wnt ligands by direct binding of Wnts thus preventing activation of Wnt signaling in the cell. The Wnt family currently comprises 19 different proteins. Wnts are known to regulate organogenesis during embryonic development and in mammals and in other species such as amphibians and birds have been implicated in cardiac morphogenesis as well. They regulate diverse cellular processes such as proliferation, differentiation and apoptosis, but the role of the Wnts in regulating such processes in the post natal heart was not known. Although various Wnts such as WntlOb and several frizzled receptors are expressed in the human heart, it was not known whether they play a role in cardiac homeostasis. The data described herein indicates that Sfrp2, a known modulator of Wnt signaling exerts prosurvival action on cardiomyocytes. The data demonstrate that Sfrp2 increases as well as nuclear βcatenin within the hypoxic cardiomyocyte in a dose dependent manner, βcatenin when activated translocates to the nucleus and initiates transcription of a wide variety of genes; thus the nuclear fraction

represents a more accurate measure of activated βcatenin. Sfrpl has previously been shown to potentiate Wnt signaling by directly binding to Frizzled receptors. In hypoxic cardioinyocytes, Sfrp2 binds locally present Wnts and alters the balance of intracellular Wnt signaling within a cardiomyocyte to favor a canonical pathway. Wnt3a was found to be upregulated in hypoxic cardiomyocytes. Wnt3a increases cardiomyocyte apoptosis and Sfrp2 blocks this effect of Wnt3a. Sfrp2 may also bind directly to frizzled receptor on cardiomyocytes activating the canonical pathway. The data indicate that Sfrp2 by increasing cellular and nuclear βcatenin enhances the survival response of cardiomyocytes against hypoxia induced apoptosis. Sfrp2 also upregulated expression of Birclb, an anti-apoptotic gene belonging to the NAIP family. Sfrp2 mediated increased βcatenin activates transcription of anti-apoptotic genes such as Birclb in hypoxic cardiomyocytes. Indeed, pharmacologic inhibition of GSK3β, resulting in increased βcatenin has been found to upregulate expression of anti-apoptotic genes such as Bcl2. Sfrp2 is involved in regulating cardiomyocyte cell survival and preserving cardiac function following myocardial infarction. Sfrp2 also plays a role as an important paracrine factor mediating beneficial effects of stem cell therapy. Sfrp2 alters the local milieu around the infarct zone to favor cardiomyocyte cell survival. Simple administration of Sfrp2 protein or fragments that modulate the Wnt-βcatenin pathway achieve results similar to stem cell based cardiac therapy, and a protein based therapy has advantages over cell based cardiac therapy for acute myocardial infarction and other ischemic cardiac disorders.

Example 3: Sfrp2 maintains cells in a stem cell state

Sfrp2 was found to be strongly expressed by mouse embryonic stem cells (e.g., P19CL6 cell line which readily differentiates into cardiomyocytes under certain conditions). Sfrp2 was found to strongly inhibit differentiation of the murine embryonic P19C16 cell line. Overexpression of Sfrp2 or addition of recombinant Sfrp2 protein inhibited differentiation of these cells. This data indicates that Sfrp2, by inhibiting differentiation of stem cells and maintaining them in the undifferentiated state, plays a role in maintenance of a stem cell phenotype and self renewal of stem cells. When added to P19CL6 cells, purified Sfrp2 prevented these cells from differentiating into cardiomyocytes. This result indicates that Sfrp2 by inhibiting differentiation of embryonic stem cells and maintaining them in the undifferentiated state preserves a stem cell phenotype of such cells. Maintenance of sternness is a fundamental and essential property of stem cells. It is not only of essential biological importance but great clinical significance. For example, bone marrow transplantation involves selection and administration of hematopoietic stem cells. A composition, e.g., Sfrp2 or other paracrine factor, that maintain the sternness of embryonic and adult stem cells is useful to preserve and maintain stem cells for tissue repair and regeneration. Example 4: Identification of protective factors secreted by Akt-MSC

Microarray analysis of Akt-MSC and control MSC under normoxia or hypoxia was performed to identify transcripts that were differentially regulated between these conditions. Using this approach, 61 proteins of know paracrine function were identified, e.g., pleiotrophin, chemokine ligands 2 and 7 and various angiogenic factors such as VEGFa, angiopoietin 4 and HGF. Upregulated transcripts with unassigned function were subjected to genomic analysis using a combination of bioinformatic software programs that allows predictions of potential secreted peptides. Putative secreted proteins thus indentified were then screened using siRNA technologies in a high throughout cell-based assays to examine key physiological mechanisms involved in the cardioprotective effects of Akt-MSCs. Using this approach, secreted proteins were indentified that are overexpressed in Akt-MSCs. One of these was highly expressed in Akt-MSCs but nearly undetectable in control MSCs. Permanent clones of Akt-siRNA knock down were then established for each of these genes and conditioned medium from these cells was compared to conditioned medium from Akt-MSCs for its cytoprotective effect in cardiac myocytes in vitro by apoptosis and cell viability assays.

Subsequently, the open reading frames of these novel transcripts were cloned and expressed in E. coli as maltose binding protein (MBP) fusion proteins. Compared with MBP

alone, one of the MBP-novel fusion proteins (Protein #12; "hl2") significantly reduced the H ₂ O ₂ -induced apoptosis in H9C2 myocytes. Protein 12 was re-cloned into pET vector to allow rapid purification as a 6xHis tagged recombinant protein. Since Protein 12 is cysteine rich, purification was performed under denaturing condition and the protein was refolded by dialysis with a redox pair to promote disulfide bond formation. To test the cardioprotective effects of Protein 12, the effects of addition of this protein on H ₂ O ₂ -induced apoptosis in H9C2 myocytes was evaluated. Myocytes were treated with 100 μM H ₂ O ₂ or vehicle and the levels of apoptosis was assessed by FACS analysis following Annexin V/PI staining. H ₂ O ₂ induced high levels of early apoptosis, yielding approximately 30% Annexin V positive cells with less than 5% necrotic cells (PI positive). Pre-treatment of the cells with 1OnM of Protein 12 for 30 min reduced early apoptosis by nearly 50%. This protein significantly reduced H ₂ O ₂ induced caspase 9 activity in adult rat cardiomyocytes by 38.5%, dramatically inhibited the mitochondrial release of cytochrome C and increased the total survival rate by 28%. The data indicate that this cysteine-rich Protein 12 possesses cardio-protective effects of Akt-MSCs. A total of 5 transcripts with previously undefined function were found to account account for myocardial protection of AKT-MSCs. The open reading frame of these novel transcripts were subsequently cloned, expressed and purified from E. coli, as either fusion proteins of maltose binding protein-novel proteins or as 6xHis tagged recombinant proteins. Protein No.12, which is a cysteine-rich insoluble protein when expressed in E.coli., was then purified under denaturing condition and refolded by dialysis with a redox pair to promote disulfide bond formation. This No.12 protein was used in various assays for oxidative stress induced apoptosis in cardiomyocytes and was found to have a strong cardio-protective effect. For Human No.12, the coding sequence without the predicted N-terminal signal region (1158 base pairs) were amplified and cloned in-frame of protein translation into pMal-C vector to generate a fusion protein of maltose binding protein-Human No.12, designated as MBP-hl2 (-80 KDa). Expression was induced by IPTG in E.coli. and purification of MBP-hl2 was done by standard affinity chromatography according to New England Biolab's instructions. MBP-hl2 was further purified by FPLC system. Compared with control MBP alone, this MBP-M2 fusion protein significantly prevents H ₂ O ₂ -induced early apoptosis in H9C2 myocytes (-30% reduction of apoptosis), by Annexin V/PI double staining with FACS analysis. To gain further insight of protein No.12' function in cardiovascular biology, same coding region (1158 base pairs) were re-amplified and cloned in-frame into pET 15b vector to generate 6XHis-tagged recombinant protein, designated as His-hl2 (-40 KDa). Protein was first purified under denaturing condition

and refolded by dialysis with gradually decreasing amount of dithiothreital. Oxidized and reduced of glutathione as the 'redox pair' was added in the final step to promote disulfide bond formation. Refolded His-hl2 proteins were dialyzed extensively in PBS and were used in subsequent apoptosis assays. Fig. 7 shows the results of Annexin V/PI staining with FACS analysis in H9C2 myocytes for early apoptosis. H9C2 myoctyes were seeded at l*10 ⁴ per well in 6-well plate one day before experiment. Recombinant His-hl2 proteins were added to the cells at different concentration for 30 min first and then the cells were challenged with 100 μM OfH ₂ O ₂ for 2 hours to induce apoptosis. The apoptotic cells were calculated as the percentage of Annexin V positive cells in total cells in FACS analysis. Recombinant human IGF-I proteins were used as a positive control. Pre-incubation of this His-hl2 recombinant protein dramatically reduced subsequent H2θ ₂ -induced early apoptosis in H9C2 myocytes, resulting in a -50% reduction in annexin V positive cells, P<0.001. The effect of Human No. 12 is equivalent to human recombinant IGF-I protein at the same dose, 10 nM. An assay was carried out to evaluate caspase inhibition by recombinant His-hl2 protein in adult rat cardiomyocytes (Fig. 8). Adult rat cardiomyocytes were pre-incubated with 10 nM of recombinant His-hl2 protein for 30 min and then challenged with 100 μM Of H ₂ O ₂ for different time points. Cell lysates were used for the measurement of relative amount of active caspase with Promega's Caspase-Glo kits. His-hl2 protein significantly reduced caspase 9 activity starting from 5 hours onward, reaching highest inhibition (~40% inhibition) at 9 hour, pO.001. The absolute amount of active Caspase 3/7 is relatively lower than that of Caspase 9 in there cells, however, His-hl2 protein also significantly reduced caspase 3/7 activity at 9 hours, pθ.01.

Survival signaling mechanism of His-hl2 protein on cardiomyocytes was also evaluated. Experiments were carried out to determine whether His-hl2 exerts its protective effect for H ₂ O ₂ - induced apoptosis of cardiomyocytes, in a paracrine fashion mainly through intracellular survival signaling transduction, which positively regulates the whole machinery of apoptosis network. The expression of apoptosis-related genes was studied in rat adult cardiomyocytes after incubation of His-hl2 protein at 10 nM at various time points. Adult rat cardiomyocytes were incubated with recombinant His-hl2 protein at 10 nM final concentration for 10 min, 30 min, Ih, 2h and 3 h. Whole cell lysates were separated on 10% SDS-PAGE gels and probed with phosphor- Akt antibodies, total Akt antibody and GSK-3B antibody (Fig. 9). Lane 1, vehicle PBS control treatment; Lane 2-6, 10 min, 30 min, Ih, 2h and 3h incubation of cardiomyoctyes with

His-hl2 protein respectively. Compared with lane 1 vehicle PBS control treatment, incubation of His-hl2 protein dramatically activates phosphor Alct ^{Thr3 8} at 30 min, with the concurrent phosphorylation of Akt's substrate-GSK-3β, at the same time point. No significant changes were found in total Akt and β-tublin as loading controls. Fig. 10 shows the results of an assay to evaluate cytochrome C release. Adult rat cardiomycytes were pre-incubated with recombinant His-hl2 protein at 10 nM for 30 min, then challenged with 100 μM OfH ₂ O ₂ for 6h to induce apoptosis. Cytosolic proteins were separated by 15% SDS-PAGE gel and probed with anti-cytochrome C antibodies. Lane 1-2, vehicle PBS control treatment; Lane 3-4, H ₂ O ₂ treatment of cardiomyocytes for 6h; Lane 5-8, cardiomyoctyes pre-incubated with His-hl2 protein for 30 min and then challenged with H ₂ O ₂ for 6h. Compared with Lane 1-2 controls, H ₂ O ₂ treatment of Lane 3-4 resulted in a dramatic release of cytochrome C into cytosolic compartment of cardiomyocytes. However, preincubation of His-hl2 protein with cardiomyocytes for 30 min significantly prevented the release of cytochrome C.

Fig. 11 shows stabilization of mitochondrial Bcl-2 protein level by His-hl2 protein during cardiomyocyte apoptosis. Adult rat cardiomycytes were pre-incubated with recombinant His-hl2 protein at 10 nM for 30 min, then challenged with 100 μM Of H ₂ O ₂ for 6h to induce apoptosis. Mitochondrial proteins were separated by 12.5% SDS-PAGE gel and probe with anti- Bcl-2 antibody. Lane 1, no treatment control; Lane 2, cardiomyocytes challenged with 100 μM OfH ₂ O ₂ for 6h; Lane 3-6, cardiomyocytes pre-incubated with 10 nM of His-hl2 for 30 min and then challenged with 100 μM OfH ₂ O ₂ for 6h. Compared with Lane 1 control, H ₂ O ₂ treatment of Lane 2 resulted in a modest decrease of mitochondrial Bcl-2. Pre-incubation of cardiomyocytes with His-hl2 protein stabilized the mitochondrial Bcl-2 protein level. Sequences and GenBank Accession Number of His-hl2

No.12 has a GenBank designation human BC037293. This gene product is also know as chromosome 3 open reading frame 58 (c3orf58). Mouse No.12 homologous gene is currently unknown. The cDNA of human No.12 clone was purchased from ATCC, coding region were amplified to make the expression construct, N-teπninal signal deletion coding sequence of human No.12 were amplified and clone into pMal-C vector for fusion protein expression and purification, which were used in the initial screening studies. Human No.12 was further expressed as 6xHis tagged recombinant protein for cardio-protection studies.

Human No.12 full-length mRNA sequence (h!2: SEO ID NO 16) ^"

1 gccggagtcg gagggcgggg agctaggagg agggagctcg agagttgtgg agactagtga

61 ctgggagaag tcgcagcccg ctcaggcccg cgccttcccg ctccccgtct tcctctctca

121 cacacctact ccgccctccg ccccagcccg cgcgctagct cchtctctcg cccggggttc 181 ctgccggtag ctctccgggt cttggcgcgg cgggggcgcc ccgggggtgc cctcgccctc

241 ccgttgcggg cgggcgggcg gtatgtggcg cctggtgccc ccgaagctgg gccgcctgtc

301 ccgctcgctg aagctggcgg cgctgggcag cctgttggtg ctgatggtgc tgcactcgcc

361 gtcgctgctc gcctcttggc agcgcaacga actgaccgac cggcgcttcc tgcagctcaa

421 taagtgcccg gcgtgcttcg gcacgagctg gtgccgccgc ttcctcaacg ggcaggtggt 481 attcgaggcg tggggccgct tgcgcctgct ggacttcctc aacgtgaaga acgtgtactt

541 cgcgcagtac ggcgagcccc gcgagggcgg ccgccgccga gtggtgctca agcgcσtcgg

601 ctcgcagcgc gagctggcgc agctcgacca gagcatctgc aagcgggcca ccggccggcc

661 ccgctgcgac ctgctgcagg ccatgccccg gaccgagttc gcgcgcctca acggcgacgt

721 gcgtctgctc acgcccgagg cggtggaggg ctggtcggac ctggtgcact gcccctcgca 781 gcgccttctc gaccgcctgg tgcgccgcta cgcggagacc aaggactcgg gcagcttcct

841 gcttcgcaac ctcaaggact cggagcgcat gcagctgctg ctgaccchgg ccttcaaccc

901 cgagccgctg gtgctacaga gttttccgtc tgatgaaggt tggccatttg caaagtatct

961 tggagcttgt ggaagaatgg tggctgtaaa ttatgttgga gaagaactgt ggagttactt

1021 taatgcgcca tgggaaaaac gagttgacct cgcfctggcaa ttaatggaaa tagcagaaca 1081 gcttacaaac aatgactttg aatttgcact ctacctcctg gacgtcagct ttgacaattt

1141 tgcagttggt cctagagatg ggaaggtaat cattgtggat gctgaaaatg ttttggttgc

1201 tgacaaaaga ttaattagac aaaataaacc tgaaaattgg gatgtatggt atgaaagcaa

1261 gtttgatgac tgtgataagg aggcttgctt atcattttca aaagaaattc tttgtgctcg

1321 tgccactgtg gaccacaatt actatgctgt ttgtcagaac ctcttatcca gacatgccac 1381 ctggcgtggc acttctggag gactccttca tgatccacca agtgaaattg ccaaagatgg

1441 ccggctcgag gccttgctgg atgagtgtgc caacccaaag aagcgctatg gcagattcca

1501 ggctgcaaaa gaactgcgtg aatacctagc acaattaagt aacaacgtga ggtagtctat

1561 ggtgaacttt tctttttttc tccatttaaa cagcactggc taaaactaaa ccaccaaaaa

1621 acgatctgaa aaaatgaaat ttggaagtgt tacattcaga ggatgataaa cttgcactga 1681 tagatcttaa tgttaacatc catcaaaata agacattact tcaaaaatca catgatgctt

1741 ctgcaaataa gtatgttctt atactttgga ggcttgagct gtcatcagct gctccccact

1801 accccggaat gcttgagtgg attaatgaat attgttaagc tattggaaat gagtctgata

1861 gtacattggc ttgtgtatca aagggtactt ggtacttagt ttgcatttac tatcatgatt

1921 ttgtgaatct cttgcattta ctttgaatgt caagtcagat tggtctgttt tataggccgc 1981 tttttccttc tgatgtgtag ggttttttcc cccttttttt ttttaattaa attttgaaaa

2041 ttcaggttac tgtaggtgtt catttaaatt tttaatagtt gtcattcagt gctatttggt

2101 acatatttac tgttagggca ggattcccag gtttactgtg tttttttttt ttttttttta

2161 aagaaagcta aatattacat tatgtaaata cttcttttca ccaacttctg tagtttcacc

2221 attgcatggt gtcatttcag gttatttaac agttatatcc ctctatgcca ataattagaa 2281 gtgtacacta aacatgaagt ttggcatatg ttgcaaaatg tcattttatc tttctaaagg

2341 ctttaagaag aatatactag aatctatata ttgatgttaa ttttgattca gaaaaaaaat

2401 acaacccagt atctaaaaag tgttaactag tccaagatag taatgcatat gccaaagaaa

2461 tattacacct aatctcatgt ttagaattta aaatagaatt ggtcagctac ttahtcttac

2521 caccctactt ccagtatttt agctctgtca ttattaaatt cagatcttcc tgattatttt 2581 ttctgttgaa agttaaacta ctgctttcaa gtaatttaaa gttatcctac cttttatbca

2641 tgggtagttt tgcaaaatta acatggtagc cattgtttga atttaatcgg gcatcataac

2701 ttttcattba ttgaggaact aatcattatt actataaagc atacaaatta gccagtcagc

2761 acactttggt cttctttacc taagggttaa acatcagaac atcaaattta attatttgca

2821 tagaaatgtg tgggctcttt atataagttg actatcacta acaggtaata tttttctgtt 2881 tgaagttgtt acttttgttt acagcaaagt ttgatgtagt gtgcagtagt gagctctaga

2941 ctgatctttt tctaaatcag aaagtgatta aagtatgcac aaccaaaggc aggtttttct

3001 ttttcattta ttcagcaact atttattaag catcaactct gtgccaggca cgttactagc

3061 tgctacatac tgtctgaaca tgacatacgg ttaagtaact ttacaattat tatcaaatac 3121 ttcaatgtag atatttctta agttgaaata gcattaacta ggataatgct ttcatgttat 3181 tttattgtct tgtgatagaa attcaacttg taccatctaa aactaggttg ctataaaaat

3241 aggaggatga agtcaataaa gtttatgcca gtttaaaaac tggaaggaaa aggtaagagc

3301 tctccattat aaaatagttg cattcggtta atttttacac attagtgcat tgcgtatatc

3361 aactggccct caatgaagca tttaagtgct tggaatttta ctaaactgac ttttttgcaa

3421 ctttgggaga tttttgaggg gagtgttgaa aattgccaaa cactcacctc ttactcaaaa 3481 cttcaaataa aatacacatt ttcaagaggg agcacctttt atatttgata agttttcatt

3541 ataaacctta taataccagt cacaaagagg ttgtctgtct atggtttagc aaacatttgc

3601 ttttcttttt ggaagtgtga ttgcaattgc agaacagaaa gtgagaaaac actgccagcg

3661 gtgattgcta cttgaggtag ttttttacaa ctaccatttc ccctccatga aattatgtga

3721 aatttatttt atctttggga aaagttgaga agatagtaaa agaattagga atttaaaatt 3781 acagggaaaa atatgtaagt gaaaagcaat aaatattttg ttcactttgc tatcaagatg

3841 ttcactatca gatatttatt atatggcagc aatttatatt tttaatcatt gcccattaat

3901 agacgcagta aaatattttt gaatcagaca tttggggttt gtatgtgcat taaaattgtc

3961 ttttgtactg taagttactg ttaatttgaa tattttattg aactgtctcc ctgtgccttt

4021 ataatataaa gttgtttcta caacttttaa tgatcttaat aaagaatact ttaggaaaaa 4081 aaaaaaaaaa a

Human No.12 protein sequence (sequence in underlined type was used to generate recombinant His-hl2 protein) 0il2: SEO ID NO: 17)

MWRLVPPKLGRLSRSLKLAALGSLLVLMVLHSPSLLASWQRNEL

TDRRFLQLNKCPACFGTSWCRRFLNGQWFEAWGRLRLLDFLNVKNVYFAQYGEPREG

GRRRWLKRLGSQRELAQLDQSICKRATGRPRCDLLQAMPRTEFARLNGDVRLLTPEA VEGWSDLVHCPSQRLLDRL VRRYAETKDSGSFLLRNLKDSERMQLLLTLAFNPEPLVL

QSFPSDEGWPFAKYLGACGRMVAVNYVGEELWSYFNAPWEKRVDLAWQLMEIAEQLT N

NDFEFALYLLDVSFDNFAVGPRDGKVIIVDAENVLVADKRLIRQNKPENWDVWYESK F

DDCDKEACLSFSKEILCARATVDHNYYAVCQNLLSRHATWRGTSGGLLHDPPSEIAK D GRLEALLDECANPKKRYGRFQAAKELREYLAQLSNNVR

Other genes and gene products, the function and activity of which have previously not been known, have now been identified as having cardioprotective activity. The nucleic acid and amino acid sequences of these factors are described below.

HumanNo.l mRNA sequence (hi: SEQ IDNO:18) 1 gcatcttggc agggtccggg gacgtggact atttcgcaca ccacaccacg gggagggatt

61 tttttctatt ttccctacga aaaacagatc tttttaagga tggtgctgct ccactggtgc

121 ctgctgtggc tcctgtttcc actcagctca aggacccaga agttacccac ccgggatgag

181 gaactttttc agatgcagat ccgggacaag gcattttttc atgattcgtc agtaattcca

241 gatggagctg aaattagcag ttatctcttt agagatacac ctaaaaggta tttctttgtg 301 gttgaagaag acaatactcc attatcagtc acagtgacgc cctgtgatgc gcctttggag

361 tggaagctga gcctccagga gctgccagag gacaggagcg gggaaggctc aggtgatctg

421 gaacctcttg agcagcagaa gcagcagatc attaatgagg aaggcactga gttattctcc

481 tacaaaggca atgatgttga gtattttata tcgtctagtt ccccatccgg tttgtatcag

541 ttggatcttc tttcaacaga gaaagacaca catttcaaag tatatgccac cacaactcca 601 gaatctgatc agccataccc tgagttaccc tatgacccaa gagtagatgt gacctcactg

661 gggcgcacca cggtcacttt ggcctggaaa ccaagcccca ctgcctcttt gctgaaacaa

721 CGcattcagt actgtgtggt catcaacaaa gagcacaatt tcaaaagtct ctgtgcagtg

781 gaagcaaaac tgagtgcaga tgatgctttt atgatggcac cgaaacctgg tctggacttc

841 agcccctttg actttgccca ctttggattt ccttctgata attcaggtaa agaacgcagt 901 ttccaggcaa agccttctcc aaaactgggg cgtcatgtct actccaggcc caaggttgat

961 attcagaaaa tctgcatagg aaacaagaac atcttcaccg tctctgatct gaaacccgac

1021 acgcagtact actttgatgt atttgtggtc aacatcaaca gcaacatgag caccgcttat

1081 gtaggtacct ttgccaggac caaggaagaa gccaaacaga agacagtcga gctaaaagat

1141 gggaagataa cagatgtatt tgttaaaagg aagggagcaa agtttctacg gtttgctcca 1201 gtctcttctc accaaaaagt caccttcttt attcactctt gtctggatgc tgtccaaatc

1261 caagtgagaa gagatgggaa acttcttctg tctcagaatg tggaaggcat tcagcagttt

1321 cagcttagag gaaaacctaa agctaaatac ctcgttcgac tgaaaggaaa caagaaagga

1381 gcatctatgt tgaaaattct agctaccaca aggcctacta agcagtcatt tccctctctt

1441 cctgaagaca caagaatcaa agcctttgac aagctccgta cctgttcctc ggccaccgtg 1501 gcttggctag gcactcagga aaggaacaag ttttgcatct acaaaaaaga agtggatgat

1561 aactacaatg aagaccagaa gaaaagagag caaaaccaat gtctaggacc agatataagg

1621 aagaagtcag aaaaggtcct ctgtaaatat ttccacaghc aaaacttgca gaaagcagtg

1681 accacagaaa caattaaagg tcttcagcct ggcaaatctt acctgctgga tgtttatgtc

1741 ataggacatg gggggcactc tgtaaagtat cagagtaagg ttgtgaaaac tagaaagttc

1801 tgttagttac cttcttatag agatatatta tgtagaactc caggagggac attaaatcac 1861 tttaagtata aactgactac tcccacagtt gagagaagtt gtgacctgta cttgtactat 1921 ggaaggaagg atatcaacgt gtgtatattg atgtttatab aagtaactct tgaaggagac 1981 ttgttctagc gtgccccatg gtacctagtg tgtgtctgat gccggttggt gtcaaagata 2041 gagggcttct tgaaggaact tgccattcct tgctttgacc actgcatgaa ctgcttctaa 2101 attattttat tacctaaaaa tttaaaatat gccattcatt gcacacaccc acaaatgcaa 2161 atcattcctc tctatagatg ctaggatata tataaattat tttataaatt cttgttttaa 2221 atgtcagtgt ttctatgatt gtaaactatt aaattctttt cctattaaag tacagatcta 2281 atctaagtat tattaagttg atagccctct agtcagttat attgctattg taaattcthg 2341 tttgttgagt aaaatgttta aatactatat gtatctcatg tacaaagttg acatacatta 2401 tattcatgta cataaaatta aagagattag attataa

Human No.l protein sequence (hi: SEQ ID NO:19) MVLLHWCLLWLLFPLSSRTQKLPTRDEELFQMQIRDKAFFHDSS

VIPDGAEISSYLFRDTPKRYFFWEEDNTPLSVTVTPCDAPLEWKLSLQELPEDRSGE GSGDLEPLEQQKQQIINEEGTELFSYKGNDVEYFISSSSPSGLYQLDLLSTEKDTHFK

VYATTTPESDQPYPELPYDPRVDVTSLGRTTVTLAWKPSPTASLLKQPIQYCWINKE HNFKSLCAVEAKLSADDAFMMAPKPGLDFSPFDFAHFGFPSDNSGKERSFQAKPSPKL GRHVYSRPKVDIQKICIGNKNIFTVSDLKPDTQYYFDVFWNINSNMSTAYVGTFART KEEAKQKTVELKDGKITDVFVKRKGAKFLRFAPVSSHQKVTFFIHSCLDAVQIQVRRD GKLLLSQNVEGIQQFQLRGKPKAKYLVRLKGNKKGASMLKILATTRPTKQSFPSLPED

TRIKAFDKLRTCSSATVAWLGTQERNKFCIYKKEVDDNYNEDQKKREQNQCLGPDIR K

KSEKVLCKYFHSQNLQKAVTTETIKGLQPGKSYLLDVYVIGHGGHSVKYQSKWKTRK FC

Human No.5 mRNA sequence (h5: SEO ID NO:20

1 agcgggatag cccgcggccg cgcctgcccg ctcgcacccc tctcccgcgc ccggttctcc 61 ctcgcagcac ctcgaagtgc gcccctcgcc ctcctgctcg cgccccgccg ccatggctgc

121 ctcccccgcg cggcctgctg tcctggccct gaccgggctg gcgctgctcc tgctcctgtg

181 ctggggccca ggtggcataa gtggaaataa actcaagctg atgcttcaaa aacgagaagc

241 acctgttcca actaagacta aagtggccgt tgatgagaat aaagccaaag aattccttgg

301 cagcctgaag cgccagaagc ggcagctgtg ggaccggact cggcccgagg tgcagcagtg 361 gtaccagcag tttctctaca tgggctttga cgaagcgaaa tttgaagatg acatcaccta

421 ttggcttaac agagatcgaa atggacatga atactatggc gattactacc aacgtcacta

481 tgatgaagac tctgcaattg gtccccggag cccctacggc tttaggcatg gagccagcgt

541 caactacgat gactactaac catgacttgc cacacgctgt acaagaagca aatagcgatt

601 ctcttcatgt atctcctaat gccttacact acttggtttc tgatttgctc tatttcagca 661 gatcttttct acctactttg tgtgatcaaa aaagaagagt taaaacaaca catgtaaatg

721 ccttttgata tttcatggga atgcctctca tttaaaaata gaaataaagc attttgttaa

781 aaagaaaaaa aaaaaaaaaa

Human No.5 protein sequence (h5; SEQ ID NO:21)

MAASPARPAVLALTGLALLLLLCWGPGGISGNKLKLMLQKREAP

VPTKTKVAVDENKAKEFLGSLKRQKRQLWDRTRPEVQQWYQQFLYMGFDEAKFEDDI T YWLNRDRNGHEYYGDYYQRHYDEDSAIGPRSPYGFRHGASVNYDDY

HumanNo.8 mRNA sequence (h8: SEO IDNO:22~)

1 cactgggaga cagtccactt aaatgcagct ccagggttgc gaggcaccca ccagcatcat 61 tccccatgcg aggtggcaaa tgcaacatgc tctccagttt ggggtgtcta cttctctgtg

121 gaagtattac actagccctg ggaaatgcac agaaattgcc aaaaggtaaa aggccaaacc

181 tcaaagtcca catcaatacc acaagtgact ccatcctctt gaagttcttg cgtccaagtc

241 caaatgtaaa gcttgaaggt cttctcctgg gatatggcag caatgtatca ccaaaccagt

301 acttccctct tcccgctgaa gggaaattca cagaagctat agttgatgca gagccgaaat 361 atctgatagt tgtgcgacct gctccacctc caagtcaaaa gaagtcatgt tcaggtaaaa

421 ctcgttctcg caaacctctg cagctggtgg ttggcactct gacaccgagc tcagtcttcc

481 tgtcctgggg tttcctcatc aacccacacc atgactggac attgccaagt cactgtccca

541 atgacagatt ttatacaatt cgctatcgag aaaaggataa agaaaagaag tggatttttc

601 aaatctgtcc agccactgaa acaattgtgg aaaacctaaa gcccaacaca gtttatgaat 661 ttggagtgaa agacaatgtg gaaggtggaa tttggagtaa gattttcaat cacaagactg

721 ttgttggaag taaaaaagta aatgggaaaa tccaaagtac ctatgaccaa gaccacacag

781 tgccagcata tgtcccaagg aaacbaatcc caataacaat catcaagcaa gtgattcaga

841 atgttactca caaggattca gctaaatccc cagaaaaagc bccactggga ggagtgatac

901 tagtccacct tattattcca ggtcttaatg aaactactgt aaaacttcct gcatccctaa 961 tgtttgagat ttcagatgca ctcaagacac aattagctaa gaatgaaacc ttggcattac

1021 ctgccgaatc taaaacacca gaggttgaaa aaatctcagc acgacccaca acagtgactc

1081 ctgaaacagt tccaagaagc actaaaccca ctacgtctag tgcattagat gtttcagaaa

1141 caacactggc ttcaagtgaa aagccatgga ttgtgcctac agctaaaata tctgaagatt

1201 ccaaagttct gcagcctcaa actgcaactt atgatgtttt ctcaagccct acaacatcag 1261 atgagcctga gatatcagat tcctacacag caacaagtga tcgtattctg gattctatcc

1321 cacctaaaaσ ttctagaact cttgaacagc caagggcaac actggctcca agtgaaacac

1381 catttgttcc tcaaaaactg gaaatcttta ccagtccaga aatgcagcct acgacacctg

1441 ctccccagca aactacatct atcccttcta cacctaaacg acgcccccgg cccaaaccgc

1501 caagaaccaa acctgaaaga accacaagtg ccggaacaat tacacctaaa atttctaaaa 1561 gccctgaacc tacatggaca acaccggctc ccggtaaaac acaatttatt tctctgaaac

1621 ctaaaatccc tctcagccca gaagtgacac acaccaaacc tgctcccaag cagacaccac

1681 gtgctcctcc taagccaaaa acatcaccac gcccaagaat cccacaaaca caaccagttc

1741 ctaaggtgcG ccagcgtgtt actgcaaaac caaaaacgtc accaagtcca gaagtgtcat

1801 acaccacacc tgctccaaaa gatgtgctcc ttcctcataa accataccct gaggtctctc 1861 agagcgaacc tgctcctcta gagacacgag gcatcccttt tatacccatg atttccccaa

1921 gtcctagtca agaggaacta cagaccactc tggaagaaac agaccaatcc acccaagaac

1981 ctttcacaac taagattcca cgaacaactg aactagcaaa gacaactcag gcgccacaca

2041 gattttatac tactgtgagg cccagaacat ctgacaagcc acacatcaga cctggggtca 2101 agcaagcacc caggccatca ggtgctgata gaaatgtatc agtggactct acccacccca 2161 ctaaaaagcG agggactcgc cgcccaccct tgccacccag acctacacac ccacgaagaa 2221 aacctttacc accaaataat gtcactggaa agccaggaag tgcaggaatc atttcatcag 2281 gcccaataac tacaccaccc ctgaggtcaa cacccaggcc tactggaact cccttggaga 2341 gaatagagac agatataaag caaccaacag ttcctgcctc tggagaagaa ctggaaaata 2401 taactgactt tagctcaagc ccaacaagag aaactgatcc tcttgggaag ccaagattca 2461 aaggacctca tgtgcgatac atccaaaagc ctgacaacag tccctgctcc attactgact 2521 ctgtcaaacg gttccccaaa gaggaggcca cagaggggaa tgccaccagc ccaccacaga 2581 acccacccac caacctcact gtggtcaccg tggaagggtg cccctcattt gtcatcttgg 2641 actgggaaaa gccactaaat gacactgtca ctgaatatga agttatatcc agagaaaatg 2701 ggtcattcag tgggaagaac aagtccattc aaatgacaaa tcagacattt tccacagtag 2761 aaaatctgaa accaaacacg agttatgaat tccaggtgaa acccaaaaac ccgcttggtg 2821 aaggcccggt cagcaacaca gtggcattca gtactgaatc agcggaccca agagtgagtg 2881 agccagtttc tgcaggaaga gatgccatct ggactgaaag accctttaat tcagactctt 2941 actcagagtg taagggcaaa caatatgtca aaaggacatg gtataaaaaa tttgtaggag

3001 tgcagctgtg caactctctc agatacaaga tttacttgag cgactccctc acaggaaaat 3061 tttataacat aggtgatcag aggggccatg gagaagatca ctgccagttt gtggattcat

3121 ttttagatgg acgcactggg cagcaactca cttctgacca gttaccaatc aaagaaggtt 3181 atttcagagc agttcgccag gaacctgtcc aatttggaga aataggtggt cacacccaaa 3241 tcaattatgt tcagtggtat gaatgtggga ctacaattcc tggaaaatgg tagatgctgc 3301 acaaagttac cttctgtttc atcattgcaa acaaaaatca ttgaaaatac tatgccgcat

3361 tcatttaaag ctattttgtt tactatgtat aaaagtctac aatctaatta atagcaatac

3421 tagatgttta ttattagaaa agattgctga gagtatttat caggttttac aaagtcattt

3481 taagaaagca agatactgat gttaacagaa taacattttt ggggaagctg gctccctatt

3541 catggtattt taagagatca tttgtatatt atttatcaca ctgttgtaat gatgttttga 3601 gatactttta taacaaaatt aacatcaaaa aggtatatac tttttaaaaa aaatttactt

3661 ttattgatgt gtactcttcc tattgatgag ttaattccat aaatctctac ttagtttaac

3721 ttattggatc aaattatctt cagcatgtat atctggggaa aaaaggtccg aattttcaca

3781 tttatattta aacttcaatt ttttatattt aaacttcaat tttttagcaa cagctgaata

3841 gcttbgcgga ggagtttaat agttacacat tcatgctaat atacatttcc tttaaacatc 3901 cacaaattct taaaaagatt gaatcagtaa atttcatttc agctaaaaat ggagtctaat

3961 atattgtttc aaaagataca tttttaccca ccataaatgt tacaatatct gaatatgctt

4021 tgtcaaacta tccctttatg caatcgtctt catatbgttt ttatgattct aatcaagctg

4081 tatgtagaga ctgaatgtga agtcaagtct gagcacaaaa agataatgca caatgagatt

4141 gcctaccatt ttataggata tttactatgt atttatacgt taagacctct atgaatgaat 4201 gtatcagaga atgtctttgt aactaactgt ttaattcaat ctgtaataaa aatctaacta

4261 actaactcat ttatttctat taaaaaggta ttgtccttta ggcggggaat gggaatcctt

4321 gctgcactgt tgcagtcatt ctgaaaggac ctttccctgt acttaccttt caacatgctt

4381 caatcttatc aacgctacat tttgtatttt tcaaacaggt ataaattctg caataaagag

4441 atgtagtttt tttttaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 4501 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaa

Human No.8 protein sequence (h8: SEO ID NO:23)

MRGGKCNMLSSLGCLLLCGSITLALGNAQKLPKGKRPNLKVHIN

TTSDSILLKFLRPSPNVKLEGLLLGYGSNVSPNQYFPLPAEGKFTEAIVDAEPKYLI V VRPAPPPSQKKSCSGKTRSRKPLQL WGTLTPSSVFLSWGFLINPHHDWTLPSHCPND RFYTIRYREKDKEKKWIFQICPATETIVENLKPNTVYEFGVKDNVEGGIWSKIFNHKT WGSKKWGKIQSTYDQDHTVPAYVPRKLIPITIIKQVIQNVTHKDSAKSPEKAPLGG VILVHLIIPGLNETTVKLPASLMFEISDALKTQLAKNETLALPAESKTPEVEKISARP

TTVTPETVPRSTKPTTSSALDVSETTLASSEKPWIVPTAKISEDSKVLQPQTATYDV F SSPTTSDEPEISDSYTATSDRILDSIPPKTSRTLEQPRATLAPSETPFVPQKLEIFTS PEMQPTTPAPQQTTSIPSTPKRRPRPKPPRTKPERTTSAGTITPKISKSPEPTWTTPA PGKTQFISLKPKIPLSPEVTHTKPAPKQTPRAPPKPKTSPRPRIPQTQPVPKVPQRVT AKPKTSPSPEVSYTTPAPKDVLLPHKPYPEVSQSEPAPLETRGIPFIPMISPSPSQEE

LQTTLEETDQSTQEPFTTKIPRTTELAKTTQAPHRFYTTVRPRTSDKPHIRPGVKQA P RPSGADRNVSVDSTHPTKKPGTRRPPLPPRPTHPRRKPLPPNNVTGKPGSAGIISSGP ITTPPLRSTPRPTGTPLERIETDIKQPTVPASGEELENITDFSSSPTRETDPLGKPRF KGPHVRYIQKPDNSPCSITDSVKRFPKEEATEGNATSPPQNPPTNLTWTVEGCPSFV ILDWEKPLNDTVTEYEVISRENGSFSGKNKSIQMTNQTFSTVENLKPNTSYEFQVKPK

NPLGEGPVSNTVAFSTESADPRVSEPVSAGRDAIWTERPFNSDSYSECKGKQYVKRT W YKKFVGVQLCNSLRYKIYLSDSLTGKFYNIGDQRGHGEDHCQFVDSFLDGRTGQQLTS DQLPIKEGYFRAVRQEPVQFGEIGGHTQINYVQWYECGTTIPGKW

HumanNo.13 mRNA sequence 0.13; SEO IDNO:24)

1 ctccggtgag ttttgtggcg ggaagcttct gcgctggtgc ttagtaaccg actttcctcc 61 ggactcctgc acgacctgct cctacagccg gcgatccact cccggctgtt cccccggagg

121 gtccagaggc ctttcagaag gagaaggcag ctctgtttct ctgcagagga gtagggtcct

181 ttcagccatg aagcatgtgt tgaacctcta cctgttaggt gtggtactga ccctactctc

241 catcttcgtt agagtgatgg agtccctaga gggchtacta gagagcccat cgcctgggac

301 ctcctggacc accagaagcc aactagccaa cacagagccc accaagggcc ttccagacca 361 tccatccaga agcatgtgat aagacctcct tccatactgg ccatattttg gaacactgac

421 ctagacatgt ccagatggga gtcccattcc tagcagacaa gctgagcacc gttgtaacca

481 gagaactatt actaggcctt gaagaacctg tctaactgga tgctcattgc ctgggcaagg

541 cctgtttagg ccggttgcgg tggctcatgc ctgtaatcct agcactttgg gaggctgagg

601 tgggtggatc acctgaggtc aggagttcga gaccagcctc gccaacatgg cgaaacccca 661 tctctactaa aaatacaaaa gttagctggg tgtggtggca gaggcctgta atcccagctc

721 ctbgggaggc tgaggcggga gaattgcttg aacccgggga cggaggttgc agtgagccga

781 gatcgcactg ctgtacccag cctgggccac agtgcaagac tccatctcaa aaaaaaaaaa

841 aaaaaaaaaa aaaaaaaaa Human No.13 protein sequence (hl3; SEO ID NO: 25)

MKHVLNL YLLGWLTLLSIFVRVMESLEGLLESPSPGTSWTTRSQLANTEPTKGLPDHPSRSM

OTHER EMBODIMENTS Although particular embodiments have been disclosed herein in detail, this has been done by way of example for purposes of illustration only, and is not intended to be limiting with respect to the scope of the appended claims, which follow. In particular, it is contemplated by the inventors that various substitutions, alterations, and modifications may be made to the invention without departing from the spirit and scope of the invention as defined by the claims. The choice of nucleic acid starting material, clone of interest, or library type is believed to be a matter of routine for a person of ordinary skill in the art with knowledge of the embodiments described herein.

Other aspects, advantages, and modifications considered to be within the scope of the following claims.