US20030199464A1 - Regeneration of endogenous myocardial tissue by induction of neovascularization - Google Patents

Regeneration of endogenous myocardial tissue by induction of neovascularization Download PDF

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US20030199464A1
US20030199464A1 US10/128,738 US12873802A US2003199464A1 US 20030199464 A1 US20030199464 A1 US 20030199464A1 US 12873802 A US12873802 A US 12873802A US 2003199464 A1 US2003199464 A1 US 2003199464A1
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agent
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progenitor cells
cardiomyocyte
endothelial progenitor
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US10/128,738
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Silviu Itescu
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Columbia University in the City of New York
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Priority to US10/128,738 priority Critical patent/US20030199464A1/en
Priority to EP11157749A priority patent/EP2366706A1/fr
Priority to JP2003587162A priority patent/JP4897199B2/ja
Priority to EP10164397.1A priority patent/EP2292631B1/fr
Priority to AU2003231090A priority patent/AU2003231090B2/en
Priority to CA2482996A priority patent/CA2482996C/fr
Priority to CNB038147157A priority patent/CN100379751C/zh
Priority to US10/512,518 priority patent/US7887796B2/en
Priority to PCT/US2003/012768 priority patent/WO2003090512A2/fr
Priority to ZA200408777A priority patent/ZA200408777B/en
Priority to EP03724217A priority patent/EP1501852A4/fr
Priority to US10/693,480 priority patent/US8663652B2/en
Publication of US20030199464A1 publication Critical patent/US20030199464A1/en
Assigned to TRUSTEES OF COLUMBIA UNIVERSITY IN THE CITY OF NEW YORK, THE reassignment TRUSTEES OF COLUMBIA UNIVERSITY IN THE CITY OF NEW YORK, THE ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: ITESCU, SILVIU
Priority to IL164671A priority patent/IL164671A/en
Priority to US11/648,769 priority patent/US8242091B2/en
Priority to AU2009213032A priority patent/AU2009213032B2/en
Priority to JP2011021983A priority patent/JP5619644B2/ja
Priority to JP2013249643A priority patent/JP5911841B2/ja
Priority to US14/196,711 priority patent/US20150056161A1/en
Priority to IL235149A priority patent/IL235149B/en
Priority to JP2016014321A priority patent/JP2016155804A/ja
Abandoned legal-status Critical Current

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Definitions

  • Vascular network formation is the end result of a complex process that begins in the pre-natal period with induction of vasculogenesis by hemangioblasts—cells derived from the human ventral aorta which give rise to both endothelial and hematopoietic elements (8-11).
  • CXC chemokines containing the ELR motif regulate migration of human bone marrow-derived endothelial progenitor cells to sites of tissue ischemia.
  • selective bone marrow homing and engraftment of hematopoietic progenitors depends on CXCR4 binding to SDF-1 expressed constitutively in the bone marrow (28-30)
  • interruption of CXCR4/SDF-1 interactions could redirect trafficking of human bone marrow-derived endothelial progenitor cells to sites of tissue ischemia, thereby augmenting therapeutic vasculogenesis.
  • CXC chemokines, including IL-8, Gro-alpha, and SDF-1 play a central role in regulating human adult bone marrow-dependent vasculogenesis.
  • This invention provides a method of treating a disorder of a subject's heart involving loss of cardiomyocytes which comprises administering to the subject an amount of an agent effective to cause cardiomyocyte proliferation within the subject's heart so as to thereby treat the disorder.
  • This invention further provides the instant method wherein the agent is human endothelial progenitor cells.
  • This invention further provides the instant method comprising administering an effective amount of a second agent that increases the cardiomyocyte proliferation caused by the human endothelial progenitor cells.
  • This invention also provides a method of determining the susceptibility of a cardiomyocyte in a subject to apoptosis comprising:
  • FIGS. 1 A- 1 D IL-8/Gro-Alpha CXC Chemokines Regulate Migration Of Human Endothelial progenitor cells (angioblasts) To Myocardial Tissue In Vivo And Subsequent Development Of Vasculogenesis.
  • FIGS. 2 A- 2 C Blocking CXCR4/SDF-1 Interactions Redirects Intravenously Injected Human Endothelial progenitor cells From Bone Marrow To Ischemic Myocardium.
  • (B) and (C) depict the effects of mAbs against CXCR4, SDF-1 or CD34 on trafficking of human CD34+endothelial progenitor cells (angioblasts) to rat bone marrow and myocardium following LAD ligation.
  • Co-administration of anti-CXCR4 or anti-SDF-1 significantly reduced trafficking of intravenously injected human CD34+ cells to rat bone marrow at 48 hours and increased trafficking to ischemic myocardium, whereas anti-CD34 mAb had no effect (results are expressed as mean+sem of bone marrow and cardiac studies performed in three LAD-ligated animals at 48 hours after injection).
  • FIGS. 3 A- 3 F Redirected Trafficking Of Human Endothelial progenitor cells (angioblasts) To The Site Of Infarction Induces Vasculogenesis And Protects Cardiomyocytes against Apoptosis.
  • the infarct zones of rats receiving either 10 3 or 10 5 endothelial progenitor cells (angioblasts) show myocardial scars composed of paucicellular, dense fibrous tissue stained blue by trichrome (x400).
  • the infarct zones of rats injected with 10 5 endothelial progenitor cells plus anti-CXCR4 mAb show significant increase in cellularity of granulation tissue, minimal matrix deposition and fibrosis, and numerous medium-sized capillaries of human origin.
  • the infarct zones of rats injected with 2 ⁇ 10 5 endothelial progenitor cells show a similar reduction in fibrous tissue and increase in medium-sized capillaries, and an additional increase in large-sized vessels of human origin.
  • (B) and (C) show the relationship between the number of human CD117 bright endothelial progenitor cells injected intravenously (10 3 , 10 5 , 10 5 plus anti-CXCR4 mAb, and 2 ⁇ 10 5 ) and development of rat infarct bed vasculogenesis at two weeks, defined as the mean number of capillaries/high power field (hpf) with medium- or large-sized lumen diameter (respectively, 0.02 mm mean diameter with 3-6 contiguous endothelial lining cells and 0.05 mm mean diameter with >6 contiguous endothelial lining cells). Results are expressed as the mean+sem of at least 15 hpf in three separate experiments.
  • (D) shows that co-administration of anti-CXCR4 mAb together with the highest concentration of endothelial progenitor cells (angioblasts), 2 ⁇ 10 5 , resulted in a further 23% increase in growth of large-lumen capillaries. More strikingly, there was a further 2-fold increase in capillary numbers when 2 ⁇ 10 5 endothelial progenitor cells were injected intravenously after direct intracardiac delivery of 1.0 ⁇ g/ml SDF-1 into infarcted hearts (p ⁇ 0.01) (results are expressed as mean+sem of three separate experiments).
  • (E) shows that at 2 weeks the numbers of apoptotic myocytes at the peri-infarct rim, defined by concomitant staining with anti-desmin mAb and DNA end-labeling using TUNEL technique, are significantly reduced in rats receiving either 2.0 ⁇ 10 5 endothelial progenitor cells or 10 5 endothelial progenitor cells together with anti-CXCR4 mAb in comparison to rats receiving 10 3 or 10 5 endothelial progenitor cells (p ⁇ 0.01) (results are expressed as mean+sem of three separate experiments).
  • (F) shows that co-administration of anti-CXCR4 mAb or intracardiac injection of SDF-1 resulted in further reductions in cardiomyocyte apoptosis of 65% and 76%, respectively, at two weeks (both p ⁇ 0.001) (results are expressed as mean+sem of three separate experiments).
  • FIGS. 4 A- 4 H Infarct Bed Vasculogenesis Improves Long-Term Myocardial Function Through Mechanisms Involving Both Cardiomyocyte Protection and Proliferation/Regeneration.
  • (A) and (B) show the relationship between the number of human CD117 bright endothelial progenitor cells (angioblasts) injected intravenously (10 3 , 10 5 , 10 5 plus anti-CXCR4 mAb, and 2 ⁇ 10 5 ) and improvement in myocardial function at 15 weeks, defined as mean improvement in left ventricular ejection fraction (LVEF) (A) and mean reduction in left ventricular area at end-systole (LVAs)
  • Bar graph shows that the group of animals receiving 2 ⁇ 10 5 human endothelial progenitor cells has a significantly higher index of cell cycling cardiomyocytes at the peri-infarct region than saline controls or sham operated animals (both p ⁇ 0.01). No difference between the groups is seen at sites distal to the infarct.
  • (E) shows that the index of cell-cycling cardiomyocytes at the peri-infarct rim was increased by a further 1.9-fold when 2 ⁇ 10 5 human endothelial progenitor cells were intravenously co-administered together with SDF-1 injected directly into the ischemic myocardium alone (p ⁇ 0.01), or an 8-fold cumulative increase in cell-cycling cardiomyocytes at two weeks compared with LAD-ligated controls receiving saline (results are expressed as mean+sem of three separate experiments).
  • (F) shows that intravenous co-administration of anti-CXCR4 mAb, or intracardiac co-administration of SDF-1, but not IL-8, results in 2.8 to 4-fold greater LVEF improvement, determined by echocardiography, compared with intravenous injection of 2 ⁇ 10 5 endothelial progenitor cells alone (p ⁇ 0.01) (results are expressed as mean+sem of three separate experiments).
  • (G) shows that at 15 weeks the mean proportion of scar/normal left ventricular myocardium in rats receiving either or 10 5 endothelial progenitor cells together with anti-CXCR4 mAb was significantly reduced in comparison to rats receiving either 10 3 or 10 5 endothelial progenitor cells (angioblasts) alone, or saline (p ⁇ 0.01).
  • the group receiving 2.0 ⁇ 10 5 endothelial progenitor cells demonstrated still 38% greater reduction in the ratio of scar/muscle tissue (results are expressed as mean+sem of three separate experiments).
  • FIG. 1 Sections of rat hearts stained with Masson's trichrome at 15 weeks after LAD ligation and injection of 2.0 ⁇ 10 6 G-CSF mobilized human cells containing 10 3 (left) or 2.0 ⁇ 10 5 (right) CD117 bright endothelial progenitor cells.
  • Hearts of rats receiving 10 3 endothelial progenitor cells had greater loss of anterior wall mass, collagen deposition (lighter gray), and septal hypertrophy compared with hearts of rats receiving 2.0 ⁇ 10 5 endothelial progenitor cells.
  • FIG. 5 This figure shows mRNA expression of three genes in the ischemic rat hearts at various time points. You will see that at 48 hours and 2 weeks after LAD ligation and ischemia, HBP23 (the rat homologue of human PAG/NKEF-A,B,C, all of which are part of the family of peroxiredoxins (Prx)) is decreased, and vitamin D3 upregulated protein VDUP-1 is increased. The early (48 hour) reduction in PRX and increase in VDUP-1 results in a compensatory increase in thiol reductase thioredoxin (TRX). Note that endothelial progenitor cell therapy reverses this pattern of mRNA expression.
  • HBP23 the rat homologue of human PAG/NKEF-A,B,C, all of which are part of the family of peroxiredoxins (Prx)
  • Prx peroxiredoxins
  • FIG. 6 DNA sequence (SEQ ID NO:1) corresponding to mRNA encoding VDUP-1.
  • FIG. 7 This figure shows catalytic DNA 5′-AT-3′ cleavage sites on VDUP-1 DNA (SEQ ID NO:3) corresponding to the coding region of VDUP-1 mRNA. Cleavage site pairs are uppercase.
  • FIG. 8 This figure shows catalytic DNA 5′-GC-3′ cleavage sites on VDUP-1 DNA (SEQ ID NO:3) corresponding to the VDUP-1 mRNA. Cleavage site pairs are uppercase.
  • FIG. 9 This figure shows catalytic DNA 5′-GT-3′ cleavage sites on VDUP-1 DNA (SEQ ID NO:3) corresponding to the VDUP-1 mRNA. Cleavage site pairs are uppercase.
  • FIG. 10 This figure shows catalytic DNA 5′-AC-3′ cleavage sites on VDUP-1 DNA (SEQ ID NO:3) corresponding to the VDUP-1 mRNA. Cleavage site pairs are uppercase.
  • FIG. 11 This figure shows sites which can be cleaved by a hammerhead ribozyme in VDUP-1 DNA (SEQ ID NO:3) corresponding to the VDUP-1 mRNA coding region.
  • Uppercase “T” represents cleavage site.
  • VEGF vascular endothelial growth factor
  • VEGF-R vascular endothelial growth factor receptor
  • FGF fibroblast growth factor
  • IGF Insulin-like growth factor
  • SCF stem cell factor
  • G-CSF granulocyte colony stimulating factor
  • M-CSF macrophage colony stimulating factor
  • GM-CSF granulocyte-macrophage colony stimulating factor
  • MCP monocyte chemoattractant protein.
  • angioblasts is synonymous with the term “endothelial progenitor cells”.
  • CXC chemokine refers to the structure of the chemokine. Each “C” represents a cysteine and “X” represents any amino acid. “CXCR4” refers to CXC receptor 4.
  • CC chemokine refers to the structure of the chemokine.
  • Each “C” represents a cysteine.
  • Catalytic shall mean the functioning of an agent as a catalyst, i.e. an agent that increases the rate of a chemical reaction without itself undergoing a permanent structural change.
  • Nucleic acid shall include without limitation any nucleic acid, including, without limitation, DNA, RNA, oligonucleotides, or polynucleotides, and analogs or derivatives thereof.
  • the nucleotides that form the nucleic acid may be nucleotide analogs or derivatives thereof.
  • the nucleic acid may incorporate non nucleotides.
  • nucleotides shall include without limitation nucleotides and analogs or derivatives thereof.
  • nucleotides may comprise the bases A, C, G, T and U, as well as derivatives thereof. Derivatives of these bases are well known in the art, and are exemplified in PCR Systems, Reagents and Consumables (Perkin Elmer Catalogue 1996-1997, Roche Molecular Systems, Inc., Branchburg, N.J., U.S.A.).
  • “Proliferation” and “regeneration” are used synonymously when referring to cardiomyocytes in this application. “Proliferation” in respect to cardiomyocytes, shall mean a fold increase in proportion of cardiomyocytes entering the cell cycle relative to untreated rat heart.
  • Trafficking means the blood-borne migration of cells, in particular angioblast/endothelial progenitor cells.
  • This invention provides a method of treating a disorder of a subject's heart involving loss of cardiomyocytes which comprises administering to the subject an amount of an agent effective to cause cardiomyocyte proliferation within the subject's heart so as to thereby treat the disorder.
  • the agent is human endothelial progenitor cells.
  • the endothelial progenitor cells are bone marrow-derived. In another they are derived from cord blood, or embryonic or fetal sources. Effective amounts of endothelial progenitor cells sufficient to cause cardiomyocyte proliferation can be done based on animal data using routine computational methods. In one embodiment the effective amount is about 1.5 ⁇ 10 5 endothelial progenitor cells per kg body mass to about 3 ⁇ 10 5 per kg body mass. In another embodiment the effective amount is about 3 ⁇ 10 5 per kg body mass to about 4.5 ⁇ 10 5 endothelial progenitor cells per kg body mass.
  • the effective amount is about 4.5 ⁇ 10 5 per kg body mass to about 5.5 ⁇ 10 5 endothelial progenitor cells per kg body mass. In another embodiment the effective amount is about 5.5 ⁇ 10 5 per kg body mass to about 7 ⁇ 10 5 endothelial progenitor cells per kg body mass. In another embodiment the effective amount is about 7 ⁇ 10 5 per kg body mass to about 1 ⁇ 10 6 endothelial progenitor cells per kg body mass. In another embodiment the effective amount is about 1 ⁇ 10 6 per kg body mass to about 1.5 ⁇ 10 6 endothelial progenitor cells per kg body mass.
  • the effective amount of human endothelial progenitor cells is between about 1.5 ⁇ 10 6 and 4.5 ⁇ 10 6 endothelial progenitor cells per kg of the subject's body mass and In a preferred embodiment the effective amount is about 5 ⁇ 10 5 endothelial progenitor cells per kg of the subject's body mass.
  • the endothelial progenitor cells are allogeneic with respect to the subject.
  • the subject is an adult or an embryo or a fetus.
  • the endothelial progenitor cells are derived from cloned autologous embryonic stem cells.
  • the agent induces expression of a mRNA encoding a peroxiredoxin.
  • the expression of peroxiredoxin mRNA may be increased, for example, by administration of 2(3)-t-butyl-4-hydroxyanisole (BHA) (see 106) which has been shown to increase expression of Peroxiredoxin-1 when administered by diet.
  • BHA 2(3)-t-butyl-4-hydroxyanisole
  • Peroxiredoxin mRNA expression such as that of thiol peroxidases, may also be induced by heme, cadmium or cobalt (see 108).
  • Peroxiredoxins include, but are not limited to, PAG, HBP23, MSP23, NKEF.
  • the agent induces expression of a mRNA encoding NF-E2-related factor 2 (Nrf2).
  • Cytomplasmic NF-E2-related factor 2 (Nrf2) expression can be indirectly increased by raising free Nrf2 levels. Since Nrf2 is tightly bound to keap1 in the cytoplasm then reducing expression of keap1 mRNA is a suitable target e.g. by keap1 antisense oligonucleotides or catalytic nucleic acids (see 109 for keap1). Also, blocking the interaction between Nrf2 and Keap1 by inhibiting the interaction of the Neh2 domain of Nrf2 and the DGR domain of keap1, e.g.
  • the agent induces dissociation of a Nrf2 protein from a Keap-1.
  • the agent inhibits association of a Nrf2 protein with a Keap-1.
  • the agent inhibits association of a thiol reductase thioredoxin with a VDUP-1 protein.
  • the agent inhibits c-Abl tyrosine kinase activation.
  • the agent is STI-571.
  • the agent is a CXC chemokine.
  • the agent is Stromal-Derived Factor-1, Il-8 or Gro-Alpha.
  • the amount of CXC chemokine administered is between 0.2 and 5 ⁇ g/ml at a max volume of 10 ml for a 70 kg human subject. In a preferred embodiment the amount is about 1 ⁇ g/ml.
  • the agent is an inhibitor of plasminogen activator inhibitor-1.
  • the agent is an antibody directed against an epitope of CXCR4.
  • the amount of antibody directed against an epitope of CXCR4 is between 25 and 75 ⁇ g/ml, at a max volume of 10 ml for a 70 kg human subject. A simple calculation is performed for subjects of different mass. In a preferred embodiment the amount is about 5 ⁇ g/ml.
  • the instant method further comprises administering an effective amount of a second agent that increases the cardiomyocyte proliferation caused by the human endothelial progenitor cells.
  • Effective amounts of the second agent are amounts sufficient to enhance or accelerate cardiomyocyte proliferation in the presence of administered endothelial progenitor cells.
  • the endothelial progenitor cells express CD117, CD34, AC133 or a high level of intracellular GATA-2 activity.
  • the administering comprises injecting directly into the subject's peripheral circulation, heart muscle, left ventricle, right ventricle, coronary artery, cerebro-spinal fluid, neural tissue, ischemic tissue, or post-ischemic tissue.
  • the second agent is an antisense oligonucleotide which specifically inhibits translation of Vitamin D3 Up-Regulated Protein-1 (VDUP-1) mRNA.
  • VDUP-1 Vitamin D3 Up-Regulated Protein-1
  • VDUP-1 protein (SEQ ID NO:2) expression
  • Antisense oligonucleotides are small fragments of DNA and derivatives thereof complementary to a defined sequence on a specified mRNA.
  • a VDUP-1 antisense oligonucleotide specifically binds to targets on the VDUP-1 mRNA (SEQ ID NO:1) molecule and in doing so inhibits the translation thereof into VDUP-1 protein (SEQ ID NO:2).
  • Antisense oligonucleotide molecules synthesized with a phosphorothioate backbone have proven particularly resistant to exonuclease damage compared to standard deoxyribonucleic acids, and so they are used in preference.
  • a phosphorothioate antisense oligonucleotide for VDUP-1 mRNA can be synthesized on an Applied Biosystems (Foster City, Calif.) model 380B DNA synthesizer by standard methods. E.g. Sulfurization can be performed using tetraethylthiuram disulfide/acetonitrile.
  • oligodeoxynucleotides can be base deblocked in ammonium hydroxide at 60° C. for 8 h and purified by reversed-phase HPLC [0.1M triethylammonium bicarbonate/acetonitrile; PRP-1 support]. Oligomers can be detritylated in 3% acetic acid and precipitated with 2% lithiumperchlorate/acetone, dissolved in sterile water and reprecipitated as the sodium salt from 1 M NaCl/ethanol. Concentrations of the full length species can be determined by UV spectroscopy.
  • Any other means for such synthesis known in the art may additionally or alternatively be employed. It is well known to use similar techniques to prepare oligonucleotides such as the phosphorothioates and alkylated derivatives.
  • Hybridization of antisense oligonucleotides with VDUP-1 mRNA interferes with one or more of the normal functions of VDUP-1 1 mRNA.
  • the functions of mRNA to be interfered with include all vital functions such as, for example, translocation of the RNA to the site of protein translation, translation of protein from the RNA, splicing of the RNA to yield one or more mRNA species, and catalytic activity which may be engaged in by the RNA.
  • Preferred modified oligonucleotide backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3′-alkylene phosphonates, 5′-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3′-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphates and borano-phosphates having normal 3′-5′ linkages, 2′-5′ linked analogs of these, and those having inverted polarity wherein one or more internucleotide linkages is a 3′ to 3′, 5′ to 5′ or 2′ to 2′ linkage.
  • oligonucleotides having inverted polarity comprise a single 3′ to 3′ linkage at the 3′-most internucleotide linkage i.e. a single inverted nucleoside residue which may be abasic (the nucleobase is missing or has a hydroxyl group in place thereof) are included.
  • Various salts, mixed salts and free acid forms are also included.
  • messenger RNA includes not only the information to encode a protein using the three letter genetic code, but also associated ribonucleotides which form a region known to such persons as the 5′-untranslated region, the 3′-untranslated region, the 5′ cap region and intron/exon junction ribonucleotides.
  • antisense oligonucleotides or the catalytic nucleic acids described below may be formulated in accordance with this invention which are targeted wholly or in part to these associated ribonucleotides as well as to the informational ribonucleotides.
  • the antisense oligonucleotides may therefore be specifically hybridizable with a transcription initiation site region, a translation initiation codon region, a 5′ cap region, an intron/exon junction, coding sequences, a translation termination codon region or sequences in the 5′- or 3′-untranslated region.
  • the catalytic nucleic acids may specifically cleave a transcription initiation site region, a translation initiation codon region, a 5′ cap region, an intron/exon junction, coding sequences, a translation termination codon region or sequences in the 5′- or 3′-untranslated region.
  • the translation initiation codon is typically 5′-AUG (in transcribed mRNA molecules; 5′-ATG in the corresponding DNA molecule).
  • 5′-AUG in transcribed mRNA molecules
  • 5′-ATG in the corresponding DNA molecule
  • a minority of genes have a translation initiation codon having the RNA sequence 5′-GUG, 5′UUG or 5′-CUG, and 5′-AUA, 5′-ACG and 5′-CUG have been shown to function in vivo.
  • the term “translation initiation codon” can encompass many codon sequences, even though the initiator amino acid in each instance is typically methionine in eukaryotes.
  • translation initiation codon refers to the codon or codons that are used in vivo to initiate translation of an mRNA molecule transcribed from a gene encoding VDUP-1, regardless of the sequence(s) of such codons.
  • a translation termination codon of a gene may have one of three sequences, i.e., 5′-UAA, 5′-UAG and 5′-UGA (the corresponding DNA sequences are 5′-TAA, 5′-TAG and 5′-TGA, respectively).
  • the term “translation initiation codon region” refers to a portion of such an mRNA or gene that encompasses from about 25 to about 50 contiguous nucleotides in either direction (i.e., 5′ or 3′) from a translation initiation codon. This region is one preferred target region.
  • translation termination codon region refers to a portion of such an mRNA or gene that encompasses from about 25 to about 50 contiguous nucleotides in either direction (i.e., 5′ or 3′) from a translation termination codon. This region is also one preferred target region.
  • Other preferred target regions include the 5′ untranslated region (5′UTR), known in the art to refer to the portion of an mRNA in the 5′ direction from the translation initiation codon, and thus including nucleotides between the 5′ cap site and the translation initiation codon of an mRNA or corresponding nucleotides on the gene, and the 3′ untranslated region (3′UTR), known in the art to refer to the portion of an mRNA in the 3′ direction from the translation termination codon, and thus including nucleotides between the translation termination codon and 3′ end of an mRNA or corresponding nucleotides on the gene.
  • 5′UTR 5′ untranslated region
  • 3′UTR 3′ untranslated region
  • mRNA splice sites may also be preferred target regions, and are particularly useful in situations where aberrant splicing is implicated in disease, or where an overproduction of a particular mRNA splice product is implicated in disease. Aberrant fusion junctions due to rearrangements or deletions may also be preferred targets.
  • antisense oligonucleotides can be chosen which are sufficiently complementary to the target, i.e., hybridize sufficiently well and with sufficient specificity, to give the desired disruption of the function of the molecule.
  • “Hybridization”, in the context of this invention means hydrogen bonding, also known as Watson-Crick base pairing, between complementary bases, usually on opposite nucleic acid strands or two regions of a nucleic acid strand. Guanine and cytosine are examples of complementary bases which are known to form three hydrogen bonds between them. Adenine and thymine are examples of complementary bases which form two hydrogen bonds between them.
  • Specifically hybridizable and “complementary” are terms which are used to indicate a sufficient degree of complementarity such that stable and specific binding occurs between the DNA or RNA target and the catalytic nucleic acids.
  • catalytic nucleic acids are synthesized once cleavage target sites on the VDUP-1 mRNA molecule.
  • antisense oligonucleotides or catalytic nucleic acids or analogs thereof it is preferred to administer antisense oligonucleotides or catalytic nucleic acids or analogs thereof to mammals suffering from cardiovascular disease, in either native form or suspended in a carrier medium in amounts and upon treatment schedules which are effective to therapeutically treat the mammals to reduce the detrimental effects of cardiovascular disease.
  • One or more different catalytic nucleic acids or antisense oligonucleotides or analogs thereof targeting different sections of the nucleic acid sequence of VDUP-1 mRNA may be administered together in a single dose or in different doses and at different amounts and times depending upon the desired therapy.
  • the catalytic nucleic acids or antisense oligonucleotides can be administered to mammals in a manner capable of getting the oligonucleotides initially into the blood stream and subsequently into cells, or alternatively in a manner so as to directly introduce the catalytic nucleic acids or antisense oligonucleotides into the cells or groups of cells, for example cardiomyocytes, by such means by electroporation or by direct injection into the heart.
  • Antisense oligonucleotides whose presence in cells can inhibit transcription or protein synthesis can be administered by intravenous injection, intravenous drip, subcutaneous, intraperitoneal or intramuscular injection, orally or rectally.
  • Doses of the oligonucleotides or analogs thereof of the present invention in a pharmaceutical dosage unit will be an efficacious, nontoxic quantity administered to a human patient in need of cardiomyocyte regeneration (or inhibition of VDUP-1 expression) from 1-6 or more times daily or every other day. Dosage is dependent on severity and responsiveness of the effects of abnormal cardiovascular disease to be treated, with course of treatment lasting from several days to months or until a cure is effected or a reduction of the effects is achieved. Oral dosage units for human administration generally use lower doses. The actual dosage administered may take into account the size and weight of the patient, whether the nature of the treatment is prophylactic or therapeutic in nature, the age, weight, health and sex of the patient, the route of administration, and other factors.
  • the second agent is a pro-angiogenic agent.
  • the pro-angiogenic agent is vascular endothelial growth factor, fibroblast growth factor or angiopoietin.
  • the second agent induces expression of a pro-angiogenic factor.
  • the second agent is Hypoxia Inducible Factor-1.
  • the second agent is a catalytic nucleic acid which specifically inhibits translation of Vitamin D3 Up-Regulated Protein-1 mRNA.
  • the catalytic nucleic acid comprises deoxyribonucleotides.
  • the catalytic nucleic acid comprises ribonucleotides.
  • Catalytic nucleic acid molecules can cleave Vitamin D3 Up-Regulated Protein-1 (VDUP-1) mRNA (corresponding DNA shown in SEQ ID NO:1, FIG. 5) at each and any of the consensus sequences therein. Since catalytic ribo- and deoxyribo-nucleic acid consensus sequences are known, and the VDUP-1 Protein mRNA sequence is known, one of ordinary skill could readily construct a catalytic ribo- or deoxyribo nucleic acid molecule directed to any of the VDUP-1 protein mRNA consensus sequences based on the instant specification. In preferred embodiments of this invention the catalytic deoxyribonucleic acids include the 10-23 structure.
  • catalytic ribonucleic acids examples include hairpin and hammerhead ribozymes.
  • the catalytic ribonucleic acid molecule is formed in a hammerhead (50) or hairpin motif (51,52,53), but may also be formed in the motif of a hepatitis delta virus (54), group I intron (60), RNaseP RNA (in association with an RNA guide sequence) (55,56) or Neurospora VS RNA (57,58,59).
  • catalytic nucleic acids can be designed based on the consensus cleavage sites 5′-purine:pyrimidine-3′ in the VDUP-1 mRNA sequence (104) (see FIGS. 6 - 10 ) for cleavage sites on DNA corresponding to the mRNA encoding VDUP-1 (SEQ ID NO:2). Those potential cleavage sites located on an open loop of the mRNA according to RNA folding software e.g. RNADRaw 2.1 are particularly preferred as targets (61).
  • the DNA based catalytic nucleic acids can utilize the structure where two sequence-specific arms are attached to a catalytic core based on the VDUP-1 mRNA sequence.
  • catalytic DNA structure is detailed in (62) and (63).
  • Commercially available mouse brain polyA-RNA (Ambion) can serve as a template in the in vitro cleavage reaction to test the efficiency of the catalytic deoxyribonucleic acids.
  • Catalytic RNA is described above, is designed similarly.
  • Hammerhead ribozymes can cleave any 5′-NUH-3′ triplets of a mRNA, where U is conserved and N is any nucleotide and H can be C,U,A, but not G.
  • the sites which can be cleaved by a hammerhead ribozyme in human VDUP-1 mRNA coding region are shown in FIG. 10.
  • Cleaving of VDUP-1 mRNA with catalytic nucleic acids interferes with one or more of the normal functions of VDUP-1 mRNA.
  • the functions of mRNA to be interfered with include all vital functions such as, for example, translocation of the RNA to the site of protein translation, translation of protein from the RNA, splicing of the RNA to yield one or more mRNA species, and catalytic activity which may be engaged in by the RNA.
  • the nucleotides may comprise other bases such as inosine, deoxyinosine, hypoxanthine may be used.
  • isoteric purine 2′deoxy-furanoside analogs, 2′-deoxynebularine or 2′deoxyxanthosine, or other purine or pyrimidine analogs may also be used.
  • inosine may be used to reduce hybridization specificity
  • diaminopurines may be used to increase hybridization specificity.
  • Adenine and guanine may be modified at positions N3, N7, N9, C2, C4, C5, C6, or C8 and still maintain their hydrogen bonding abilities.
  • Cytosine, thymine and uracil may be modified at positions N1, C2, C4, C5, or C6 and still maintain their hydrogen bonding abilities.
  • Some base analogs have different hydrogen bonding attributes than the naturally occurring bases. For example, 2-amino-2′-dA forms three (3), instead of the usual two (2), hydrogen bonds to thymine (T).
  • Examples of base analogs that have been shown to increase duplex stability include, but are not limited to, 5-fluoro-2′-dU, 5-bromo-2′-dU, 5-methyl-2′-dC, 5-propynyl-2′-dC, 5-propynyl-2′-dU, 2-amino-2′-dA, 7-deazaguanosine, 7-deazadenosine, and N2-Imidazoylpropyl-2′-dG.
  • Nucleotide analogs may be created by modifying and/or replacing a sugar moiety.
  • the sugar moieties of the nucleotides may also be modified by the addition of one or more substituents.
  • one or more of the sugar moieties may contain one or more of the following substituents: amino, alkylamino, araalkyl, heteroalkyl, heterocycloalkyl, aminoalkylamino, O, H, an alkyl, polyalkylamino, substituted silyl, F, Cl, Br, CN, CF 3 , OCF 3 , OCN, O-alkyl, S-alkyl, SOMe, SO 2 Me, ONO 2 , NH-alkyl, OCH 2 CH ⁇ CH 2 , OCH 2 CCH, OCCHO, allyl, O-allyl, NO 2 , N 3 , and NH 2 .
  • the 2′ position of the sugar may be modified to contain one of the following groups: H, OH, OCN, O-alkyl, F, CN, CF 3 , allyl, O-allyl, OCF 3 , S-alkyl, SOMe, SO 2 Me, ONO 2 , NO 2 , N 3 , NH 2 , NH-alkyl, or OCH ⁇ CH 2 , OCCH, wherein the alkyl may be straight, branched, saturated, or unsaturated.
  • the nucleotide may have one or more of its sugars modified and/or replaced so as to be a ribose or hexose (i.e. glucose, galactose) or have one or more anomeric sugars.
  • the nucleotide may also have one or more L-sugars.
  • the sugar may be modified to contain one or more linkers for attachment to other chemicals such as fluorescent labels.
  • the sugar is linked to one or more aminoalkyloxy linkers.
  • the sugar contains one or more alkylamino linkers. Aminoalkyloxy and alkylamino linkers may be attached to biotin, cholic acid, fluorescein, or other chemical moieties through their amino group.
  • Nucleotide analogs or derivatives may have pendant groups attached.
  • Pendant groups serve a variety of purposes which include, but are not limited to, increasing cellular uptake of the oligonucleotide, enhancing degradation of the target nucleic acid, and increasing hybridization affinity.
  • Pendant groups can be linked to any portion of the oligonucleotide but are commonly linked to the end(s) of the oligonucleotide chain. Examples of pendant groups include, but are not limited to: acridine derivatives (i.e.
  • cross-linkers such as psoralen derivatives, azidophenacyl, proflavin, and azidoproflavin; artificial endonucleases; metal complexes such as EDTA-Fe(II), o-phenanthroline-Cu(I), and porphyrin-Fe(II); alkylating moieties; nucleases such as amino-1-hexanolstaphylococcal nuclease and alkaline phosphatase; terminal transferases; abzymes; cholesteryl moieties; lipophilic carriers; peptide conjugates; long chain alcohols; phosphate esters; amino; mercapto groups; radioactive markers; nonradioactive markers such as dyes; and polylysine or other polyamines.
  • cross-linkers such as psoralen derivatives, azidophenacyl, proflavin, and azidoproflavin
  • artificial endonucleases such as EDTA-Fe(II), o
  • the nucleic acid comprises an oligonucleotide conjugated to a carbohydrate, sulfated carbohydrate, or gylcan.
  • Conjugates may be regarded as a way as to introduce a specificity into otherwise unspecific DNA binding molecules by covalently linking them to a selectively hybridizing oligonucleotide.
  • the catalytic nucleic acid binding domains i.e. the non-catalytic domains
  • antisense oligonucleotide may comprise modified bonds.
  • internucleosides bonds of the oligonucleotide may comprise phosphorothioate linkages.
  • the nucleic acid may comprise nucleotides having moiety may be modified by replacing one or both of the two bridging oxygen atoms of the linkage with analogues such as —NH, —CH 2 , or —S. Other oxygen analogues known in the art may also be used.
  • the phosphorothioate bonds may be stereo regular or stereo random.
  • the oligonucleotide moiety may have one or more of its sugars modified or replaced so as to be ribose, glucose, sucrose, or galactose, or any other sugar.
  • the phosphorothioate oligonucleotide may have one or more of its sugars substituted or modified in its 2′ position, i.e. 2′allyl or 2′-O-allyl.
  • An example of a 2′-O-allyl sugar is a 2′-O-methylribonucleotide.
  • the phosphorothioate oligonucleotide may have one or more of its sugars substituted or modified to form an ⁇ -anomeric sugar.
  • a catalytic nucleic acid may include non-nucleotide substitution.
  • the non-nucleotide substitution includes either abasic nucleotide, polyether, polyamine, polyamide, peptide, carbohydrate, lipid or polyhydrocarbon compounds.
  • abasic or “abasic nucleotide” as used herein encompasses sugar moieties lacking a base or having other chemical groups in place of base at the 1′ position.
  • Determining the effective amount of the instant nucleic acid molecules can be done based on animal data using routine computational methods.
  • the effective amount contains between about 10 ng and about 100 ⁇ g of the instant nucleic acid molecules per kg body mass. In another embodiment, the effective amount contains between about 100 ng and about 10 ⁇ g of the nucleic acid molecules per kg body mass. In a further embodiment, the effective amount contains between about 1 ⁇ g and about 5 ⁇ g, and in a further embodiment about 2 ⁇ g, of the nucleic acid molecules per kg body mass.
  • the second agent is cardiomyocyte progenitor cells.
  • the second agent is skeletal muscle progenitor cells. Either of theses cell types can be derived from embryonic, fetal or adult subjects.
  • the second agent promotes trafficking of the endothelial progenitor cells to the subject's heart.
  • the second agent that promotes trafficking is an antibody directed against an epitope of CXCR4.
  • the second agent that promotes trafficking is a CC chemokine.
  • the CC chemokine is RANTES, EOTAXIN, monocyte chemoattractant protein-1 (MCP-1), MCP-2, MCP-3, or MCP.
  • the second agent that promotes trafficking is a CXC chemokine.
  • the CXC chemokine is Interleukin-8, Gro-Alpha, or Stromal-Derived Factor-1.
  • This invention also provides a method of determining the susceptibility of a cardiomyocyte in a subject to apoptosis comprising:
  • This invention also provides a method of determining the susceptibility of a cardiomyocyte in a subject to apoptosis comprising:
  • a ratio that is at least two standard deviations above that found in control patients with normal hearts is considered a high ratio.
  • Quantitation of expression of protein in cardiomyocytes is performed using routine methods known to those of skill in the art, for example, northern and western blots.
  • the subject of any of the above methods is a mammal and in a preferred further embodiment the mammal is a human being.
  • the subject has a cardiovascular disease.
  • the subject has congestive heart failure, has suffered a myocardial infarct, has suffered myocardial ischemia, has angina, or has a cardiomyopathy.
  • LAD coronary artery ligation resulted in trafficking of intravenously injected human endothelial progenitor cells to the site of ischemic myocardium, it was also accompanied by increased distribution of human cells to rat bone marrow.
  • FIG. 2 a at 2-14 days after intravenous injection of 2 ⁇ 10 6 human CD34+ cells bone marrow from LAD-ligated rats contained 5-8 fold higher levels of human CD117 bright endothelial progenitor cells compared with bone marrow from normal rats, p ⁇ 0.001.
  • co-administration of mAbs against either human CXCR4 or rat SDF-1 significantly inhibited migration of intravenously administered human endothelial progenitor cells to ischemic rat bone marrow compared with anti-CD34 control antibody (both p ⁇ 0.001).
  • co-administration of mAbs against either human CXCR4 or rat SDF-1 increased trafficking of CD34+ human endothelial progenitor cells to ischemic rat myocardium by means of 24% and 17%, respectively (both p ⁇ 0.001), FIG. 2 c.
  • Induction of neovascularization at two weeks was measured by performing quantitative analysis of medium- and large-sized capillaries, defined, respectively, as having 3-6 or >6 contiguous endothelial lining cells.
  • Medium-sized capillaries had mean lumen diameter of 0.020 mm+0.002, while large-sized capillaries had mean lumen diameter of 0.053 mm+0.004 (p ⁇ 0.001).
  • large-lumen capillaries overlapped in size with arterioles which could be distinguished by a thin layer containing 2-3 smooth muscle cells of rat origin, as determined by positive staining with desmin and rat MHC class I mAbs. As shown in FIGS.
  • both the group receiving 2 ⁇ 10 5 endothelial progenitor cells and the one receiving 10 5 endothelial progenitor cells plus anti-CXCR4 mAb demonstrated 1.7-fold higher numbers of medium-sized capillaries compared with the other two groups (p ⁇ 0.01).
  • the group receiving 2 ⁇ 10 5 endothelial progenitor cells additionally demonstrated 3.3-fold higher numbers of large-lumen capillaries compared with the groups receiving 10 3 or 10 5 endothelial progenitor cells (p ⁇ 0.01), and 2-fold higher numbers of large-lumen capillaries compared with the group receiving 10 5 endothelial progenitor cells plus anti-CXCR4 mAb (p ⁇ 0.01).
  • FIG. 1 As shown in FIG.
  • FIG. 3 e the number of apoptotic cardiomyocytes at the infarct rim was significantly reduced in both rats receiving 10 5 endothelial progenitor cells plus anti-CXCR4 mAb and those receiving 2 ⁇ 10 5 endothelial progenitor cells compared with the groups receiving either 10 3 or 10 5 endothelial progenitor cells alone (both p ⁇ 0.001).
  • co-administration of anti-CXCR4 mAb or intracardiac injection of SDF-1 resulted in further reductions in cardiomyocyte apoptosis of 65% and 76%, respectively, FIG. 3 f (both p ⁇ 0.001).
  • FIGS. 4 a and b We next examined the effect of increasing the number of human endothelial progenitor cells trafficking to ischemic myocardium on long-term myocardial function, defined as the degree of improvement in left ventricular ejection fraction (LVEF) and reduction in left ventricular end-systolic area (LVAs) at 15 weeks after intravenous injection, FIGS. 4 a and b . No improvement in these parameters was observed in the groups receiving 10 3 or 10 5 endothelial progenitor cells in comparison to rats receiving saline alone.
  • LVEF left ventricular ejection fraction
  • LVAs left ventricular end-systolic area
  • rats receiving 10 5 endothelial progenitor cells plus anti-CXCR4 mAb demonstrated significant improvement in these parameters, 22+2% mean recovery in LVEF and 24+4% mean reduction in LVAs (both p ⁇ 0.001). Even more strikingly, the group receiving 2 ⁇ 10 5 endothelial progenitor cells had a mean recovery in LVEF of 34+4% and a mean reduction in LVAs of 37+6% (both p ⁇ 0.001), or 50% further improvement in both parameters.
  • the number of cardiomyocytes progressing through cell cycle at the peri-infarct region of rats receiving 2 ⁇ 10 5 human endothelial progenitor cells was 40-fold higher than that at sites distal to the infarct, where myocyte DNA activity was no different than in sham-operated rats. As shown in FIG.
  • animals receiving 2 ⁇ 10 5 human endothelial progenitor cells had a 20-fold higher number of cell-cycling cardiomyocytes at the peri-infarct rim than that found in non-infarcted hearts (1.19+0.2% vs 0.06+0.03%, p ⁇ 0.01) and 3.5-fold higher than in the same region in LAD-ligated controls receiving saline (1.19+0.2% vs 0.344+0.1%, p ⁇ 0.01).
  • intracardiac injection of SDF-1 in combination with intravenous injection of 2 ⁇ 10 5 human endothelial progenitor cells resulted in approximately an 8-fold cumulative increase in cell-cycling cardiomyocytes at two weeks compared with LAD-ligated controls receiving saline, and translated into over 4-fold greater LVEF improvement, determined by echocardiography, compared with intravenous injection of 2 ⁇ 10 5 endothelial progenitor cells alone (FIG. 4 f , p ⁇ 0.01).
  • Co-administration of anti-CXCR4 mAb augmented LVEF improvement by 2.8-fold (p ⁇ 0.01) while intracardiac injection of IL-8 conferred no additive benefit.
  • FIG. 4 h The overall effects of medium- and large-size neovasculature combining to both protect against myocyte apoptosis and induce myocyte proliferation/regeneration are shown dramatically in FIG. 4 h where, in contrast to saline controls, injection with 2 ⁇ 10 5 endothelial progenitor cells resulted in almost complete salvage of the anterior myocardium, normal septal size and minimal collagen deposition.
  • E2 catalytic nucleic acid
  • Vitamin D3 Up-Regulated Proteinl VDUP1
  • H 2 O 2 hydrogen peroxide
  • the cell cycle inhibitory activity of p21Cip1/WAF1 is intimately correlated with its nuclear localization and participation in quaternary complexes of cell cycle regulators by binding to G1 cyclin-CDK through its N-terminal domain and to proliferating cell nuclear antigen (PCNA) through its C-terminal domain (68-71).
  • PCNA cell nuclear antigen
  • the latter interaction blocks the ability of PCNA to activate DNA polymerase, the principal replicative DNA polymerase (72).
  • For a growth-arrested cell to subsequently enter an apoptotic pathway requires signals provided by specific apoptotic stimuli in concert with cell-cycle regulators.
  • caspase-mediated cleavage of p21 together with upregulation of cyclin A-associated cdk2 activity, have been shown to be critical steps for induction of cellular apoptosis by either deprivation of growth factors (73) or hypoxia of cardiomyocytes (74).
  • the apoptosis signal-regulating kinase 1 (ASK1) is a pivotal component in the mechanism of cytokine- and stress-induced apoptosis (75,76). Under basal conditions, resistance to ASK1-mediated apoptosis appears to be the result of complex formation between ASK1, cytoplasmic p21Cip1/WAF1 (77), and the thiol reductase thioredoxin (TRX) (78). Intact cytoplasmic expression of p21Cip1/WAF1 appears to be important for both prevention of apoptosis in response to ASK1 (75) and in maintaining a state of terminal differentiation (77).
  • the reduced form of TRX binds to the N-terminal portion of ASK1 and is a physiologic inhibitor of ASK1-mediated cellular apoptosis (78).
  • the recently-identified protein VDUP1 has been shown to compete with ASK1 for binding of the reduced form of TRX (78,79), resulting in augmention of ASK1-mediated apoptosis (80). This indicates that ASK1-mediated cellular apoptosis is increased by processes that result in a net dissociation of TRX from ASK1, such as either generation of TRX-VDUP1 complexes or generation of oxidised TRX by changes in cellular redox status accompanying oxidative stress.
  • TRX and glutathione constitute the major cellular reducing systems that maintain the thiol-disulfide status of the cytosol (81).
  • the redox-active/dithiol active site of TRX is highly conserved across all species, Trp-Cys-Gly-Pro-Cys-Lys.
  • the two cysteine residues at the active site, Cys-32 and Cys-35 undergo reversible oxidation-reduction reactions catalyzed by a NADPH-dependent enzyme TRX reductase. These reactions involve electron transfer via disulfide bridges formed with members of a family of antioxidant enzymes known as peroxiredoxins (Prxs), which show peroxidase activity (82,83).
  • Prxs peroxiredoxins
  • Prxs are distinct from other peroxidases in that they have no cofactors, such as metals or prosthetic groups. Prxs generally have two conserved cysteines at the N- and C-terminal regions (84), and their antioxidant effects are coupled with the physiological electron donor activity of the TRX system (82,85,86). Prxs with 95-97% sequence homology have been identified in rats (Heme-binding protein 23, HBP23) (87), mice (mouse macrophage stress protein 23, MSP23) (88) and humans (proliferation-associated gene product, PAG (89) and human natural killer cell-enhancing factor A (90)).
  • Prxs are members of a repertoire of oxidative stress responsive genes whose expression is regulated by NF-E2-related factor 2 (Nrf2) which binds to an anti-oxidant responsive element (ARE) present in the promoter of each (91). These include glutathione-S-transferase, heme oxygenase-1, and TRX. Under basal conditions, Nrf2 is bound to a specific protein, Keap1, in the cytosol (92). However, under conditions of oxidative stress Nrf2 dissociates from Keap1 and translocates to the nucleus where it induces transcriptional activation of the anti-oxidant genes containing ARE motifs.
  • Nrf2 NF-E2-related factor 2
  • ARE anti-oxidant responsive element
  • Nrf2 nuclear translocation of Nrf2 and subsequent ARE activation appear to be dependent on pathways activated by phosphatidylinositol 3-kinase (PI3 kinase) (93).
  • PI3 kinase phosphatidylinositol 3-kinase
  • hemin is a potent inducer of Nrf2 dissociation from Keap1, resulting in TRX gene transcription through the ARE (94).
  • Prxs During periods of rapid changes in cellular redox Prxs presumably serve to maintain the cytosolic levels of reduced TRX by accepting electrons from the oxidized form of TRX. This homeostatic mechanism likely enables maintenance of sufficient levels of reduced TRX to ensure adequate binding to ASK1 and prevention of cellular apoptosis. If the endogenous Prx system is overloaded, as might occur during changes in cellular redox when excess oxidized TRX is generated, cellular apoptosis will occur through the unopposed effects of ASK1. To counteract this, transcriptional activation of Prxs must occur following oxidative stress via nuclear translocation of Nrf2. This can be achieved either by Nrf2 dissociation from Keap1 via hemin- and PI3 kinase-dependent mechanisms (93,94), or by increasing Nrf2 mRNA and protein expression as occurs following increase in oxygen tension (95,96).
  • the Prx gene products specifically bind the SH3 domain of c-Abl, a non-receptor tyrosine kinase, inhibiting its activation by various stimuli, including agents that damage DNA (97).
  • c-Abl activation through the SH3 domain induces either arrest of the cell cycle in phase G1 or cellular apoptosis (98).
  • Cell cycle arrest is dependent on the kinase activity of c-Abl (96) and is mediated by the ability of c-Abl to downregulate the activity of the cyclin-dependent kinase Cdk2 and induce the expression of p21 (99).
  • c-Abl The apoptotic effects of c-Abl are dependent on the ability of nuclear c-Abl to phosphorylate p73, a member of the p53 family of tumor-suppressor proteins which can induce apoptosis (100,101). Recently, it has been shown that cytoplasmic, rather than nuclear, forms of c-Abl are activated by H 2 O 2 and that this results in mitochondrial localization of c-Abl, c-Abl dependent cytochrome c release, and cellular apoptosis following oxidative stress (102,103).
  • the PAG gene product By associating with c-Abl in vivo, the PAG gene product (and presumably the other Prxs) can inhibit tyrosine phosphorylation induced by c-Abl overexpression and rescue cells from both the cytostatic and pro-apoptotic effects of the activated c-Abl gene product (97).
  • Nrf2-dependent oxidative stress responsive genes are downregulated following myocardial ischemia likely reflects direct effects of hemin and oxygen deprivation.
  • the end result of Prx downregulation in the ischemic heart would be augmentation in ASK1-dependent cellular apoptosis as well as Abl-dependent apoptosis and cell cycle arrest.
  • the observed parallel increase in VDUP1 expression would further augment ASK1-dependent cellular apoptosis.
  • the ratio in expression of PAG or other Prx mRNA or protein to VDUP1 mRNA or protein can form the basis of a diagnostic assay to predict the degree of risk for cardiomyocyte apoptosis and cell cycle arrest after ischemia, as well as enable monitoring of the response to specific therapy after myocardial ischemia that protects cardiomyocytes against apototic death and enhances myocardial proliferation/regeneration.
  • Nrf2 mRNA or causing dissociation of Nrf2 protein from Keap1, or preferably cause both to occur simultaneously in the setting of myocardial ischemia in order to increase transcription and activity of members of a repertoire of oxidative stress responsive genes whose expression is regulated by binding of Nrf2 to an anti-oxidant responsive element (ARE) in their promoters, including the Prxs, TRX and glutathione-S-transferase would result in both protection of cardiomyocytes against apoptosis as well as induce cardiomyocyte cell cycle progression following oxidative stress.
  • ARE anti-oxidant responsive element
  • VDUP1 Reducing the expression of VDUP1 following myocardial ischemia would protect the ischemic myocardium against apoptosis by reducing binding of TRX to VDUP1, and consequently increasing TRX-ASK1 interactions.
  • Neovascularization of the myocardium by either bone marrow-derived endothelial progenitors or any other process, is an example of one method which causes induction of Prx expression and reduction in VDUP1 expression after myocardial ischemia, and results in both protection against redox-mediated apoptosis and induction of myocardial proliferation/regeneration.
  • Single-donor leukopheresis products were obtained from humans treated with recombinant G-CSF 10 mg/kg (Amgen, CA) sc daily for four days. Donors were healthy individuals undergoing standard institutional procedures of bone marrow mobilization, harvesting and isolation for allogeneic stem cell transplants. Mononuclear cells were separated by Ficoll-Hypaque, and highly-purified CD34+ cells (>98% positive) were obtained using magnetic beads coated with anti-CD34 monoclonal antibody (mAb) (Miltenyi Biotech, CA).
  • mAb anti-CD34 monoclonal antibody
  • CD34 cells were stained with fluorescein-conjugated mAbs against CD34 and CD117 (Becton Dickinson, CA), AC133 (Miltenyi Biotech, CA), CD54 (Immunotech, CA), CD62E (BioSource, MA), VEGFR-2, Tie-2, vWF, eNOS, CXCR1, CXCR2, and CXCR4 (all Santa Cruz Biotech, CA), and analyzed by four-parameter fluorescence using FACScan (Becton Dickinson, CA).
  • Cells positively selected for CD34 expression were also stained with phycoerythrin (PE)-conjugated anti-CD117 mAb (Becton Dickinson, CA), and sorted for bright and dim fluorescence using a Facstar Plus (Becton Dickinson) and a PE filter.
  • Intracellular staining for GATA-2 was performed by permeabilizing one million cells from each of the brightly and dimly fluorescing cell populations using a Pharmingen Cytofix/CytopermTM kit, incubating for 30 minutes on ice with 10 ⁇ l of fluorochrome-conjugated mAbs against both CD117 and CD34 surface antigens (Becton Dickinson, CA).
  • CD34+CD117 brigh t cells were plated in 48-well chemotaxis chambers fitted with membranes (8 mm pores) (Neuro Probe, MD). After incubation for 2 hours at 37° C., chambers were inverted and cells were cultured for 3 hours in medium containing IL-8, SDF-1 alpha/beta, and SCF at 0.2, 1.0 and 5.0 ⁇ g/ml. The membranes were fixed with methanol and stained with LeukostatTM (Fischer Scientific, Ill.). Chemotaxis was calculated by counting migrating cells in 10 high-power fields.
  • CD34+ cells obtained from a single donor after G-CSF mobilization were injected into the tail vein 48 hours after LAD ligation either alone or together with 50 ⁇ g/ml monoclonal antibody (mAb) with known functional inhibitory activity against either human CXCR1, human CXCR2, human CXCR4, rat SDF-1 (all R & D Systems, MN), human CD34 (Pharmingen, CA), or rat IL-8 (ImmunoLaboratories, Japan). Controls received either isotype control antibodies at the same concentration or saline after LAD ligation.
  • mAb monoclonal antibody
  • Quantitation of myocardial infiltration after injection of human cells was performed by assessment of DiI fluorescence in hearts from rats sacrificed 2 days after injection (expressed as number of DiI-positive cells per high power field, minimum 5 fields examined per sample). Quantitation of rat bone marrow infiltration by human cells was performed in 12 rats at baseline, days 2, 7, and 14 by flow cytometric and RT-PCR analysis of the proportion of HLA class I-positive cells relative to the total rat bone marrow population.
  • Poly(A)+ mRNA was extracted by standard methods from the hearts of 3 normal and 12 LAD-ligated rats. RT-PCR was used to quantify myocardial expression of rat IL-8 and Gro-alpha mRNA at baseline and at 6, 12, 24 and 48 hours after LAD ligation after normalizing for total rat RNA as measured by GAPDH expression.
  • cDNA was amplified in the polymerase chain reaction (PCR) using Taq polymerase (Invitrogen, Carlsbad, Calif., U.S.A.), radiolabeled dideoxy-nucleotide ([a32P]-ddATP: 3,000 Ci/mmol, Amersham, Arlington Heights, Ill.), and primers for rat Cinc (rat homologue of human IL-8/Gro-alpha and GAPDH (Fisher Genosys, CA).
  • Taq polymerase Invitrogen, Carlsbad, Calif., U.S.A.
  • radiolabeled dideoxy-nucleotide [a32P]-ddATP: 3,000 Ci/mmol, Amersham, Arlington Heights, Ill.
  • primers for rat Cinc rat homologue of human IL-8/Gro-alpha and GAPDH (Fisher Genosys, CA).
  • Primer pairs (sense/antisense) for rat Cinc and GAPDH were, gaagatagattgcaccgatg (SEQ ID NO:4)/catagcctctcacatttc SEQ ID NO:5), gcgcccgtccgccaatgagctgcgc SEQ ID NO:6)/cttggggacacccttcagcatctttgg SEQ ID NO:7), and ctctacccacggcaagttcaa SEQ ID NO:8)/gggatgaccttgcccacagc SEQ ID NO:9), respectively.
  • rat IL-8/Gro-alpha Serum levels of rat IL-8/Gro-alpha were measured at baseline and at 6, 12, 24 and 48 hours after LAD ligation in four rats by a commercial ELISA using polyclonal antibodies against the rat IL-8/Gro homologue Cinc (ImmunoLaboratories, Japan). The amount of protein in each serum sample was calculated according to a standard curve of optical density (OD) values constructed for known levels of rat IL-8/Gro-alpha protein.
  • OD optical density
  • Anti-Cinc antibodies were also used according to the manufacturer's instructions at 1:200 dilution in immunohistochemical studies to identify the cellular source of Cinc production in rat myocardium after LAD ligation. Positively-staining cells were visualized as brown through the Avidin/Biotin system described below.
  • Staining was performed by immunoperoxidase technique using an Avidin/Biotin Blocking Kit, a rat-absorbed biotinylated anti-mouse IgG, and a peroxidase-conjugate (all Vector Laboratories Burlingame, Calif.).
  • Capillary density was determined at 2 weeks post infarction from sections labeled with anti-CD31 mAb, and confirmed with anti-factor VIII mAb, and compared to the capillary density of the unimpaired myocardium. Values are expressed as the number of CD31-positive capillaries per HPF (400x).
  • Cardiomyocyte DNA synthesis and cell cycling was determined by dual staining of rat myocardial tissue sections obtained from LAD-ligated rats at two weeks after injection of either saline or CD34+ human cells, and from healthy rats as negative controls, for cardiomyocyte-specific troponin I and human- or rat-specific Ki-67. Briefly, paraffin embedded sections were microwaved in a 0.1M EDTA buffer, and stained with either a primary monoclonal antibody against rat Ki-67 at 1:3000 dilution (gift of Giorgio Catoretti, Columbia University) or human Ki-67 at 1:300 dilution (Dako, CA) and incubated overnight at 4 degrees C.
  • TUNEL dexynucleotidyl transferase
  • tissue sections were then digested with Proteinase K (10 ⁇ g/ml in Tris/HCL) for 30 minutes at 37° C.
  • the slides were then washed 3 times in PBS and incubated with 50 ⁇ l of the TUNEL reaction mixture (TdT and fluorescein-labeled dUTP) and incubated in a humid atmosphere for 60 minutes at 37° C.
  • TdT was eliminated from the reaction mixture.
  • the sections were then incubated for 30 minutes with an antibody specific for fluorescein-conjugated alkaline phosphatase (AP) (Boehringer Mannheim, Mannheim, Germany).
  • AP fluorescein-conjugated alkaline phosphatase
  • the TUNEL stain was visualized with a substrate system in which nuclei with DNA fragmentation stained blue, (BCIP/NBT substrate system, Dako, Carpinteria, Calif.). The reaction was terminated following three minutes of exposure with PBS. To determine the proportion of blue-staining apoptotic nuclei within myocytes, tissue was counterstained with a monoclonal antibody specific for desmin. Endogenous peroxidase was blocked by using a 3% hydrogen perioxidase solution in PBS for 15 minutes, followed by washing with 20% goat serum solution. An anti-troponin I antibody (Accurate Chemicals, CT) was incubated overnight (1:200) at 40 degrees C.
  • BCIP/NBT substrate system Dako, Carpinteria, Calif.
  • Tissue sections were examined microscopically at 200 ⁇ magnification. Within each 200 ⁇ field 4 regions were examined, containing at least 250 cells per region and cumulatively approximating 1 mm 2 of tissue, at both the peri-infarct site and distally to this site. Stained cells at the edges of the tissue were not counted. Results were expressed as the mean number of apoptotic myocytes per mm 2 at each site examined.
  • Echocardiographic studies were performed using a high frequency liner array transducer (SONOS 5500, Hewlett Packard, Andover, Mass.). 2D images were obtained at mid-papillary and apical levels. End-diastolic (EDV) and end-systolic (ESV) left ventricular volumes were obtained by bi-plane area-length method, and % left ventricular ejection fraction was calculated as [(EDV ⁇ ESV)/EDV] ⁇ 100.
  • SONOS 5500 High frequency liner array transducer
  • ESV End-diastolic
  • ESV end-systolic
  • messenger RNA was isolated from each heart, and 1 ⁇ g was used for first-strand cDNA synthesis with random primers.
  • the subtractive hybridization was performed with the PCR-select cDNA subtraction kit (CLONTECH), following the manufacturer's recommendations.
  • CLONTECH PCR-select cDNA subtraction kit
  • the two cDNA libraries were digested with RsaI. Digestion products of the “tester” library were ligated to a specific adapter (T7 promoter), then hybridized with a 30-fold excess of the “driver” library for subtraction. After hybridization, the remaining products were further amplified by PCR.
  • the ischemic tissue is the “tester” and the normal tissue is the “driver.”
  • the “tester” and the “driver” are switched to determine the genes that are down-regulated in the ischemic sample.
  • Nrf2 Transcription factor Nrf2 coordinately regulates a group of oxidative stress-inducible genes in macrophages. J Biological Chem 2000, 275:16023-16029
  • Keap1 represses nuclear activation of antioxidant responsive elements by Nrf2 through binding to the amino-terminal Neh2 domain. Genes Dev. Jan. 1, 1999;13(1):76-86.
  • Jost, C. A., Marin, M. C. & Kaelin, W. J. p73 is a human p53-related protein that can induce apoptosis. Nature 389, 191-194 (1997).

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CA2482996A CA2482996C (fr) 2002-04-23 2003-04-23 Proliferation de cardiomyocytes promus sdf-1 pour le traitement de troubles cardiomyocytes induits par la mort
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US20050265980A1 (en) * 2004-05-14 2005-12-01 Becton, Dickinson And Company Cell culture environments for the serum-free expansion of mesenchymal stem cells
US20060035290A1 (en) * 2004-08-13 2006-02-16 Medtronic, Inc. Isolation of endothelial progenitor cell subsets and methods for their use
US20060111290A1 (en) * 2000-06-05 2006-05-25 The Trustees Of Columbia University In The City Of New York Use of SDF-1 or G-CSF to improve myocardial function after ischemic injury
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CA2482996A1 (fr) 2003-11-06
CN1662548A (zh) 2005-08-31
US20050233992A1 (en) 2005-10-20
WO2003090512A2 (fr) 2003-11-06
US20070172467A1 (en) 2007-07-26
IL164671A (en) 2014-11-30
EP2292631B1 (fr) 2017-02-15
US20150056161A1 (en) 2015-02-26
US7887796B2 (en) 2011-02-15
CN100379751C (zh) 2008-04-09
EP2366706A1 (fr) 2011-09-21
EP2292631A1 (fr) 2011-03-09
AU2003231090B2 (en) 2009-10-01
WO2003090512A3 (fr) 2004-11-04
AU2009213032A1 (en) 2009-10-08
ZA200408777B (en) 2007-04-25
JP5911841B2 (ja) 2016-05-11
EP1501852A4 (fr) 2008-10-01
IL164671A0 (en) 2005-12-18
AU2009213032B2 (en) 2013-06-27
EP1501852A2 (fr) 2005-02-02
JP2005534290A (ja) 2005-11-17
JP2011137011A (ja) 2011-07-14
IL235149B (en) 2019-03-31
US8663652B2 (en) 2014-03-04
JP2016155804A (ja) 2016-09-01
US20040247564A1 (en) 2004-12-09
US8242091B2 (en) 2012-08-14
JP2014088390A (ja) 2014-05-15
AU2003231090A1 (en) 2003-11-10
JP4897199B2 (ja) 2012-03-14
JP5619644B2 (ja) 2014-11-05
CA2482996C (fr) 2017-02-07

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