Throughout this application, various publications are referenced in parentheses by arabic numbers. Full citations for these references may be found at the end of the specification immediately preceding the claims. The disclosures of these publications in their entireties are hereby incorporated by reference into this application to more fully describe the state of the art to which this invention pertains.
Healing of a myocardial infarct is complicated by the need for viable myocytes at the peri-infarct rim to undergo compensatory hypertrophy in order to increase pump function in response to the loss of infarcted tissue (1,2). This initiates a process termed cardiac remodelling which is characterized by apoptotic loss of hypertrophied myocytes, expansion of the initial infarct area, progressive collagen replacement, and heart failure (3-6). We have recently put forward the hypothesis that hypertrophied cardiac myocytes undergo apoptosis because the endogenous capillary network cannot provide the compensatory increase in perfusion required for cell survival (7).
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).
Cells which can differentiate into endothelial elements also exist in adult bone marrow (12-14) and can induce vasculogenesis in ischemic tissues (15-17). In the adult, new blood vessel formation can occur either through angiogenesis from pre-existing mature endothelium or vasculogenesis mediated by bone marrow-derived endothelial precursors. Recently, we identified a specific population of endothelial progenitor cells (angioblasts) derived from human adult bone marrow which has phenotypic and functional characteristics of embryonic angioblasts (7). We showed that intravenous administration of these cells resulted in selective homing to ischemic myocardium, induction of infarct bed vasculogenesis, prevention of peri-infarct myocyte apoptosis, and significant improvement in myocardial function (7).
We recently discovered that CXC chemokines containing the ELR motif regulate migration of human bone marrow-derived endothelial progenitor cells to sites of tissue ischemia. Moreover, since selective bone marrow homing and engraftment of hematopoietic progenitors depends on CXCR4 binding to SDF-1 expressed constitutively in the bone marrow (28-30), we demonstrated that 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. Our results indicated that CXC chemokines, including IL-8, Gro-alpha, and SDF-1, play a central role in regulating human adult bone marrow-dependent vasculogenesis.
Recent observations have suggested that a second compensatory response of viable cardiomyocytes is to proliferate and regenerate following injury (18,19). We have previously shown that pro-angiogenic fatcors, such as endothelial progenitor cells at a minimum concentration can induce vasculogenesis. Here we disclose the surprising result that careful dosing of endothelial progenitor cells/other pro-angiogenic agents can induce cardiomyocyte proliferation also, and this proliferation can be enhanced by manipulating CXCR4/stromal derived factor-1 interactions. Moreover, we show that the mechanisms for this enhanced proliferation is via modulation of mRNA expression of a number of stress-inducible genes involved in cardiomyocyte apoptosis and cell-cycling.
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:
(a) quantitating the expression a peroxiredoxin in the cardiomyocyte;
(b) quantitating the expression of Vitamin D3 Up-Regulated Protein-1 in the cardiomyocyte; and
BRIEF DESCRIPTION OF THE FIGURES
(c) determining the ratio of peroxiredoxin expression: Vitamin D3 Up-Regulated Protein-1 expression, wherein a low ratio indicates a high susceptibility of the cardiomyocyte to apoptosis and a high ratio indicates a low susceptibility of the cardiomyocyte to apoptosis in the subject.
FIGS. 1A-1D: IL-8/Gro-Alpha CXC Chemokines Regulate Migration Of Human Endothelial progenitor cells (angioblasts) To Myocardial Tissue In Vivo And Subsequent Development Of Vasculogenesis.
(A) DiI-labelled human endothelial progenitor cells (angioblasts) (>98% CD34+purity) injected intravenously into nude rats infiltrate rat myocardium after coronary artery ligation and infarction but not after sham operation at 48 hours.
(B) Migration of human endothelial progenitor cells (angioblasts) to ischemic rat myocardium is inhibited by mAbs against either rat IL-8 or the IL-8/Gro-alpha chemokine family receptors CXCR1 and CXCR2 (all p<0.01), but not against VEGF or its receptor Flk-1 (results are expressed as mean+sem of three separate experiments).
(C) Masson's trichrome stain of rat myocardial infarct bed at two weeks after LAD ligation demonstrating diffuse increase in matrix deposition and few capillaries in representative animal injected with saline (x400), diffuse increase in capillaries (arrowheads) and reduction in matrix deposition in representative animal injected with human bone marrow-derived endothelial progenitor cells (x400), and reduction in capillary numbers in representative animal injected with human endothelial progenitor cells (angioblasts) together with mAb against human CXCR1/2 (x400).
(D) Intracardiac injection of IL-8 or SDF-1 at 1 μg/ml significantly increases in vivo chemotaxis of DiI-labelled human endothelial progenitor cells (angioblasts) (98% CD34+purity) into non-ischemic rat heart in comparison with injection of saline or stem cell factor (SCF), p<0.01 (results are expressed as mean+sem of three separate experiments). Below is shown representative fluorescence microscopy of intravenously-injected DiI-labelled human endothelial progenitor cells infiltrating non-ischemic rat heart after intracardiac injection with saline, IL-8 or SDF-1.
FIGS. 2A-2C: Blocking CXCR4/SDF-1 Interactions Redirects Intravenously Injected Human Endothelial progenitor cells From Bone Marrow To Ischemic Myocardium.
(A) the proportion of human CD34+CD117bright endothelial progenitor cells (angioblasts) in rat bone marrow 2-14 days after intravenous injection is significantly increased following ischemia induced by LAD ligation (results are expressed as mean+sem of bone marrow studies in three animals at each time point).
(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. 3A-3F. Redirected Trafficking Of Human Endothelial progenitor cells (angioblasts) To The Site Of Infarction Induces Vasculogenesis And Protects Cardiomyocytes Against Apoptosis.
(A) Myocardial infarct bed two weeks post-LAD ligation from representative animals in each group stained with Masson's trichrome (upper panel) or immunoperoxidase after binding of anti-CD31 mAb (lower panel). The infarct zones of rats receiving either 103 or 105 endothelial progenitor cells (angioblasts) show myocardial scars composed of paucicellular, dense fibrous tissue stained blue by trichrome (x400). In contrast, the infarct zones of rats injected with 105 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 2×105 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 CD117bright endothelial progenitor cells injected intravenously (103, 105, 105 plus anti-CXCR4 mAb, and 2×105) 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.
(B) the groups receiving either 2×105 endothelial progenitor cells (angioblasts) or 105 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).
(C) the group receiving 2×105 endothelial progenitor cells (angioblasts) additionally demonstrated 3.3-fold higher numbers of large-lumen capillaries compared with the groups receiving 103 or 105 endothelial progenitor cells (p<0.01), and 2-fold higher numbers of large-lumen capillaries compared with the group receiving 105 endothelial progenitor cells plus anti-CXCR4 mAb (p<0.01).
(D) shows that co-administration of anti-CXCR4 mAb together with the highest concentration of endothelial progenitor cells (angioblasts), 2×105, 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 2×105 endothelial progenitor cells were injected intravenously after direct intracardiac delivery of 1.0 μg/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 2.0×105 endothelial progenitor cells or 105 endothelial progenitor cells together with anti-CXCR4 mAb in comparison to rats receiving 103 or 105 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. 4A-4H. 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 CD117bright endothelial progenitor cells (angioblasts) injected intravenously (103, 105, 105 plus anti-CXCR4 mAb, and 2×105) 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)
(B). No significant improvement in these parameters was observed in the groups receiving 103 or 105 endothelial progenitor cells in comparison to rats receiving saline alone. In contrast, rats receiving 105 endothelial progenitor cells plus anti-CXCR4 mAb demonstrated significant recovery in LVEF and reduction in LVAs (both p<0.001). The group receiving 2×105 endothelial progenitor cells demonstrated still 50% greater recovery in LVEF and reduction in LVAs (both p<0.001).
(C) Section from infarct of representative animal receiving 2×105 endothelial progenitor cells (angioblasts) showing a high frequency of cardiomyocytes staining positively for both cardiac-specific troponin I and rat-specific Ki-67 (arrows). Note the proximity of Ki-67-positive cardiomyocytes to capillaries (arrowheads).
(D) Section from infarct of representative animal receiving 2×105 endothelial progenitor cells showing “finger” of cardiomyocytes of rat origin, as determined by expression of rat MHC class I molecules, extending from the peri-infarct region into the infarct zone. These cellular islands contain a high frequency of myocytes staining positively for both cardiac-specific troponin I and rat-specific Ki-67 (arrows). Sections from infarcts of representative animals receiving saline do not show same frequency of dual staining myocytes. Bar graph shows that the group of animals receiving 2×105 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 2×105 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 2×105 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 105 endothelial progenitor cells together with anti-CXCR4 mAb was significantly reduced in comparison to rats receiving either 103 or 105 endothelial progenitor cells (angioblasts) alone, or saline (p<0.01). The group receiving 2.0×105 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).
(H) Sections of rat hearts stained with Masson's trichrome at 15 weeks after LAD ligation and injection of 2.0×106 G-CSF mobilized human cells containing 103 (left) or 2.0×105 (right) CD117bright endothelial progenitor cells. Hearts of rats receiving 103 endothelial progenitor cells had greater loss of anterior wall mass, collagen deposition (lighter gray), and septal hypertrophy compared with hearts of rats receiving 2.0×105 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.
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.
As used herein, “VEGF” is defined as vascular endothelial growth factor. “VEGF-R” is defined as vascular endothelial growth factor receptor. “FGF” is defined as fibroblast growth factor. “IGF” is defined as Insulin-like growth factor. “SCF” is defined as stem cell factor. “G-CSF” is defined as granulocyte colony stimulating factor. “M-CSF” is defined as macrophage colony stimulating factor. “GM-CSF” is defined as granulocyte-macrophage colony stimulating factor. “MCP” is defined as monocyte chemoattractant protein.
As used herein “angioblasts” is synonymous with the term “endothelial progenitor cells”.
As used herein, “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.
As used herein, “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. For example, 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.
In one embodiment the agent is human endothelial progenitor cells. In one embodiment 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 1.5×105 endothelial progenitor cells per kg body mass to about 3×105 per kg body mass. In another embodiment the effective amount is about 3×105 per kg body mass to about 4.5×105 endothelial progenitor cells per kg body mass. In another embodiment the effective amount is about 4.5×105 per kg body mass to about 5.5×105 endothelial progenitor cells per kg body mass. In another embodiment the effective amount is about 5.5×105 per kg body mass to about 7×105 endothelial progenitor cells per kg body mass. In another embodiment the effective amount is about 7×105 per kg body mass to about 1×106 endothelial progenitor cells per kg body mass. In another embodiment the effective amount is about 1×106 per kg body mass to about 1.5×106endothelial progenitor cells per kg body mass. In one embodiment the effective amount of human endothelial progenitor cells is between about 1.5×106 and 4.5×106 endothelial progenitor cells per kg of the subject's body mass and In a preferred embodiment the effective amount is about 5×105 endothelial progenitor cells per kg of the subject's body mass.
In one embodiment the endothelial progenitor cells are allogeneic with respect to the subject. In differing embodiments the subject is an adult or an embryo or a fetus. In another embodiment the endothelial progenitor cells are derived from cloned autologous embryonic stem cells.
In one embodiment 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. Alternatively, local control of O2 can have the same effect (see 107). 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.
In another embodiment 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. by using the entire Neh2 domain of nrf2, amino acids 1-73, or only the hydrophilic region, amino acids 33-73 (see 109) will increase free cytoplasmic Nrf-2. In another embodiment the agent induces dissociation of a Nrf2 protein from a Keap-1. In another embodiment the agent inhibits association of a Nrf2 protein with a Keap-1. In another embodiment the agent inhibits association of a thiol reductase thioredoxin with a VDUP-1 protein. In another embodiment the agent inhibits c-Abl tyrosine kinase activation. In a further embodiment the agent is STI-571.
In one embodiment the agent is a CXC chemokine. In further embodiments the agent is Stromal-Derived Factor-1, Il-8 or Gro-Alpha. In one embodiment the amount of CXC chemokine administered is between 0.2 and 5 μg/ml at a max volume of 10 ml for a 70 kg human subject. In a preferred embodiment the amount is about 1 μg/ml. In one embodiment the agent is an inhibitor of plasminogen activator inhibitor-1. In another embodiment the agent is an antibody directed against an epitope of CXCR4. In one embodiment the amount of antibody directed against an epitope of CXCR4 is between 25 and 75 μg/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 μg/ml.
In a further embodiment 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. In further embodiments the endothelial progenitor cells express CD117, CD34, AC133 or a high level of intracellular GATA-2 activity. In one embodiment 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.
In one embodiment the second agent is an antisense oligonucleotide which specifically inhibits translation of Vitamin D3 Up-Regulated Protein-1 (VDUP-1) mRNA.
Therapeutically useful targeted inhibition of VDUP-1 protein (SEQ ID NO:2) expression can be achieved through the use of antisense oligonucleotides. 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. Following cleavage from controlled pore glass support, 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. Also, 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.
Representative U.S. patents that teach the preparation of the above phosphorus-containing linkages include, but are not limited to, U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799; 5,587,361; 5,194,599; 5,565,555; 5,527,899; 5,721,218; 5,672,697 and 5,625,050, certain of which are commonly owned with this application, and each of which is herein incorporated by reference.
In accordance with this invention, persons of ordinary skill in the art will understand that 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. Thus, 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. Similarly, 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. As is known in the art, the translation initiation codon is typically 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. Thus, the term “translation initiation codon” can encompass many codon sequences, even though the initiator amino acid in each instance is typically methionine in eukaryotes. It is also known in the art that eukaryotic genes may have two or more alternative translation initiation codons, any one of which may be preferentially utilized for translation initiation in a particular cell type or tissue, or under a particular set of conditions. In the context of the invention, “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. It is also known in the art that 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. Similarly, the term “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. The open reading frame or “coding region,” which is known in the art to refer to the region between the translation initiation codon and the translation termination codon, is also a region which may be targeted effectively. 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. 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.
Once the target site or sites have been identified, 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. Similarly, catalytic nucleic acids are synthesized once cleavage target sites on the VDUP-1 mRNA molecule.
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. Human pharmacokinetics of certain antisense oligonucleotides have been studied. See (105) incorporated by reference in its entirety. It is within the scale of a person's skill in the art to determine optimum dosages and treatment schedules for such treatment regimens.
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.
In another embodiment the second agent is a pro-angiogenic agent. In further embodiments the pro-angiogenic agent is vascular endothelial growth factor, fibroblast growth factor or angiopoietin. In another embodiment the second agent induces expression of a pro-angiogenic factor. In a further embodiment the second agent is Hypoxia Inducible Factor-1.
In another embodiment the second agent is a catalytic nucleic acid which specifically inhibits translation of Vitamin D3 Up-Regulated Protein-1 mRNA. In a further embodiment the catalytic nucleic acid comprises deoxyribonucleotides. In another embodiment 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. Examples of catalytic ribonucleic acids include hairpin and hammerhead ribozymes. In preferred embodiments of this invention, 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).
To target the VDUP-1 mRNA (SEQ ID NO:1), 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. Further examples of catalytic DNA structure are 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. For example, 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. In addition, isoteric purine 2′deoxy-furanoside analogs, 2′-deoxynebularine or 2′deoxyxanthosine, or other purine or pyrimidine analogs may also be used. By carefully selecting the bases and base analogs, one may fine tune the hybridization properties of the oligonucleotide. For example, inosine may be used to reduce hybridization specificity, while 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. For example, 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, CF3, OCF3, OCN, O-alkyl, S-alkyl, SOMe, SO2Me, ONO2, NH-alkyl, OCH2CH═CH2, OCH2CCH, OCCHO, allyl, O-allyl, NO2, N3, and NH2. For example, the 2′ position of the sugar may be modified to contain one of the following groups: H, OH, OCN, O-alkyl, F, CN, CF3, allyl, O-allyl, OCF3, S-alkyl, SOMe, SO2Me, ONO2, NO2, N3, NH2, NH-alkyl, or OCH═CH2, OCCH, wherein the alkyl may be straight, branched, saturated, or unsaturated. In addition, 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.
Representative U.S. patents that teach the preparation of such modified bases/nucleosides/nucleotides include, but are not limited to, U.S. Pat. Nos. 6,248,878, and 6,251,666 which are herein incorporated by reference.
The sugar may be modified to contain one or more linkers for attachment to other chemicals such as fluorescent labels. In an embodiment, the sugar is linked to one or more aminoalkyloxy linkers. In another embodiment, 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. 2-methoxy-6-chloro-9-aminoacridine); 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. In one example, 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) or antisense oligonucleotide may comprise modified bonds. For example 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, —CH2, 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. Alternatively, 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. Further, the phosphorothioate oligonucleotide may have one or more of its sugars substituted or modified to form an α-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. The term “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. In one embodiment, the effective amount contains between about 10 ng and about 100 μg of the instant nucleic acid molecules per kg body mass. In another embodiment, the effective amount contains between about 100 ng and about 10 μg of the nucleic acid molecules per kg body mass. In a further embodiment, the effective amount contains between about 1 μg and about 5 μg, and in a further embodiment about 2 μg, of the nucleic acid molecules per kg body mass.
In another embodiment the second agent is cardiomyocyte progenitor cells. In another embodiment the second agent is skeletal muscle progenitor cells. Either of theses cell types can be derived from embryonic, fetal or adult subjects.
In one embodiment the second agent promotes trafficking of the endothelial progenitor cells to the subject's heart.
In a further embodiment the second agent that promotes trafficking is an antibody directed against an epitope of CXCR4.
In another further embodiment the second agent that promotes trafficking is a CC chemokine. In further embodiments the CC chemokine is RANTES, EOTAXIN, monocyte chemoattractant protein-1 (MCP-1), MCP-2, MCP-3, or MCP.
In another further embodiment the second agent that promotes trafficking is a CXC chemokine. In further embodiments 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:
(a) quantitating the amount of a mRNA encoding peroxiredoxin in the cardiomyocyte;
(b) quantitating the amount of a mRNA encoding Vitamin D3 Up-Regulated Protein-1 in the cardiomyocyte; and
(c) determining the ratio of the amount of mRNA encoding peroxiredoxin: amount of mRNA encoding Vitamin D3 Up-Regulated Protein-1, wherein a low ratio indicates a high susceptibility of the cardiomyocyte to apoptosis and a high ratio indicates a low susceptibility of the cardiomyocyte to apoptosis in the subject.
This invention also provides a method of determining the susceptibility of a cardiomyocyte in a subject to apoptosis comprising:
(a) quantitating the expression of a peroxiredoxin protein in the cardiomyocyte;
(b) quantitating the expression of Vitamin D3 Up-Regulated Protein-1 in the cardiomyocyte; and
(c) determining the ratio of the peroxiredoxin protein expression: Vitamin D3 Up-Regulated Protein-1 expression, wherein a low ratio indicates a high susceptibility of the cardiomyocyte to apoptosis and a high ratio indicates a low susceptibility of the cardiomyocyte to apoptosis in the subject.
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.
In one embodiment the subject of any of the above methods is a mammal and in a preferred further embodiment the mammal is a human being. In one embodiment the subject has a cardiovascular disease. In further embodiments the subject has congestive heart failure, has suffered a myocardial infarct, has suffered myocardial ischemia, has angina, or has a cardiomyopathy.
- EXPERIMENTAL RESULTS
This invention will be better understood by reference to the Experimental Details which follow, but those skilled in the art will readily appreciate that the specific experiments detailed are only illustrative of the invention as described more fully in the claims which follow thereafter.
CXC Chemokines Regulate Endothelial Progenitor Cell Migration to the Heart
We investigated the in vivo role of CXC receptor-ligand interactions in mediating chemotaxis of human Endothelial progenitor cell to ischemic tissue and subsequent induction of vasculogenesis, using myocardial infarction in the LAD-ligated nude rat model. As shown in FIG. 1a, DiI-labelled human CD34+ cells obtained by G-CSF mobilization (>98% CD34 purity, containing 6-12% CD117bright endothelial progenitor cells) were selectively detected in infarcted myocardium after intravenous injection, but not in myocardium from sham-operated rats. Co-administration of blocking mAbs against rat Cinc (the rat homologue of human IL-8 and Gro-alpha), or against the human surface receptors for these pro-angiogenic chemokines, CXCR1 or CXCR2, reduced myocardial trafficking of human bone marrow-derived CD34+ cells at 48 hours by 40-60% relative to control antibodies (p<0.01), FIG. 1b. By two weeks, rats receiving human CD34+ cells demonstrated significantly increased infarct bed microvascularity in comparison to rats receiving saline, FIG. 1c, and this was reduced by 50% when anti-CXCR1/2 mAbs were co-administered. Since we have previously shown that the vasculogenic properties of CD34+ cells are abolished after depletion of the minor CD117bright angioblast fraction (7), these results indicate that by regulating angioblast migration to ischemic tisssues CXC chemokines influence the development of vasculogenesis at these sites. In contrast, although direct intracardiac injection of IL-8 or SDF-1 at 1.0 μg/ml into non-infarcted hearts resulted in 2.3 and 2.5-fold increases in myocardial infiltration by human CD34+ cells at 48 hours (both p<0.01), FIG. 1d, no vasculogenesis was observed at two weeks under these conditions. Together, these results indicate that following chemokine-induced migration to the infarct zone, differentiation of endothelial progenitor cell to mature endothelial cells and induction of vasculogenesis require additional factors, as yet undefined, which are produced under ischemic conditions.
Inhibiting CXCR4/SDF-1 Interactions Redirects Endothelial Progenitor Cells to the Heart
Although 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. As shown in FIG. 2a, at 2-14 days after intravenous injection of 2×106 human CD34+ cells bone marrow from LAD-ligated rats contained 5-8 fold higher levels of human CD117bright endothelial progenitor cells compared with bone marrow from normal rats, p<0.001. This was presumably due to the proliferative effects of factors in ischemic serum since we have previously shown that culture for 2 days with ischemic serum increases proliferation of CD34+CD117bright human endothelial progenitor cells by 4-5 fold (7). Because the migration of actively cycling CD34+ cells to bone marrow is promoted by SDF-1 produced constitutively by marrow stromal cells (31), we investigated whether the distribution of human CD34+CD117bright endothelial progenitor cells to ischemic rat bone marrow involved SDF-1/CXCR4 interactions. As shown in FIG. 2b, 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). Moreover, 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. 2c.
Capillary Lumen Size is Dependent on Absolute Angioblast Numbers
We next examined the relationship between angioblast number, myocardial neovascularization, and protection against myocyte apoptosis. Two days following LAD ligation animals were intravenously injected with G-CSF mobilized CD34+ human cells reconstituted with varying proportions of CD117bright endothelial progenitor cells (103, 105, 105 plus anti-CXCR4 mAb, 2×105, and 2×105 plus anti-CXCR4 mAb). Similar numbers of DiI-labelled human cells were detected in the infarct zone 48 hours after injecting each cellular population, data not shown. 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). Notably, 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. 3a-c, both the group receiving 2×105 endothelial progenitor cells and the one receiving 105 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 2×105 endothelial progenitor cells additionally demonstrated 3.3-fold higher numbers of large-lumen capillaries compared with the groups receiving 103 or 105 endothelial progenitor cells (p<0.01), and 2-fold higher numbers of large-lumen capillaries compared with the group receiving 105 endothelial progenitor cells plus anti-CXCR4 mAb (p<0.01). As shown in FIG. 3d, co-administration of anti-CXCR4 mAb together with the highest concentration of endothelial progenitor cells, 2×105, 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 2×105 endothelial progenitor cells were injected intravenously after direct intracardiac delivery of 1.0 μg/ml SDF-1 into infarcted hearts (p<0.01). Since no similar increase in peri-infarct capillary numbers was seen after intracardiac delivery of IL-8, we interpret these results to indicate that the endogenous IL-8 concentrations in ischemic rat hearts were sufficient to saturate angioblast CXCR1/2 receptors in vivo, whereas intracardiac injection of SDF-1 resulted in a shift in the balance of SDF-1 expression between bone marrow and heart, and consequently resulted in redirected angioblast trafficking to the ischemic heart.
As shown in FIG. 3e, the number of apoptotic cardiomyocytes at the infarct rim was significantly reduced in both rats receiving 105 endothelial progenitor cells plus anti-CXCR4 mAb and those receiving 2×105 endothelial progenitor cells compared with the groups receiving either 103 or 105 endothelial progenitor cells alone (both p<0.001). Moreover, 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. 3f (both p<0.001). Together, these data indicate that post-infarct cardioprotection against myocyte apoptosis is dependent on myocardial neovascularization induced by a critical number of intravenously injected endothelial progenitor cells. This threshold can apparently be lowered by strategies that prevent endothelial progenitor cell redistribution to the bone marrow, such as interrupting CXCR4/SDF-1 interactions, or enhanced SDF-1 expression in the ischemic myocardium.
Capillary Lumen Size as Determinant of Improvement in Cardiac Function
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. 4a and b. No improvement in these parameters was observed in the groups receiving 103 or 105 endothelial progenitor cells in comparison to rats receiving saline alone. In contrast, rats receiving 105 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 2×105 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. These results were very surprising to us since both groups of animals had demonstrated the same degree of neovascularization involving medium-sized capillaries at two weeks together with similar levels of protection against early apoptosis of cardiomyocytes. This suggested that the additional functional long-term improvement in rats receiving 2×105 human endothelial progenitor cells was related to the early development of large-sized capillaries and was mediated through a different mechanism than protection against myocyte apoptosis.
Large Capillaries Induce Sustained Regeneration of Endogenous Myocytes
Although myocyte hypertrophy and increase in nuclear ploidy have generally been considered the primary mammalian cardiac responses to ischemia, damage, and overload (1,2) recent observations have suggested that human cardiomyocytes have the capacity to proliferate and regenerate in response to injury (18,19). Therefore, we investigated whether the additive improvement in cardiac function observed after injection of 2×105 human endothelial progenitor cells involved induction of cardiomyocyte proliferation and/or regeneration. At two weeks after LAD ligation rats receiving 2×105 human endothelial progenitor cells demonstrated numerous “fingers” of cardiomyocytes of rat origin, as determined by expression of rat MHC class I molecules, extending from the peri-infarct region into the infarct zone. Similar extensions were seen less frequently in animals receiving 103 and 105 endothelial progenitor cells, and very rarely in those receiving saline. As shown in FIG. 4c, the islands of cardiomyocytes at the peri-infarct rim in animals receiving 2×105 human endothelial progenitor cells contained a high frequency of rat myocytes with DNA activity, as determined by dual staining with mAbs reactive against cardiomyocyte-specific troponin I and rat Ki-67. In contrast, in animals receiving saline there was a high frequency of cells with fibroblast morphology and reactivity with rat Ki-67, but not troponin I, within the infarct zone. The number of cardiomyocytes progressing through cell cycle at the peri-infarct region of rats receiving 2×105 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. 4d, animals receiving 2×105 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). When 2×105 human endothelial progenitor cells were intravenously injected after direct intracardiac delivery of 1.0 μg/ml SDF-1 into infarcted hearts, the number of cell-cycling cardiomyocytes at the peri-infarct rim was increased by a further 1.9-fold compared with intravenous injection of 2×105 human endothelial progenitor cells alone (FIG. 4 e, p<0.01). Thus, intracardiac injection of SDF-1 in combination with intravenous injection of 2×105 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 2×105 endothelial progenitor cells alone (FIG. 4f, 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.
Quantitation of the ratio of fibrous tissue to myocytes at 15 weeks demonstrated significantly reduced proportions of scar/normal left ventricular myocardium in both the group receiving 2×105 endothelial progenitor cells and the one receiving 105 endothelial progenitor cells plus anti-CXCR4 mAb, respectively 13% and 21% compared with 37-46% for each of the other groups (p<0.01), FIG. 4g. Since both groups had nearly identical levels of protection against early cardiomyocyte apoptosis, we infer that the additional 38% reduction in scar/myocyte ratio seen in the group injected with 2×105 endothelial progenitor cells actually reflects proliferation/regeneration of endogenous rat cardiomyocytes induced by nutrient supply from large-vessel neovascularization. This presumably accounts for the increase in functional improvement seen in this group. We conclude that the ratio of scar size to left ventricular muscle mass reflects, in part, positive effects imparted by the ability of the residual myocardium to proliferate and regenerate, in addition to negative effects of the initial infarct size and positive effects of anti-apoptotic, cardioprotective mechanisms. 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. 4h where, in contrast to saline controls, injection with 2×105 endothelial progenitor cells resulted in almost complete salvage of the anterior myocardium, normal septal size and minimal collagen deposition.
PAI-1-Inhibiting Catalytic Nucleic Acid Augments Human Angioblast-Dependent Cardiomyocyte Regeneration
We investigated whether possible neovascularization induced by a catalytic nucleic acid (designated E2) capable of inhibiting expression of PAI-1 was associated with cardiomyocyte regeneration. Injection of E2 alone did not induce cardiomyocyte regeneration despite the increase in neovascularization. Combining E2 injection with intravenously delivered human endothelial progenitor cells strikingly increased the degree of cardiomyocyte regeneration, to levels 7.5-fold higher than in saline controls (p<0.01). A scrambled DNA control enzyme (E0) had no such effect. Moreover, whereas E2 alone did not improve myocardial function, as determined by recovery in left ventricular ejection fraction at two weeks combining E2 with human endothelial progenitor cells resulted in almost doubling of the positive effect of endothelial progenitor cells alone on cardiac functional recovery. These results emphasize the importance of cardiomyocyte regeneration as the primary mechanism by which cardiac function is improved after infarction. Since combining E2 with human endothelial progenitor cells resulted in 62% greater numbers of large capillaries at the peri-infarct rim than use of either approach alone, these results indicate that angioblast-induced cardiomyocyte regeneration and improvement in cardiac function can be optimized by use of synergistic approaches, such as strategies that inhibit PAI-1 expression, which augment neovascularization either directly or through angioblast-dependent processes.
Gene Effects on Myocyte Proliferation
We hypothesized that myocardial neovascularization induces the signals required to elicit myocyte proliferation, and therapeutic intervention mimicking this could have striking implications for repair and regeneration of hearts damaged by episodes of ischemia or other insults.
To begin approaching this complex problem, we have employed the technique of cDNA Subtractive Hybridization. This technique enables comparison of the pattern of gene expression between two different sets of conditions. Our initial approach was to compare which genes are differentially expressed between hearts from normal rats and rats who have undergone left anterior descending (LAD) coronary artery ligation 48 hours earlier. We hypothesized that whatever the altered pattern of gene over- or underexpression after 48 hours of ischemia, neovascularization would result in a reversal in the pattern towards that seen in the non-ischemic rat heart.
Using cdNA subtractive hybridization we observed a striking reciprocal change in expression of a group of genes whose function is linked through their regulation of cellular apoptosis and cell cycle progression following oxidative stress and other inducers of DNA damage. Whereas expression of certain antioxidant genes such as superoxide dismutase was upregulated in ischemic tissue, the antioxidant stress responsive genes induced by hemin, notably heme binding protein 23 (HBP23) and glutathione-S-transferase, were downregulated. Moreover, a recently-identified protein, Vitamin D3 Up-Regulated Proteinl (VDUP1), whose mRNA expression is induced by hydrogen peroxide (H2O2) following oxidative stress and whose function counterbalances that of HBP23, was upregulated in ischemic hearts. These findings are particularly striking when considered in the context of previous observations that the presence of deficits in antioxidants and increased oxidative stress accompanying myocardial infarction appear to be directly implicated in the pathogenesis of post-infarct heart failure (74,75).
As shown in FIG. 5, using RT-PCR, the reciprocal changes in mRNA expression of HBP23 and VDUP1 in ischemic vs normal rat hearts were confirmed. Moreover, mRNA expression of these two genes returned to normal in rat tissue two weeks after intravenous injection of human adult bone marrow-derived progenitors and infarct zone neovascularization. In contrast, in LAD-ligated rat hearts receiving saline, no change in mRNA expression of these genes was observed at two weeks in comparison to the pattern observed at 48 hours. To comprehend the relationship between the effects of neovascularization on these altered expression patterns of HBP23 and VDUP1 following ischemia, and the observed protection against cardiomyocyte apoptosis together with induction of cardiomyocyte proliferation/regeneration, it is important to understand in detail the molecular effects of the products encoded by these genes on cellular apoptosis and cell cycle progression.
When cells proliferate, the mitotic cycle progression is tightly regulated by an intricate network of positive and negative signals. Progress from one phase of the cell cycle to the next is controlled by the transduction of mitogenic signals to cyclically expressed proteins known as cyclins and subsequent activation or inactivation of several members of a conserved family of serine/threonine protein kinases known as the cyclin-dependent kinases (cdks) (67). Growth arrest observed with such diverse processes as DNA damage, terminal differentiation, and replicative senescence is due to negative regulation of cell cycle progression by two functionally distinct families of Cdk inhibitors, the Ink4 and Cip/Kip families (64). 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). 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. For example, 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). Moreover, the reduced form of TRX, but not the oxidized form, 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 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. Although the precise extracellular signalling pathways have not been elucidated, nuclear translocation of Nrf2 and subsequent ARE activation appear to be dependent on pathways activated by phosphatidylinositol 3-kinase (PI3 kinase) (93). In addition, hemin is a potent inducer of Nrf2 dissociation from Keap1, resulting in TRX gene transcription through the ARE (94).
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).
In addition to directly interacting with TRX, the Prx gene products (PAG, HBP23, MSP23, NKEF, etc) 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). 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 H2O2 and that this results in mitochondrial localization of c-Abl, c-Abl dependent cytochrome c release, and cellular apoptosis following oxidative stress (102,103). 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).
Our finding that 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. Thus, 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.
Reversing the reduced expression of the Prxs following myocardial ischemia would increase Prxs in the heart in order to protect the ischemic myocardium against apoptosis through both c-Abl inhibition and reduction of oxidised TRX, and to enable cardiomyocyte proliferation/regeneration by inhibiting the effects of c-Abl on cell cycle progression from G1 to S phase.
Increasing 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.
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.
Small molecules which specifically inhibit binding of Nrf2 to Keap1 would be expected to have similar protective effects against cardiomyocyte apoptosis and to induce myocardial proliferation/regeneration after ischemia. Similarly, small molecules that specifically inhibit binding of TRX to VDUP1, would be expected to have similar protective effects against cardiomyocyte apoptosis after ischemia.
- Methods and Materials
Use of small molecules to specifically inhibit c-Abl tyrosine kinase activation after myocardial ischemia would be expected to have similar protective effects against cardiomyocyte apoptosis and to induce myocardial proliferation/regeneration after ischemia. A specific example of a small molecule to inhibit c-Abl tyrosine kinase activation is STI-571. Use of this or related molecules after myocardial infarction would protect against cardiomyocyte apoptosis and induce myocardial proliferation/regeneration.
Purification and Characterization of Cytokine-Mobilized Human CD34+ Cells:
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). Purified 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/Cytoperm™ kit, incubating for 30 minutes on ice with 10 μl of fluorochrome-conjugated mAbs against both CD117 and CD34 surface antigens (Becton Dickinson, CA). After resuspension in 250 μl of Cytofix/Cytoperm™ solution for 20 minutes at 4 degrees C., cells were incubated with a fluorochrome-labeled mAb against GATA-2 (Santa Cruz Biotech, CA) or IgG control for 30 minutes at 4 degrees C., and analyzed by three-parameter flow cytometry.
Chemotaxis of Human Bone-Marrow Derived Endothelial Progenitors:
Highly-purified CD34+CD117bright cells (>98% purity) 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 μg/ml. The membranes were fixed with methanol and stained with Leukostat™ (Fischer Scientific, Ill.). Chemotaxis was calculated by counting migrating cells in 10 high-power fields.
Animals, Surgical Procedures, Injection of Human Cells, and Quantitation of Cellular Migration into Tissues:
Rowett (rnu/rnu) athymic nude rats (Harlan Sprague Dawley, Indianapolis, Ind.) were used in studies approved by the “Columbia University Institute for Animal Care and Use Committee”. After anesthesia, a left thoracotomy was performed, the pericardium was opened, and the left anterior descending (LAD) coronary artery was ligated. Sham-operated rats had a similar surgical procedure without having a suture placed around the coronary artery. For studies on cellular migration, 2.0×106 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 μg/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. Prior to injection, 2.0×106 human cells were incubated with 2.5 μg/mL of the fluorescent carbocyanine DiI dye (Molecular Probes) for 5 minutes at 37° C. and 15 minutes at 4° C. After washing in PBS, DiI-labeled human cells were resuspended in saline and injected intravenously. 2.0×106 CD34+ human cells were also injected into the tail vein of sham-operated or LAD-ligated rats receiving three intramyocardial injections of 1.0 μg/ml recombinant human IL-8, SDF-1, SCF or saline. Each group consisted of 6-10 rats. 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. For studies on neoangiogenesis and effects on myocardial viability and function, 2.0×106 DiI-labelled human CD34+ cells obtained from a single donor after G-CSF mobilization were reconstituted with 103, 105, or 2.0×105 immunopurified CD34+CD117bright cells, and injected into the rat tail vein 48 hours after LAD ligation, in the presence or absence of a mAb with known inhibitory activity against CXCR4. Each group consisted of 6-10 rats. Histologic and functional studies were performed at 2 and 15 weeks.
Measurement of Rat CXC Chemokine mRNA and Protein Expression:
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. After priming with oligo (dT) 15-mer and random hexamers, and reverse transcribed with Moloney murine lymphotrophic virus reverse transcriptase (Invitrogen, Carlsbad, Calif., U.S.A.), 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). 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)/cttggggacacccttcagcatcttttgg SEQ ID NO:7), and ctctacccacggcaagttcaa SEQ ID NO:8)/gggatgaccttgcccacagc SEQ ID NO:9), respectively. The labelled samples were loaded into 2% agarose gels, separated by electrophoresis, and exposed for radiography for 6 h at −70° C. 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. 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.
Histology and Measurement of Infarct Size:
Following excision at 2 and 15 weeks, left ventricles from each experimental animal were sliced at 10-15 transverse sections from apex to base. Representative sections were fixed in formalin and stained for routine histology (H&E) to determine cellularity of the myocardium, expressed as cell number per high power field (HPF) (600×). A Masson trichrome stain was performed, which labels collagen blue and myocardium red, to evaluate collagen content on a semiquantitative scale (0-3+), with 1+ light blue, 2+ light blue and patches of dark blue, and 3+ dark blue staining. This enabled measurement of the size of the myocardial scar using a digital image analyzer. The lengths of the infarcted surfaces, involving both epicardial and endocardial regions, were measured with a planimeter digital image analyzer and expressed as a percentage of the total ventricular circumference. Final infarct size was calculated as the average of all slices from each heart. All studies were performed by a blinded pathologist. Infarct size was expressed as percent of total left ventricular area. Final infarct size was calculated as the average of all slices from each heart.
Ouantitation of Capillary Density:
In order to quantitate capillary density and species origin of the capillaries, additional sections were stained freshly with mAbs directed against rat or human CD31 (Serotec, UK, and Research Diagnostics, NJ, respectively), factor VIII (Dako, CA), and rat or human MHC class I (Accurate Chemicals, CT). Arterioles were differentiated from large capillaries by the presence of a smooth muscle layer, identified by staining sections with a monoclonal antibody against muscle-specific desmin (Dako, CA). 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).
Quantitation of Cardiomyocyte Proliferation:
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. Following washes, sections were incubated with a species-specific secondary antibody conjugated with alkaline phosphatase at 1:200 dilution (Vector Laboratories Burlingame, Calif.) for 30 minutes and positively-staining nuclei were visualized as blue with a BCIP/NBT substrate kit (Dako, CA). Sections were then incubated overnight at 4 degrees C. with a monoclonal antibody against cardiomyocyte-specific troponin I (Accurate Chemicals, CT) and positively-staining cells were visualized as brown through the Avidin/Biotin system described above. Cardiomyocytes progressing through cell cycle in the infarct zone, peri-infarct region, and area distal to the infarct were calculated as the proportion of troponin I-positive cells per high power field co-expressing Ki-67.
Measurement of Myocyte Apoptosis by DNA End-Labeling of Paraffin Tissue Sections:
For in situ detection of apoptosis at the single cell level we used the TUNEL method of DNA end-labeling mediated by dexynucleotidyl transferase (TdT) (Boehringer Mannheim, Mannheim, Germany). Rat myocardial tissue sections were obtained from LAD-ligated rats at two weeks after injection of either saline or CD34+ human cells, and from healthy rats as negative controls. Briefly, tissues were deparaffinized with xylene and rehydrated with graded dilutions of ethanol and two washes in phosphate-buffered saline (PBS). The tissue sections were then digested with Proteinase K (10 μg/ml in Tris/HCL) for 30 minutes at 37° C. The slides were then washed 3 times in PBS and incubated with 50 μl of the TUNEL reaction mixture (TdT and fluorescein-labeled dUTP) and incubated in a humid atmosphere for 60 minutes at 37° C. For negative controls TdT was eliminated from the reaction mixture. Following 3 washes in PBS, the sections were then incubated for 30 minutes with an antibody specific for fluorescein-conjugated alkaline phosphatase (AP) (Boehringer Mannheim, Mannheim, Germany). 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. Following 3 washes sections were then treated with an anti-rabbit IgG, followed by a biotin conjugated secondary antibody for 30 minutes (Sigma, Saint Louis, Mo.). An avidin-biotin complex (Vector Laboratories, Burlingame, Calif.) was then added for an additional 30 minutes and the myocytes were visualized brown following 5 minutes exposure in DAB solution mixture (Sigma, Saint Louis, Mo.). 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 mm2 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 mm2 at each site examined.
Analysis of Myocardial Function:
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.
CDNA Subtractive Hybridization:
Briefly, messenger RNA was isolated from each heart, and 1 μg 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. After second-strand synthesis, 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. In the forward subtraction, which determines the genes that are overexpressed in the ischemic sample, the ischemic tissue is the “tester” and the normal tissue is the “driver.” In the reverse subtraction, the “tester” and the “driver” are switched to determine the genes that are down-regulated in the ischemic sample.
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aaactaaccc ctctttttct ccaaaggagt gcttgtggag atcggatctt ttctccagca 60
attgggggaa agaaggcttt ttctctgact tcgcttagtg taaccagcgg cgtatatttt 120
ttaggcgcct tttcgaaaac ctagtagtta atattcattt gtttaaatct tattttattt 180
ttaagctcaa actgcttaag aataccttaa ttccttaaag tgaaataatt ttttgcaaag 240
gggtttcctc gatttggagc tttttttttc ttccaccgtc atttctaact cttaaaacca 300
actcagttcc atc atg gtg atg ttc aag aag atc aag tct ttt gag gtg 349
Met Val Met Phe Lys Lys Ile Lys Ser Phe Glu Val
1 5 10
gtc ttt aac gac cct gaa aag gtg tac ggc agt ggc gag aag gtg gct 397
Val Phe Asn Asp Pro Glu Lys Val Tyr Gly Ser Gly Glu Lys Val Ala
15 20 25
ggc cgg gtg ata gtg gag gtg tgt gaa gtt act cgt gtc aaa gcc gtt 445
Gly Arg Val Ile Val Glu Val Cys Glu Val Thr Arg Val Lys Ala Val
30 35 40
agg atc ctg gct tgc gga gtg gct aaa gtg ctt tgg atg cag gga tcc 493
Arg Ile Leu Ala Cys Gly Val Ala Lys Val Leu Trp Met Gln Gly Ser
45 50 55 60
cag cag tgc aaa cag act tcg gag tac ctg cgc tat gaa gac acg ctt 541
Gln Gln Cys Lys Gln Thr Ser Glu Tyr Leu Arg Tyr Glu Asp Thr Leu
65 70 75
ctt ctg gaa gac cag cca aca ggt gag aat gag atg gtg atc atg aga 589
Leu Leu Glu Asp Gln Pro Thr Gly Glu Asn Glu Met Val Ile Met Arg
80 85 90
cct gga aac aaa tat gag tac aag ttc ggc ttt gag ctt cct cag ggg 637
Pro Gly Asn Lys Tyr Glu Tyr Lys Phe Gly Phe Glu Leu Pro Gln Gly
95 100 105
cct ctg gga aca tcc ttc aaa gga aaa tat ggg tgt gta gac tac tgg 685
Pro Leu Gly Thr Ser Phe Lys Gly Lys Tyr Gly Cys Val Asp Tyr Trp
110 115 120
gtg aag gct ttt ctt gac cgc ccg agc cag cca act caa gag aca aag 733
Val Lys Ala Phe Leu Asp Arg Pro Ser Gln Pro Thr Gln Glu Thr Lys
125 130 135 140
aaa aac ttt gaa gta gtg gat ctg gtg gat gtc aat acc cct gat tta 781
Lys Asn Phe Glu Val Val Asp Leu Val Asp Val Asn Thr Pro Asp Leu
145 150 155
atg gca cct gtg tct gct aaa aaa gaa aag aaa gtt tcc tgc atg ttc 829
Met Ala Pro Val Ser Ala Lys Lys Glu Lys Lys Val Ser Cys Met Phe
160 165 170
att cct gat ggg cgg gtg tct gtc tct gct cga att gac aga aaa gga 877
Ile Pro Asp Gly Arg Val Ser Val Ser Ala Arg Ile Asp Arg Lys Gly
175 180 185
ttc tgt gaa ggt gat gag att tcc atc cat gct gac ttt gag aat aca 925
Phe Cys Glu Gly Asp Glu Ile Ser Ile His Ala Asp Phe Glu Asn Thr
190 195 200
tgt tcc cga att gtg gtc ccc aaa gct gcc att gtg gcc cgc cac act 973
Cys Ser Arg Ile Val Val Pro Lys Ala Ala Ile Val Ala Arg His Thr
205 210 215 220
tac ctt gcc aat ggc cag acc aag gtg ctg act cag aag ttg tca tca 1021
Tyr Leu Ala Asn Gly Gln Thr Lys Val Leu Thr Gln Lys Leu Ser Ser
225 230 235
gtc aga ggc aat cat att atc tca ggg aca tgc gca tca tgg cgt ggc 1069
Val Arg Gly Asn His Ile Ile Ser Gly Thr Cys Ala Ser Trp Arg Gly
240 245 250
aag agc ctt cgg gtt cag aag atc agg cct tct atc ctg ggc tgc aac 1117
Lys Ser Leu Arg Val Gln Lys Ile Arg Pro Ser Ile Leu Gly Cys Asn
255 260 265
atc ctt cga gtt gaa tat tcc tta ctg atc tat gtt agc gtt cct gga 1165
Ile Leu Arg Val Glu Tyr Ser Leu Leu Ile Tyr Val Ser Val Pro Gly
270 275 280
tcc aag aag gtc atc ctt gac ctg ccc ctg gta att ggc agc aga tca 1213
Ser Lys Lys Val Ile Leu Asp Leu Pro Leu Val Ile Gly Ser Arg Ser
285 290 295 300
ggt cta agc agc aga aca tcc agc atg gcc agc cga acc agc tct gag 1261
Gly Leu Ser Ser Arg Thr Ser Ser Met Ala Ser Arg Thr Ser Ser Glu
305 310 315
atg agt tgg gta gat ctg aac atc cct gat acc cca gaa gct cct ccc 1309
Met Ser Trp Val Asp Leu Asn Ile Pro Asp Thr Pro Glu Ala Pro Pro
320 325 330
tgc tat atg gat gtc att cct gaa gat cac cga ttg gag agc cca acc 1357
Cys Tyr Met Asp Val Ile Pro Glu Asp His Arg Leu Glu Ser Pro Thr
335 340 345
act cct ctg cta gat gac atg gat ggc tct caa gac agc cct atc ttt 1405
Thr Pro Leu Leu Asp Asp Met Asp Gly Ser Gln Asp Ser Pro Ile Phe
350 355 360
atg tat gcc cct gag ttc aag ttc atg cca cca ccg act tat act gag 1453
Met Tyr Ala Pro Glu Phe Lys Phe Met Pro Pro Pro Thr Tyr Thr Glu
365 370 375 380
gtg gat ccc tgc atc ctc aac aac aat gtg cag tga gcatgtggaa 1499
Val Asp Pro Cys Ile Leu Asn Asn Asn Val Gln
gaaaagaagc agctttacct acttgtttct ttttgtctct cttcctggac actcactttt 1559
tcagagactc aacagtctct gcaatggagt gtgggtccac cttagcctct gacttcctaa 1619
tgtaggaggt ggtcagcagg caatctcctg ggccttaaag gatgcggact catcctcagc 1679
cagcgcccat gttgtgatac aggggtgttt gttggatggg tttaaaaata actagaaaaa 1739
ctcaggccca tccattttct cagatctcct tgaaaattga ggccttttcg atagtttcgg 1799
gtcaggtaaa aatggcctcc tggcgtaagc ttttcaaggt tttttggagg ctttttgtaa 1859
attgtgatag gaactttgga ccttgaactt acgtatcatg tggagaagag ccaatttaac 1919
aaactaggaa gatgaaaagg gaaattgtgg ccaaaacttt gggaaaagga ggttcttaaa 1979
atcagtgttt cccctttgtg cacttgtaga aaaaaaagaa aaaccttcta gagctgattt 2039
gatggacaat ggagagagct ttccctgtga ttataaaaaa ggaagctagc tgctctacgg 2099
tcatctttgc ttaagagtat actttaacct ggcttttaaa gcagtagtaa ctgccccacc 2159
aaaggtctta aaagccattt ttggagccta ttgcactgtg ttctcctact gcaaatattt 2219
tcatatggga ggatggtttt ctcttcatgt aagtccttgg aattgattct aaggtgatgt 2279
tcttagcact ttaattcctg tcaaattttt tgttctcccc ttctgccatc ttaaatgtaa 2339
gctgaaactg gtctactgtg tctctagggt taagccaaaa gacaaaaaaa attttactac 2399
ttttgagatt gccccaatgt acagaattat ataattctaa cgcttaaatc atgtgaaagg 2459
gttgctgctg tcagccttgc ccactgtgac ttcaaaccca aggaggaact cttgatcaag 2519
atgcccaacc ctgtgatcag aacctccaaa tactgccatg agaaactaga gggcaggtct 2579
tcataaaagc cctttgaacc cccttcctgc cctgtgttag gagataggga tattggcccc 2639
tcactgcagc tgccagcact tggtcagtca ctctcagcca tagcactttg ttcactgtcc 2699
tgtgtcagag cactgagctc cacccttttc tgagagttat tacagccaga aagtgtgggc 2759
tgaagatggt tggtttcatg t 2780
Met Val Met Phe Lys Lys Ile Lys Ser Phe Glu Val Val Phe Asn Asp
1 5 10 15
Pro Glu Lys Val Tyr Gly Ser Gly Glu Lys Val Ala Gly Arg Val Ile
20 25 30
Val Glu Val Cys Glu Val Thr Arg Val Lys Ala Val Arg Ile Leu Ala
35 40 45
Cys Gly Val Ala Lys Val Leu Trp Met Gln Gly Ser Gln Gln Cys Lys
50 55 60
Gln Thr Ser Glu Tyr Leu Arg Tyr Glu Asp Thr Leu Leu Leu Glu Asp
65 70 75 80
Gln Pro Thr Gly Glu Asn Glu Met Val Ile Met Arg Pro Gly Asn Lys
85 90 95
Tyr Glu Tyr Lys Phe Gly Phe Glu Leu Pro Gln Gly Pro Leu Gly Thr
100 105 110
Ser Phe Lys Gly Lys Tyr Gly Cys Val Asp Tyr Trp Val Lys Ala Phe
115 120 125
Leu Asp Arg Pro Ser Gln Pro Thr Gln Glu Thr Lys Lys Asn Phe Glu
130 135 140
Val Val Asp Leu Val Asp Val Asn Thr Pro Asp Leu Met Ala Pro Val
145 150 155 160
Ser Ala Lys Lys Glu Lys Lys Val Ser Cys Met Phe Ile Pro Asp Gly
165 170 175
Arg Val Ser Val Ser Ala Arg Ile Asp Arg Lys Gly Phe Cys Glu Gly
180 185 190
Asp Glu Ile Ser Ile His Ala Asp Phe Glu Asn Thr Cys Ser Arg Ile
195 200 205
Val Val Pro Lys Ala Ala Ile Val Ala Arg His Thr Tyr Leu Ala Asn
210 215 220
Gly Gln Thr Lys Val Leu Thr Gln Lys Leu Ser Ser Val Arg Gly Asn
225 230 235 240
His Ile Ile Ser Gly Thr Cys Ala Ser Trp Arg Gly Lys Ser Leu Arg
245 250 255
Val Gln Lys Ile Arg Pro Ser Ile Leu Gly Cys Asn Ile Leu Arg Val
260 265 270
Glu Tyr Ser Leu Leu Ile Tyr Val Ser Val Pro Gly Ser Lys Lys Val
275 280 285
Ile Leu Asp Leu Pro Leu Val Ile Gly Ser Arg Ser Gly Leu Ser Ser
290 295 300
Arg Thr Ser Ser Met Ala Ser Arg Thr Ser Ser Glu Met Ser Trp Val
305 310 315 320
Asp Leu Asn Ile Pro Asp Thr Pro Glu Ala Pro Pro Cys Tyr Met Asp
325 330 335
Val Ile Pro Glu Asp His Arg Leu Glu Ser Pro Thr Thr Pro Leu Leu
340 345 350
Asp Asp Met Asp Gly Ser Gln Asp Ser Pro Ile Phe Met Tyr Ala Pro
355 360 365
Glu Phe Lys Phe Met Pro Pro Pro Thr Tyr Thr Glu Val Asp Pro Cys
370 375 380
Ile Leu Asn Asn Asn Val Gln
atggtgatgt tcaagaagat caagtctttt gaggtggtct ttaacgaccc tgaaaaggtg 60
tacggcagtg gcgagaaggt ggctggccgg gtgatagtgg aggtgtgtga agttactcgt 120
gtcaaagccg ttaggatcct ggcttgcgga gtggctaaag tgctttggat gcagggatcc 180
cagcagtgca aacagacttc ggagtacctg cgctatgaag acacgcttct tctggaagac 240
cagccaacag gtgagaatga gatggtgatc atgagacctg gaaacaaata tgagtacaag 300
ttcggctttg agcttcctca ggggcctctg ggaacatcct tcaaaggaaa atatgggtgt 360
gtagactact gggtgaaggc ttttcttgac cgcccgagcc agccaactca agagacaaag 420
aaaaactttg aagtagtgga tctggtggat gtcaataccc ctgatttaat ggcacctgtg 480
tctgctaaaa aagaaaagaa agtttcctgc atgttcattc ctgatgggcg ggtgtctgtc 540
tctgctcgaa ttgacagaaa aggattctgt gaaggtgatg agatttccat ccatgctgac 600
tttgagaata catgttcccg aattgtggtc cccaaagctg ccattgtggc ccgccacact 660
taccttgcca atggccagac caaggtgctg actcagaagt tgtcatcagt cagaggcaat 720
catattatct cagggacatg cgcatcatgg cgtggcaaga gccttcgggt tcagaagatc 780
aggccttcta tcctgggctg caacatcctt cgagttgaat attccttact gatctatgtt 840
agcgttcctg gatccaagaa ggtcatcctt gacctgcccc tggtaattgg cagcagatca 900
ggtctaagca gcagaacatc cagcatggcc agccgaacca gctctgagat gagttgggta 960
gatctgaaca tccctgatac cccagaagct cctccctgct atatggatgt cattcctgaa 1020
gatcaccgat tggagagccc aaccactcct ctgctagatg acatggatgg ctctcaagac 1080
agccctatct ttatgtatgc ccctgagttc aagttcatgc caccaccgac ttatactgag 1140
gtggatccct gcatcctcaa caacaatgtg cagtga 1176
PRIMER DIRECTED TO RAT Cinc
gaagatagat tgcaccgatg 20
PRIMER DIRECTED TO RAT Cinc
catagcctct cacatttc 18
PRIMER DIRECTED TO RAT Cinc
gcgcccgtcc gccaatgagc tgcgc 25
PRIMER DIRECTED TO RAT Cinc
cttggggaca cccttcagca tcttttgg 28
PRIMER DIRECTED TO RAT Cinc
ctctacccac ggcaagttca a 21
PRIMER DIRECTED TO RAT Cinc
gggatgacct tgcccacagc 20