Gregg Rokosh

Intracoronary Administration of Cardiac Progenitor Cells Alleviates Left Ventricular Dysfunction in Rats With a 30-Day-Old Infarction

Background— Administration of cardiac progenitor cells (CPCs) 4 hours after reperfusion ameliorates left ventricular function in rats with acute myocardial infarction (MI). Clinically, however, this approach is not feasible, because expansion of autologous CPCs after acute MI requires several weeks. Therefore, we sought to determine whether CPCs are beneficial in the more clinically relevant setting of an old MI (scar). Methods and Results— One month after coronary occlusion/reperfusion, rats received an intracoronary infusion of vehicle or enhanced green fluorescent protein–labeled CPCs. Thirty-five days later, CPC-treated rats exhibited more viable myocardium in the risk region, less fibrosis in the noninfarcted region, and improved left ventricular function. Cells that stained positive for enhanced green fluorescent protein that expressed cardiomyocyte, endothelial, and vascular smooth muscle cell markers were observed only in 7 of 17 treated rats and occupied only 2.6% and 1.1% of the risk and noninfarcted regions, respectively. Transplantation of CPCs was associated with increased proliferation and expression of cardiac proteins by endogenous CPCs. Conclusions— Intracoronary administration of CPCs in the setting of an old MI produces beneficial structural and functional effects. Although exogenous CPCs can differentiate into new cardiac cells, this mechanism is not sufficient to explain the benefits, which suggests paracrine effects; among these, the present data identify activation of endogenous CPCs. This is the first report that CPCs are beneficial in the setting of an old MI when given by intracoronary infusion, the most widely applicable therapeutic approach in patients. Furthermore, this is the first evidence that exogenous CPC administration activates endogenous CPCs. These results open the door to new therapeutic applications for the use of autologous CPCs in patients with old MI and chronic ischemic cardiomyopathy.

Stromal Cell–Derived Factor-1α Confers Protection Against Myocardial Ischemia/Reperfusion Injury Role of the Cardiac Stromal Cell–Derived Factor-1α–CXCR4 Axis

Background— Stromal cell–derived factor-1&agr; (SDF-1&agr;) binding to its cognate receptor, CXCR4, regulates a variety of cellular functions such as stem cell homing, trafficking, and differentiation. However, the role of the SDF-1&agr;–CXCR4 axis in modulating myocardial ischemia/reperfusion injury is unknown. Methods and Results— In mice subjected to ischemic preconditioning, myocardial SDF-1&agr; mRNA was found to be increased 3 hours later (P<0.05). Myocardial SDF-1&agr; and CXCR4 mRNA and protein were found to be expressed in both cardiac myocytes and fibroblasts. SDF-1&agr; production increased significantly after 1 or 4 hours of hypoxia and 18 hours of reoxygenation in cultured myocytes (P<0.05) but did not change in fibroblast cultures. In isolated myocytes, CXCR4 activation by SDF-1&agr; resulted in increased phosphorylation of both ERK 1/2 and AKT and decreased phosphorylation of JNK and p38. Cultured myocytes pretreated with SDF-1&agr; were resistant to hypoxia/reoxygenation damage, exhibiting less lactate dehydrogenase release, trypan blue uptake, and apoptotic cell death (terminal deoxynucleotidyl transferase–mediated dUTP nick-end labeling assay) (P<0.05). This protective effect was blocked by the CXCR4 selective antagonist AMD3100. In vivo, administration of SDF-1&agr; before 30 minutes of coronary occlusion followed by 4 hours of reperfusion decreased infarct size (P<0.05). The decrease in infarct size with SDF-1&agr; administration also was blocked by AMD3100. Conclusions— We conclude that SDF-1&agr; and its receptor, CXCR4, constitute a paracrine or autocrine axis in cardiac myocytes that is activated in response to preconditioning and hypoxic stimuli, recruiting the antiapoptotic kinases ERK and AKT and promoting an antiapoptotic program that confers protection against ischemia/reperfusion damage.

Role of the Protein Kinase C-ε–Raf-1–MEK-1/2–p44/42 MAPK Signaling Cascade in the Activation of Signal Transducers and Activators of Transcription 1 and 3 and Induction of Cyclooxygenase-2 After Ischemic Preconditioning

Background—Although Janus kinase (JAK)–mediated Tyr phosphorylation of signal transducers and activators of transcription (STAT) 1 and 3 is essential for the upregulation of cyclooxygenase-2 (COX-2) and the cardioprotection of late preconditioning (PC), the role of Ser phosphorylation of STAT1 and STAT3 in late PC and the upstream signaling mechanisms responsible for mediating Ser phosphorylation of STAT1 and STAT3 remain unknown. Methods and Results—In mice preconditioned with six 4-minute coronary occlusion/4-minute reperfusion cycles, we found that (1) ischemic PC activates the Raf1–mitogen-activated protein kinase (MAPK)/extracellular signal–regulated kinase kinase (MEK) 1/2–p44/42 MAPK signaling pathway, induces phosphorylation of STAT1 and STAT3 on the Ser-727 residue, and upregulates COX-2 expression; (2) pSer-STAT1 and pSer-STAT3 form complexes with pTyr-p44/42 MAPKs in preconditioned myocardium, supporting the concept that Ser phosphorylation of these 2 factors is mediated by activated p44/42 MAPKs; and (3) activation of the Raf-1-MEK-1/2–p44/42 MAPK-pSer-STAT1/3 pathway and induction of COX-2 during ischemic PC are dependent on protein kinase C (PKC)-&egr; activity, as determined by both pharmacological and genetic inhibition of PKC&egr;. Conclusions—To our knowledge, this is the first study to demonstrate that ischemic PC causes Ser phosphorylation of STAT1 and STAT3 and that this event is governed by PKC&egr; via a PKC&egr;–Raf1-MEK1/2-p44/42 MAPK pathway. Furthermore, this is the first report that COX-2 expression in the heart is controlled by PKC&egr;. Together with our previous findings, the present study implies that STAT-dependent transcription of the genes responsible for ischemic PC is modulated by a dual signaling mechanism that involves both JAK1/2 (Tyr phosphorylation) and PKC&egr; (Ser phosphorylation).

Cardiac Myocyte–Specific Expression of Inducible Nitric Oxide Synthase Protects Against Ischemia/Reperfusion Injury by Preventing Mitochondrial Permeability Transition

Background— Inducible nitric oxide synthase (iNOS) is an obligatory mediator of the late phase of ischemic preconditioning, but the mechanisms of its cardioprotective actions are unknown. In addition, it remains unclear whether sustained elevation of iNOS in myocytes provides chronic protection against ischemia/reperfusion injury. Methods and Results— Constitutive overexpression of iNOS in transgenic mice (α-myosin heavy chain promoter) did not induce contractile dysfunction and did not affect mitochondrial respiration or biogenesis, but it profoundly decreased infarct size in mice subjected to 30 minutes of coronary occlusion and 24 hours of reperfusion. In comparison with wild-type hearts, isolated iNOS-transgenic hearts subjected to ischemia for 30 minutes followed by 40 minutes of reperfusion displayed better contractile recovery, smaller infarct size, and less mitochondrial entrapment of 2-deoxy-[3H]-glucose. Reperfusion-induced loss of NAD+ and mitochondrial release of cytochrome c were attenuated in iNOS-transgenic hearts, indicating reduced mitochondrial permeability transition. The NO donor NOC-22 prevented permeability transition in isolated mitochondria, and mitochondrial permeability transition–induced NAD+ loss was decreased in wild-type but not iNOS-null mice treated with the NO donor diethylene triamine/NO 24 hours before ischemia and reperfusion ex vivo. iNOS-mediated cardioprotection was not abolished by atractyloside. Reperfusion-induced production of oxygen-derived free radicals (measured by electron paramagnetic resonance spectroscopy) was attenuated in iNOS-transgenic hearts and was increased in wild-type hearts treated with the mitochondrial permeability transition inhibitor cyclosporin A. Conclusions— Cardiomyocyte-restricted expression of iNOS provides sustained cardioprotection. This cardioprotection is associated with a decrease in reperfusion-induced oxygen radicals and inhibition of mitochondrial swelling and permeability transition.

Endothelial Nitric Oxide Synthase Plays an Obligatory Role in the Late Phase of Ischemic Preconditioning by Activating the Protein Kinase Cε–p44/42 Mitogen-Activated Protein Kinase–pSer-Signal Transducers and Activators of Transcription1/3 Pathway

Background— The role of endothelial nitric oxide synthase (eNOS) in ischemic preconditioning (PC) and cardioprotection is poorly understood. We addressed this issue using a genetic, rather than pharmacological, approach. Methods and Results— In the nonpreconditioned state, eNOS−/− mice exhibited infarct sizes similar to those of wild-type mice. A sequence of six 4-minute coronary occlusion/4-minute reperfusion cycles (ischemic PC) induced late PC in wild-type mice; genetic deletion of eNOS abrogated the cardioprotection induced by late PC. In wild-type mice, ischemic PC induced membranous translocation of protein kinase C (PKC)&egr; and an increase in pSer-MEK-1/2 and pTyr-p44/42 mitogen-activated protein kinase, nuclear pSer-signal transducers and activators of transcription (STAT)1 and pSer-STAT3, and nuclear STAT1/3 DNA binding activity, followed by upregulation of cyclooxygenase-2 protein and activity 24 hours later. All of these changes were abrogated in eNOS−/− mice. The NO donor diethylenetriamine/NO recapitulated the effects of ischemic PC. Conclusions— In contrast to previous reports, we found that basal eNOS activity does not modulate infarct size in the nonpreconditioned state. However, eNOS is obligatorily required for the development of the cardioprotective effects of late PC and acts as the trigger of this process by activating the PKC&egr;-MEK-1/2-p44/42 mitogen-activated protein kinase pathway, leading to Ser-727 phosphorylation of STAT1 and STAT3 and consequent upregulation of STAT-dependent genes such as cyclooxygenase-2. The effects of eNOS-derived NO are reproduced by exogenous NO (NO donors), implying that nitrates can upregulate cardiac cyclooxygenase-2.

Long-Term Outcome of Administration of c-kit(POS) Cardiac Progenitor Cells After Acute Myocardial Infarction: Transplanted Cells Do not Become Cardiomyocytes, but Structural and Functional Improvement and Proliferation of Endogenous Cells Persist for at Least One Year.

Rationale:Cardiac progenitor cells (CPCs) improve left ventricular remodeling and function after acute or chronic myocardial infarction. However, the long-term (>5 weeks) effects, potential tumorigenicity, and fate of transplanted CPCs are unknown. Objective:To assess the outcome of CPC therapy at 1 year. Methods and Results:Female rats underwent a 90-minute coronary occlusion; 4 hours after reperfusion, they received intracoronarily vehicle or 1 million male, syngeneic CPCs. One year later, CPC-treated rats exhibited smaller scars and more viable myocardium in the risk region, along with improved left ventricular remodeling and regional and global left ventricular function. No tumors were observed. Some transplanted (Y-chromosomePOS) CPCs (or their progeny) persisted and continued to proliferate, but they failed to acquire a mature cardiomyocyte phenotype and were too few (4–8% of nuclei) to account for the benefits of CPC therapy. Surprisingly, CPC transplantation triggered a prolonged proliferative res...RATIONALE Cardiac progenitor cells (CPCs) improve left ventricular remodeling and function after acute or chronic myocardial infarction. However, the long-term (>5 weeks) effects, potential tumorigenicity, and fate of transplanted CPCs are unknown. OBJECTIVE To assess the outcome of CPC therapy at 1 year. METHODS AND RESULTS Female rats underwent a 90-minute coronary occlusion; 4 hours after reperfusion, they received intracoronarily vehicle or 1 million male, syngeneic CPCs. One year later, CPC-treated rats exhibited smaller scars and more viable myocardium in the risk region, along with improved left ventricular remodeling and regional and global left ventricular function. No tumors were observed. Some transplanted (Y-chromosome(POS)) CPCs (or their progeny) persisted and continued to proliferate, but they failed to acquire a mature cardiomyocyte phenotype and were too few (4-8% of nuclei) to account for the benefits of CPC therapy. Surprisingly, CPC transplantation triggered a prolonged proliferative response of endogenous cells, resulting in increased formation of endothelial cells and Y-chromosome(NEG) CPCs for 12 months and increased formation, for at least 7 months, of small cells that expressed cardiomyocytic proteins (α-sarcomeric actin) but did not have a mature cardiomyocyte phenotype. CONCLUSIONS The beneficial effects of CPCs on left ventricular remodeling and dysfunction are sustained for at least 1 year and thus are likely to be permanent. Because transplanted CPCs do not differentiate into mature myocytes, their major mechanism of action must involve paracrine actions. These paracrine mechanisms could be very prolonged because some CPCs engraft, proliferate, and persist at 1 year. This is the first report that transplantation of any cell type in the heart induces a proliferative response that lasts at least 1 year. The results strongly support the safety and clinical utility of CPC therapy.

A murine model of inducible, cardiac-specific deletion of STAT3: its use to determine the role of STAT3 in the upregulation of cardioprotective proteins by ischemic preconditioning.

Pharmacological studies have shown that signal transducers and activators of transcription (STATs) are necessary for the delayed cardioprotection of ischemic preconditioning (PC). However, pharmacologic STAT inhibitors are not specific; furthermore, the individual role of STAT3 in late PC remains unknown. The objectives of the study were (i) to create an inducible, cardiac-specific STAT3 knockout mouse; (ii) to verify whether STAT3 deletion has any adverse effects in the short term (~1 month); and (iii) to use this novel tool to evaluate the role of STAT3 in the PC-induced upregulation of cardioprotective and anti-apoptotic proteins. We created an inducible, cardiomyocyte-restricted STAT3 deficient mouse (MCM TG:STAT3(flox/flox)) by interbreeding STAT3(flox/flox) mice and tamoxifen-inducible MCM TG mice. Treatment of MCM TG:STAT3(flox/flox) mice with tamoxifen resulted in deletion of STAT3 specifically in cardiac myocytes, concomitant with abrogation of ischemic PC-induced Tyr-705 and Ser-727 phosphorylation of STAT3 and increased STAT3 DNA-binding activity. In vehicle-treated MCM TG:STAT3(flox/flox) mice, ischemic PC increased the expression of cardioprotective (COX-2 and HO-1) and anti-apoptotic (e.g., Mcl-1, Bcl-x(L), c-FLIP(L), c-FLIP(S)) proteins 24h later; in contrast, in tamoxifen-treated MCM TG:STAT3(flox/flox) mice this increase was completely absent. Deletion of STAT3 had no apparent adverse effects on LV structure or function after 35 days. We have developed a novel inducible, cardiomyocyte-restricted STAT3 deficient mouse that can be used to specifically interrogate the role of this transcription factor in cardiovascular pathophysiology in vivo. Our data demonstrate, for the first time, that recruitment of STAT3 plays an obligatory role in the upregulation of cardioprotective and anti-apoptotic proteins and suggest that STAT3 activation is important in inhibiting both the death receptor pathway (which is modulated by c-FLIP(L) and c-FLIP(S)) and the mitochondrial pathway (which is mediated by Mcl-1 and Bcl-x(L)).

Chronic AMD3100 antagonism of SDF-1α–CXCR4 exacerbates cardiac dysfunction and remodeling after myocardial infarction

The role of the SDF-1alpha-CXCR4 axis in response to myocardial infarction is unknown. We addressed it using the CXCR4 antagonist, AMD3100, to block SDF-1alpha interaction with CXCR4 after chronic coronary artery ligation. Chronic AMD3100 treatment decreased ejection fraction and fractional shortening in mice 20days after myocardial infarction compared with vehicle-treated mice (echocardiography). Morphometric analysis showed hearts of AMD3100-treated infarcted mice to have expanded scar, to be hypertrophic (confirmed by myocyte cross-section area) and dilated, with increased LV end systolic and end diastolic dimensions, and to have decreased scar collagen content; p-AKT levels were attenuated and this was accompanied by increased apoptosis. Despite increased injury, c-kit(pos) cardiac progenitor cells (CPCs) were increased in the risk region of AMD3100-treated infarcted mice; CPCs were CD34(neg)/CD45(neg) with the majority undergoing symmetric cell division. c-kit(pos)/MHC(pos) CPCs also increased in the risk region of the AMD3100-treated infarcted group. In this group, GSK-3beta signaling was attenuated compared to vehicle-treated, possibly accounting for increased proliferation and increased cardiac committed MHC(pos) CPCs. Increased proliferation following AMD3100 treatment was supported by increased levels of cyclin D1, a consequence of increased prolyl isomerase, Pin1, and decreased cyclin D1 phosphorylation. In summary, pharmacologic antagonism of CXCR4 demonstrates that SDF-1alpha-CXCR4 signaling plays an important role during and after myocardial infarction and that it exerts pleiotropic salubrious effects, protecting the myocardium from apoptotic cell death, facilitating scar formation, restricting CPC proliferation, and directing CPCs toward a cardiac fate.

Hepatocyte growth factor/Met gene transfer in cardiac stem cells—potential for cardiac repair

The adult heart has been recently recognized as a self-renewing organ that contains a pool of committed resident cardiac stem cells (CSCs) and cardiac progenitor cells (CPCs). These adult CSCs and CPCs can be induced by cytokines and growth factors to migrate, differentiate, and proliferate in situ and potentially replace lost cardiomyocytes. Ligand-receptor systems, such as the tyrosine kinase receptor mesenchymal–epithelial transition factor (Met) and its ligand hepatocyte growth factor (HGF), are potential candidates for boosting migration, engraftment and commitment of CSCs. Here, we discuss the possible application of HGF/Met gene therapy to enhance the ability of CSCs to promote myocardial regeneration.

Gene Transfer of Inducible Nitric Oxide Synthase Affords Cardioprotection by Upregulating Heme Oxygenase-1 Via a Nuclear Factor-κB-Dependent Pathway

Background— Although inducible nitric oxide synthase (iNOS) is known to impart powerful protection against myocardial infarction, the mechanism for this salubrious action remains unclear. Methods and Results— Adenovirus-mediated iNOS gene transfer in mice resulted 48 to 72 hours later in increased expression not only of iNOS protein but also of heme oxygenase (HO)-1 mRNA and protein; HO-2 protein expression did not change. iNOS gene transfer markedly reduced infarct size in wild-type mice, but this effect was completely abrogated in HO-1−/− mice. At 48 hours after iNOS gene transfer, nuclear factor-&kgr;B was markedly activated. In transgenic mice with cardiomyocyte-restricted expression of a dominant negative mutant of I&kgr;Bα (I&kgr;BαS32A,S36A), both basal HO-1 levels and upregulation of HO-1 by iNOS gene transfer were suppressed. Chromatin immunoprecipitation analysis of mouse hearts provided direct evidence that nuclear factor-&kgr;B subunits p50 and p65 were recruited to the HO-1 gene promoter (−468 to −459 bp) 48 hours after iNOS gene transfer. Conclusions— This study demonstrates for the first time the existence of a close functional coupling between cardiac iNOS and cardiac HO-1: iNOS upregulates HO-1 by augmenting nuclear factor-&kgr;B binding to the region of the HO-1 gene promoter from −468 to −459 bp, and HO-1 then mediates the cardioprotective effects of iNOS. These results also reveal an important role of nuclear factor-&kgr;B in both basal and iNOS-induced expression of cardiac HO-1. Collectively, the present findings significantly expand our understanding of the regulation of cardiac HO-1 and of the mechanism whereby iNOS exerts its cardioprotective actions.