Qinghui Ou

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.

Cardioprotection Afforded by Inducible Nitric Oxide Synthase Gene Therapy Is Mediated by Cyclooxygenase-2 via a Nuclear Factor-κB–Dependent Pathway

Background— Gene therapy with inducible nitric oxide synthase (iNOS) markedly reduces myocardial infarct size; this effect is associated with cyclooxygenase-2 (COX-2) upregulation and is ablated by COX-2 inhibitors. However, pharmacological inhibitors are limited by relative lack of specificity; furthermore, the mechanism whereby iNOS gene therapy upregulates COX-2 remains unknown. Accordingly, we used genetically engineered mice to test the hypothesis that the cardioprotection afforded by iNOS gene transfer is mediated by COX-2 upregulation via a nuclear factor (NF)-&kgr;B-dependent pathway. Methods and Results— Mice received an intramyocardial injection of Av3/LacZ (LacZ group) or Av3/iNOS (iNOS group); 3 days later, myocardial infarction was produced by a 30-minute coronary occlusion followed by 4 hours of reperfusion. Among Av3/LacZ-treated mice, infarct size was similar in COX-2−/− and wild-type groups. iNOS gene transfer (confirmed by iNOS immunoblotting and activity assays) markedly reduced infarct size in wild-type mice but failed to do so in COX-2−/− mice. In transgenic mice with cardiac-specific expression of a dominant-negative mutant of I&kgr;B&agr; (I&kgr;B&agr;S32A,S36A), the upregulation of phosphorylated I&kgr;B&agr;, activation of NF-&kgr;B, and cardiac COX-2 protein expression 3 days after iNOS gene therapy were abrogated, which was associated with the abolishment of the cardioprotective effects afforded by iNOS gene therapy. Conclusions— These data provide strong genetic evidence that COX-2 is an obligatory downstream effector of iNOS-dependent cardioprotection and that NF-&kgr;B is a critical link between iNOS and COX-2. Thus, iNOS imparts its protective effects, at least in part, by recruiting NF-&kgr;B, leading to COX-2 upregulation. However, COX-2 does not play an important cardioprotective role under basal conditions (when iNOS is not upregulated).

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.

Repeated Administrations of Cardiac Progenitor Cells Are Markedly More Effective Than a Single Administration: A New Paradigm in Cell Therapy

RATIONALE The effects of c-kit(POS) cardiac progenitor cells (CPCs, and adult cell therapy in general) on left ventricular (LV) function have been regarded as modest or inconsistent. OBJECTIVE To determine whether 3 CPC infusions have greater efficacy than 1 infusion. METHODS AND RESULTS Rats with a 30-day-old myocardial infarction received 1 or 3 CPC infusions into the LV cavity, 35 days apart. Compared with vehicle-treated rats, the single-dose group exhibited improved LV function after the first infusion (consisting of CPCs) but not after the second and third (vehicle). In contrast, in the multiple-dose group, regional and global LV function improved by a similar degree after each CPC infusion, resulting in greater cumulative effects. For example, the total increase in LV ejection fraction was approximately triple in the multiple-dose group versus the single-dose group (P<0.01). The multiple-dose group also exhibited more viable tissue and less scar, less collagen in the risk and noninfarcted regions, and greater myocyte density in the risk region. CONCLUSIONS This is the first demonstration that repeated CPC administrations are markedly more effective than a single administration. The concept that the full effects of CPCs require repeated doses has significant implications for both preclinical and clinical studies; it suggests that the benefits of cell therapy may be underestimated or even overlooked if they are measured after a single dose, and that repeated administrations are necessary to evaluate the effectiveness of a cell product properly. In addition, we describe a new method that enables studies of repeated cell administrations in rodents.

Repeated Administrations of Cardiac Progenitor Cells Are Superior to a Single Administration of an Equivalent Cumulative Dose

Background We have recently found that 3 repeated doses (12×106 each) of c‐kitPOS cardiac progenitor cells (CPCs) were markedly more effective than a single dose of 12×106 cells in alleviating postinfarction left ventricular dysfunction and remodeling. However, since the single‐dose group received only one third of the total number of CPCs given to the multiple‐dose group, it is unknown whether the superior therapeutic efficacy was caused by repeated treatments per se or by administration of a higher total number of CPCs. This issue has major clinical implications because multiple cell injections in patients pose significant challenges, which would be obviated by using 1 large injection. Accordingly, we determined whether the beneficial effects of 3 repeated CPC doses can be recapitulated by 1 large dose containing the same total number of cells. Methods and Results Rats with a 30‐day‐old myocardial infarction received 3 echo‐guided intraventricular infusions, 35 days apart, of vehicle‐vehicle‐vehicle, 36×106 CPCs‐vehicle‐vehicle, or 3 equal doses of 12×106 CPCs. Infusion of a single, large dose of CPCs (36×106 cells) produced an initial improvement in left ventricular function, but no further improvement was observed after the second and third infusions (both vehicle). In contrast, each of the 3 doses of CPCs (12×106) caused a progressive improvement in left ventricular function, the cumulative magnitude of which was greater than with a single dose. Unlike the single dose, repeated doses reduced collagen content and immune cell infiltration. Conclusions Three repeated doses of CPCs are superior to 1 dose even though the total number of cells infused is the same, possibly because of greater antifibrotic and anti‐inflammatory actions.

178. Effect of Heme Oxygenase-1 Overexpression on DNA Synthesis of Adult Cardiac Progenitor Cells Under Hypoxia

Although the studies in animals and humans have shown that the beneficial effects of adult c-kit+ cardiac progenitor cells (CPCs) on left ventricle function and remodeling post-myocardial infarction persist for several weeks and up to 1 year after CPC administration, the vast majority (>95%) of transplanted CPCs in animal studies die or vanish shortly after cell administration. Additionally, the less blood flow in the scar tissue after heart attack results in the oxygen reduced to 1 - 2% (hypoxia) which makes the cardiac microenvironment inadequate for cell DNA synthesis, thereby implying that gene manipulations on the transplanted CPCs that limit cell death under hypoxia may enhance the efficacy (and thus clinical applicability) of cell therapy. One of the most powerful cytoprotective proteins known is heme oxygenase-1 (HO-1). To test the hypothesis that HO-1 gene may improve newly synthesized DNA level in CPCs under hypoxia, adult CPCs were isolated and sorted from wild-type (WT), HO-1 transgenic (TG) or knockout (KO) mouse hearts. The c-kit positivity in all live CPCs was 60 - 65% detected by FACS with specific monoclonal anti-c-kit antibody in which Mast cells were used as positive control and CHO cells as negative control. HO-1 protein overexpression in HO-1TG CPCs was confirmed by FACS and immunoblotting (2.8-fold greater than that in WT CPCs); in contrast, HO-1KO CPCs had no HO-1. A hypoxia chamber system containing 1% of O2, 5% of CO2, and 94% of nitrogen was employed to mimic the damaged environment in the cardiac scar tissue. Furthermore, Bromodeoxyuridine (BrdU), an analog of thymidine that can be incorporated into the newly synthesized DNA of replicating cells in S-phase, was used to determine DNA synthesis levels in CPCs. Cells were cultured for 16 h and then labeled with BrdU for 30 min before BrdU immunofluorescent staining. Under normal oxygen tension (21% of O2), HO-1TG CPCs exhibited an significant increase in nuclear BrdU intensity relative to either WT or HO-1KO CPCs (+88% vs WT group, and +63% vs HO-1KO group), indicating that the new DNA synthesis in HO-1TG CPCs are very active during cell mitosis as compared to WT or HO-1KO CPCs. Under hypoxic condition (1% of O2), although new DNA synthesis in S-phase in all of the groups was much less active, HO-1TG CPCs showed even greater differences (+2.4-fold vs WT, +2.0-fold vs HO-1KO) in the extent of BrdU nuclear incorporation as compared with WT or HO-1KO CPC group (FigFig), providing a strong evidence that HO-1 gene plays an important role in protecting CPCs under hypoxia via promotion of new DNA synthesis in BrdU-labeled cell mitosis. These results demonstrate that HO-1 gene promotes new DNA synthesis during mitoses in adult CPCs not only under normal oxygen condition but also in the hypoxia environment, therefore HO-1TG CPCs may provide higher survival rate than WT and HO-1KO CPCs. The data support the hypothesis that HO-1 gene improves the survival and reparative ability of adult CPCs via enhancing newly synthesized DNA in replicating cells in S-phase under hypoxia. Thus, the adult CPCs carried HO-1 gene as a new approach may have potentially clinical application to increase the efficacy of cell therapy for patients with heart attack.View Large Image | Download PowerPoint Slide

Repeated Administrations of Cardiac Progenitor Cells Are Markedly More Effective Than a Single AdministrationNovelty and Significance: A New Paradigm in Cell Therapy

RATIONALE The effects of c-kit(POS) cardiac progenitor cells (CPCs, and adult cell therapy in general) on left ventricular (LV) function have been regarded as modest or inconsistent. OBJECTIVE To determine whether 3 CPC infusions have greater efficacy than 1 infusion. METHODS AND RESULTS Rats with a 30-day-old myocardial infarction received 1 or 3 CPC infusions into the LV cavity, 35 days apart. Compared with vehicle-treated rats, the single-dose group exhibited improved LV function after the first infusion (consisting of CPCs) but not after the second and third (vehicle). In contrast, in the multiple-dose group, regional and global LV function improved by a similar degree after each CPC infusion, resulting in greater cumulative effects. For example, the total increase in LV ejection fraction was approximately triple in the multiple-dose group versus the single-dose group (P<0.01). The multiple-dose group also exhibited more viable tissue and less scar, less collagen in the risk and noninfarcted regions, and greater myocyte density in the risk region. CONCLUSIONS This is the first demonstration that repeated CPC administrations are markedly more effective than a single administration. The concept that the full effects of CPCs require repeated doses has significant implications for both preclinical and clinical studies; it suggests that the benefits of cell therapy may be underestimated or even overlooked if they are measured after a single dose, and that repeated administrations are necessary to evaluate the effectiveness of a cell product properly. In addition, we describe a new method that enables studies of repeated cell administrations in rodents.

Repeated Administrations of Cardiac Progenitor Cells Are Markedly More Effective Than a Single AdministrationNovelty and Significance

RATIONALE The effects of c-kit(POS) cardiac progenitor cells (CPCs, and adult cell therapy in general) on left ventricular (LV) function have been regarded as modest or inconsistent. OBJECTIVE To determine whether 3 CPC infusions have greater efficacy than 1 infusion. METHODS AND RESULTS Rats with a 30-day-old myocardial infarction received 1 or 3 CPC infusions into the LV cavity, 35 days apart. Compared with vehicle-treated rats, the single-dose group exhibited improved LV function after the first infusion (consisting of CPCs) but not after the second and third (vehicle). In contrast, in the multiple-dose group, regional and global LV function improved by a similar degree after each CPC infusion, resulting in greater cumulative effects. For example, the total increase in LV ejection fraction was approximately triple in the multiple-dose group versus the single-dose group (P<0.01). The multiple-dose group also exhibited more viable tissue and less scar, less collagen in the risk and noninfarcted regions, and greater myocyte density in the risk region. CONCLUSIONS This is the first demonstration that repeated CPC administrations are markedly more effective than a single administration. The concept that the full effects of CPCs require repeated doses has significant implications for both preclinical and clinical studies; it suggests that the benefits of cell therapy may be underestimated or even overlooked if they are measured after a single dose, and that repeated administrations are necessary to evaluate the effectiveness of a cell product properly. In addition, we describe a new method that enables studies of repeated cell administrations in rodents.