Wen-Jian Wu

Evidence for an essential role of cyclooxygenase-2 as a mediator of the late phase of ischemic preconditioning in mice

Abstract Recent studies have demonstrated that cyclooxygenase-2 (COX-2) is an essential mediator of the cardioprotective effects of the late phase of ischemic preconditioning (PC) in rabbits. The goal of this study was to determine whether COX-2 also plays an essential role in late PC in the mouse. B6129F2/J mice underwent a 30-min coronary occlusion followed by 24 h of reperfusion. Administration of the COX-2 selective inhibitor, NS-398, 30 min prior to the 30-min occlusion (5 mg/kg i.p.) had no appreciable effect on infarct size compared with untreated controls (58.8 ± 2.1%, vs. 58.8 ± 4.3% of the risk region, respectively). When mice were preconditioned with six cycles of 4-min coronary occlusion/4-min reperfusion 24 h prior to the 30-min occlusion, infarct size was markedly reduced (19.3 ± 3.4%), indicating a late PC effect. The protective effect of late PC was completely abrogated by administration of NS-398 30 min before the 30-min coronary occlusion (67.7 ± 3.0%), but not by administration of vehicle alone (23.6 ± 3.7%). These results indicate that COX-2 mediates the late phase of ischemic PC in the mouse and imply that the role of this enzyme in cardioprotection is not species-specific.

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.

Endothelial nitric oxide synthase is not necessary for the early phase of ischemic preconditioning in the mouse.

It has been proposed that constitutive expression of endothelial NO synthase (eNOS) protects against myocardial ischemia/reperfusion injury in the naive (unstressed) state and that eNOS plays a critical role in the early phase of ischemic preconditioning (PC). We addressed these issues using both a genetic approach (i.e., eNOS null [eNOS(-/-)]) mice and a pharmacologic approach (with the NOS inhibitor N(omega)-nitro-l-arginine [L-NA]). We found that in the nonpreconditioned state, both of the available strains of eNOS(-/-) mice (C57BL6 and B6129) exhibited infarct sizes similar to the corresponding wild-type (WT) mice (63.3+/-2.2% [group I, n=15] vs. 59.7+/-1.4% [group VI, n=10] of the risk region and 60.9+/-3.6% [group IX, n=14] vs. 68.2+/-2.5% [group X, n=9], respectively). When WT mice were preconditioned with either one or six cycles of 4-min coronary occlusion (O)/reperfusion (R) 10 min prior to the 30-min O, infarct size was markedly reduced (28.5+/-3.3% [group II, one O/R cycle, n=10] and 19.7+/-2.6% [group III, six O/R cycles, n=7] of the risk region, respectively), indicating the development of a robust early PC effect. In eNOS(-/-) mice preconditioned with the same protocol, the reduction in infarct size was similar (24.9+/-2.9% and 15.3+/-2.4% of the risk region, after one [group VII, n=9] or six O/R cycles [group VIII, n=10], respectively), indicating that the PC effect was intact. When WT mice were pretreated with L-NA 30 min before sham PC (group IV, n=7) or PC (group V, six O/R cycles, n=7), infarct size was not different from untreated control and PC groups. We conclude that, in the mouse, basal eNOS activity does not modulate infarct size in the nonpreconditioned state and is not necessary for the cardioprotective effects of early PC. Early PC is not eNOS-dependent, at least in this species.

Effects of Intracoronary Infusion of Escalating Doses of Cardiac Stem Cells in Rats With Acute Myocardial Infarction

Background—Although c-kitpos cardiac stem cells (CSCs) preserve left ventricular (LV) function and structure after myocardial infarction, CSC doses have been chosen arbitrarily, and the dose–effect relationship is unknown. Methods and Results—Rats underwent a 90-minute coronary occlusion followed by 35 days of reperfusion. Vehicle or CSCs at 5 escalating doses (0.3×106, 0.75×106, 1.5×106, 3.0×106, and 6.0×106 cells/heart) were given intracoronarily 4 h after reperfusion. The lowest dose (0.3×106) had no effect on LV function and morphology, whereas 0.75, 1.5, and 3.0×106 significantly improved regional and global LV function (echocardiography and hemodynamic studies). These 3 doses had similar effects on echocardiographic parameters (infarct wall thickening fraction, LV end-systolic and end-diastolic volumes, LV ejection fraction) and hemodynamic variables (LV end-diastolic pressure, LV dP/dtmax, preload adjusted maximal power, end-systolic elastance, preload recruitable stroke work) and produced similar reductions in apoptosis, scar size, infarct wall thinning, and LV expansion index and similar increases in viable myocardium in the risk region (morphometry). Infusion of 6.0×106 CSCs markedly increased postprocedural mortality. Green fluorescent protein and 5-bromo-2′-deoxyuridine staining indicated that persistence of donor cells and formation of new myocytes were negligible with all doses. Conclusions—Surprisingly, in this rat model of acute myocardial infarction, the dose–response relationship for intracoronary CSCs is flat. A minimal dose between 0.3 and 0.75×106 is necessary for efficacy; above this threshold, a 4-fold increase in cell number does not produce greater improvement in LV function or structure. Further increases in cell dose are harmful.

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.

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.