Although transplantation of c-kit+ cardiac stem cells (CSCs) has been shown to alleviate left ventricular (LV) dysfunction induced by myocardial infarction (MI), the number of exogenous CSCs remaining in the recipient heart following transplantation and their mechanism of action remain unclear. We have previously developed a highly sensitive and accurate method to quantify the absolute number of male murine CSCs in female recipient organs after transplantation. In the present study, we used this method to monitor the number of donor CSCs in the recipient heart after intracoronary infusion. Female mice underwent a 60-min coronary occlusion followed by reperfusion; 2 days later, 100,000 c-kit+/lin- syngeneic male mouse CSCs were infused intracoronarily. Only 12.7% of the male CSCs present in the heart immediately (5 min) after infusion were still present in the heart at 24 h, and their number declined rapidly thereafter. By 35 days after infusion, only ∼1,000 male CSCs were found in the heart. Significant numbers of male CSCs were found in the lungs and kidneys, but only in the first 24 h. The number of CSCs in the lungs increased between 5 min and 24 h after infusion, indicating recirculation of CSCs initially retained in other organs. Despite the low retention and rapid disappearance of CSCs from the recipient heart, intracoronary delivery of CSCs significantly improved LV function at 35 days (Millar catheter). These results suggest that direct differentiation of CSCs alone cannot account for the beneficial effects of CSCs on LV function; therefore, paracrine effects must be the major mechanism. The demonstration that functional improvement is dissociated from survival of transplanted cells has major implications for our understanding of cell therapy. In addition, this new quantitative method of stem cell measurement will be useful in testing approaches of enhancing CSC engraftment and survival after transplantation.

. Administration of a CO-releasing molecule at the time of reperfusion reduces infarct size in vivo. Am J Physiol Heart Circ Physiol 286: H1649-H1653, 2004. First published January 2, 2004 10.1152/ajpheart.00971.2003.-Although carbon monoxide (CO) has traditionally been viewed as a toxic gas, increasing evidence suggests that it plays an important homeostatic and cytoprotective role. Its therapeutic use, however, is limited by the side effects associated with CO inhalation. Recently, transition metal carbonyls have been shown to be a safe and effective means of transporting and releasing CO groups in vivo. The goal of the present study was to test whether a water-soluble CO-releasing molecule, tricarbonylchloro(glycinato) ruthenium (II) (CORM-3), reduces infarct size in vivo when given in a clinically relevant manner, i.e., at the time of reperfusion. Mice were subjected to a 30-min coronary artery occlusion followed by 24 h of reperfusion and were given either CORM-3 (3.54 mg/kg as a 60-min intravenous infusion starting 5 min before reperfusion) or equivalent doses of inactive CORM-3, which does not release CO. CORM-3 had no effect on arterial blood pressure or heart rate. The region at risk did not differ in control and treated mice (44.5 Ϯ 3.5% vs. 36.5 Ϯ 1.6% of the left ventricle, respectively). However, infarct size was significantly smaller in treated mice [25.8 Ϯ 4.9% of the region at risk (n ϭ 13) vs. 47.7 Ϯ 3.8% (n ϭ 14), P Ͻ 0.05]. CORM-3 did not increase carboxyhemoglobin levels in the blood. These results suggest that a novel class of drugs, CO-releasing molecules, can be useful to limit myocardial ischemia-reperfusion injury in vivo.carbon monoxide-releasing molecules; myocardial ischemia; reperfusion injury; transition metal carbonyls MAMMALIAN TISSUES continually produce carbon monoxide (CO) as a result of the breakdown of heme by heme oxygenase (HO) (13). Although CO has been traditionally regarded as toxic, recent evidence has revealed that this gas exerts pleiotropic homeostatic effects. Specifically, CO has been shown to promote vasorelaxation (12, 17) and to inhibit proliferation of smooth muscle cells (21), apoptosis (2), transplant rejection (4), inflammation (14, 15), platelet aggregation, microvascular thrombosis (3), cytokine production (9, 18), and oxidative stress (16). CO, delivered either as a gas or via CO-releasing molecules (CORMs), has also been shown to alleviate hypoxia/ reoxygenation injury in isolated cells and ischemia-reperfusion injury in isolated hearts (4) and in the liver (1). Although the mechanism(s) underlying the cytoprotective actions of CO has not been elucidated, evidence suggests that this gas exerts some of its effects via activation of the guanylate cyclase/ cGMP pathway (10, 22) and the p38 MAPK-dependent pathway (15).In view of the mounting evidence supporting a salubrious role of CO in a variety of pathophysiological conditions, much interest has focused on harnessing the actions of this molecule for therapeutic purposes. Thus far, most studies in vivo ha...

Although transplantation of c-kit+ cardiac stem cells (CSCs) alleviates post-myocardial infarction left ventricular dysfunction, there are no reliable methods that enable measurement of the absolute number of CSCs that persist in the recipient heart. To overcome this limitation, we developed a highly sensitive and accurate method to quantify the absolute number of murine CSCs after transplantation. This method has two unique features: i) real-time PCR-based detection of a novel male-specific, multiple-copy gene, Rbmy, which significantly increases the sensitivity of detection of male donor cells in a female recipient, and ii) an internal standard, which permits quantification of the absolute number of CSCs as well as the total number of cells in the recipient organ. Female C57BL/6 mice underwent coronary occlusion and reperfusion; 2 days later, 105 male mouse CSCs were injected intramyocardially. Tissues were analyzed by real-time PCR at serial time points. In the risk region, >75% of CSCs present at 5 min were lost in the ensuing 24 h; only 7.6±2.1% of the CSCs present at 5 min could still be found at 7 days after transplantation and only 2.8±0.5% (i.e., 1,224±230 cells/heart) at 35 days. Thus, even after direct intramyocardial injection, the total number of CSCs that remain in the murine heart is minimal (at 24 h, ~10% of the cells injected; at 35 days, ~1%). This new quantitative method of stem cell detection, which enables measurement of absolute cell number, should be useful to optimize cell-based therapies, not only for CSCs but also for other stem cells and other organs.

There is little doubt that the discovery of ischemic preconditioning (PC) has been one of the fundamental milestones in the field of ischemic biology in the past 20 years. The purpose of this article is to review the pathophysiology and molecular basis of the late phase of myocardial PC. The exploitation of late PC for the development of novel gene therapy strategies aimed at inducing a permanently preconditioned cardiac phenotype (prophylactic cardioprotection) will also be discussed. Deciphering the mechanism of late PC has not only conceptual interest but also a considerable therapeutic implications, since transfer of the genes that underlie late PC would be expected to replicate the salubrious effects of this response of the heart to stress.

BackgroundThere is mounting interest in using c-kit positive human cardiac stem cells (c-kitpos hCSCs) to repair infarcted myocardium in patients with ischemic cardiomyopathy. A recent phase I clinical trial (SCIPIO) has shown that intracoronary infusion of 1 million hCSCs is safe. Higher doses of CSCs may provide superior reparative ability; however, it is unknown if doses >1 million cells are safe. To address this issue, we examined the effects of 20 million hCSCs in pigs.MethodsRight atrial appendage samples were obtained from patients undergoing cardiac surgery. The tissue was processed by an established protocol with eventual immunomagnetic sorting to obtain in vitro expanded hCSCs. A cumulative dose of 20 million cells was given intracoronarily to pigs without stop flow. Safety was assessed by measurement of serial biomarkers (cardiac: troponin I and CK-MB, renal: creatinine and BUN, and hepatic: AST, ALT, and alkaline phosphatase) and echocardiography pre- and post-infusion. hCSC retention 30 days after infusion was quantified by PCR for human genomic DNA. All personnel were blinded as to group assignment.ResultsCompared with vehicle-treated controls (n=5), pigs that received 20 million hCSCs (n=9) showed no significant change in cardiac function or end organ damage (assessed by organ specific biomarkers) that could be attributed to hCSCs (P>0.05 in all cases). No hCSCs could be detected in left ventricular samples 30 days after infusion.ConclusionsIntracoronary infusion of 20 million c-kit positive hCSCs in pigs (equivalent to ~40 million hCSCs in humans) does not cause acute cardiac injury, impairment of cardiac function, or liver and renal injury. These results have immediate translational value and lay the groundwork for using doses of CSCs >1 million in future clinical trials. Further studies are needed to ascertain whether administration of >1 million hCSCs is associated with greater efficacy in patients with ischemic cardiomyopathy.