Summary Background C-kit+ lineage− cardiac stem cells (CSCs) improve postinfarction left ventricular (LV) dysfunction in animals; however, their efficacy in humans is unknown. Methods In February 2009, we began SCIPIO (Stem Cell Infusion in Patients with Ischemic CardiOmyopathy), a Phase I, randomized, open-label trial of CSCs in patients with postinfarction LV dysfunction (ejection fraction [EF] ≤ 40%) who underwent coronary bypass surgery. Autologous CSCs were isolated from the right atrial appendage and re-infused intracoronarily 4 ± 1 months after surgery; controls received no treatment. In Stage A, 9 treated and 4 control patients were consecutively enrolled to assess the feasibility and short-term safety of CSCs. Then, in Stage B, patients were randomized to the treated or control arm in a 2:3 ratio using a block randomization scheme and a block size of five. Primary (safety) and secondary (efficacy) endpoints were assessed at serial times after enrollment. Findings Autologous CSCs were successfully isolated and expanded in 80 out of 81 patients. In 16 treated patients, no CSC-related adverse effects have been observed. LVEF (3D echocardiography) increased from 30.3 ± 1.9% before CSC infusion to 38.5 ± 2.8% at 4 months after infusion, (P=0.001, n=14). This was associated with an improvement in regional wall motion score index (echocardiography) (1.91 ± 0.09 vs. 1.73 ± 0.09, P=0.005), NYHA functional class (2.19 ± 0.16 vs. 1.63 ± 0.16, P=0.003), and quality of life (MLHFQ score, 46.44 ± 5.22 vs. 26.69 ± 4.92, P<0.0001). In contrast, in 7 control patients, none of these variables changed appreciably during the corresponding time-interval. Importantly, the salubrious effects of CSCs were even more pronounced at 1 year (e.g., LVEF increased by 12.3 ± 2.1% vs. pre-CSCs, P=0.0007, n=8), suggesting that CSCs continue to improve LV function beyond the first 4 months. In the 7 treated patients in whom cardiac magnetic resonance (cMR) imaging could be performed, infarct size decreased by 7.8 ± 1.7 g (23.8%) at 4 months (P=0.004) and 9.8 ± 3.5 g (30.3%) at 1 year (P=0.04). Interpretation These initial results in humans are very encouraging, and suggest that infusion of autologous CSCs is effective in improving LV systolic function and reducing infarct size in patients with heart failure.

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. (Circulation. 2010;121:293-305.)Key Words: regeneration Ⅲ progenitor cells Ⅲ myocardial infarction Ⅲ reperfusion Ⅲ stem cells C ell-based therapies have the potential to alleviate left ventricular (LV) dysfunction and remodeling after acute myocardial infarction (MI) in experimental animal models 1 and in humans. 2,3 Among the various cells used, c-kitpositive (c-kit pos ) cardiac progenitor cells (CPCs) are attractive because they normally reside in the heart and presumably are responsible for replenishing the pool of cardiac myocytes and coronary vessels under normal conditions. 4 -6 The practical utility of CPCs is further supported by the fact that these cells can be isolated from small fragments of cardiac tissue and expanded for subsequent autologous administration. 5,6 Clinical Perspective on p 305Transplantation of autologous or syngeneic CPCs has been found to be effective in limiting LV dysfunction and remodeling in rodent models of acute MI, both in the setting of a permanent coronary occlusion 4 -6 and in that of a transient occlusion followed by reperfusion. 7 Clinically, how...

Background-Stromal cell-derived factor-1␣ (SDF-1␣) 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␣-CXCR4 axis in modulating myocardial ischemia/reperfusion injury is unknown. Methods and Results-In mice subjected to ischemic preconditioning, myocardial SDF-1␣ mRNA was found to be increased 3 hours later (PϽ0.05). Myocardial SDF-1␣ and CXCR4 mRNA and protein were found to be expressed in both cardiac myocytes and fibroblasts. SDF-1␣ production increased significantly after 1 or 4 hours of hypoxia and 18 hours of reoxygenation in cultured myocytes (PϽ0.05) but did not change in fibroblast cultures. In isolated myocytes, CXCR4 activation by SDF-1␣ resulted in increased phosphorylation of both ERK 1/2 and AKT and decreased phosphorylation of JNK and p38. Cultured myocytes pretreated with SDF-1␣ 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) (PϽ0.05). This protective effect was blocked by the CXCR4 selective antagonist AMD3100. In vivo, administration of SDF-1␣ before 30 minutes of coronary occlusion followed by 4 hours of reperfusion decreased infarct size (PϽ0.05). The decrease in infarct size with SDF-1␣ administration also was blocked by AMD3100. Conclusions-We conclude that SDF-1␣ 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.

Background Inappropriately sustained inflammation is a hallmark of chronic ischemic heart failure (HF); however, the pathophysiological role of T-lymphocytes is unclear. Methods and Results Permanent coronary ligation was performed in adult C57BL/6 mice. As compared with sham-operated mice, mice with HF (8 w after ligation) exhibited the following features: 1) significant (p<0.05) expansion of circulating CD3+CD8+ cytotoxic and CD3+CD4+ helper (Th) T-lymphocytes, together with increased Th1, Th2, Th17, and regulatory T-cell (Treg) CD4+ subsets; 2) significant expansion of CD8+ and CD4+ T-cells in failing myocardium, with increased Th1, Th2, Th17, and Treg CD4+ subsets, marked reduction of the Th1/Th2 ratio, augmentation of the Th17/Treg ratio, and upregulation of Th2 cytokines; and 3) significantly increased Th1, Th2, Th17 cells, and Tregs, in the spleen and mediastinal lymph nodes, with increased expansion of splenic antigen-experienced effector and memory CD4+ T cells. Antibody-mediated CD4+ T-cell depletion in HF mice (starting 4 w after ligation) reduced cardiac infiltration of CD4+ T-cells and prevented progressive LV dilatation and hypertrophy whereas adoptive transfer of splenic CD4+ T-cells (and, to a lesser extent, cardiac CD3+ T-cells) from donor mice with HF induced long-term LV dysfunction, fibrosis, and hypertrophy in naïve recipient mice. Conclusions CD4+ T-lymphocytes are globally expanded and activated in chronic ischemic HF, with Th2 (vs Th1) and Th17 (vs Treg) predominance in failing hearts, and with expansion of memory T-cells in the spleen. Cardiac and splenic T-cells in HF are primed to induce cardiac injury and remodeling, and retain this memory upon adoptive transfer.

Background-Heme oxygenase-1 (HO-1) is an inducible stress-response protein that imparts antioxidant and antiapoptotic effects. However, its pathophysiological role in cardiac remodeling and chronic heart failure (HF) is unknown. We hypothesized that induction of HO-1 in HF alleviates pathological remodeling. Methods and Results-Adult male nontransgenic and myocyte-restricted HO-1 transgenic mice underwent either sham operation or coronary ligation to induce HF. Four weeks after ligation, nontransgenic HF mice exhibited postinfarction left ventricular (LV) remodeling and dysfunction, hypertrophy, fibrosis, oxidative stress, apoptosis, and reduced capillary density, associated with a 2-fold increase in HO-1 expression in noninfarcted myocardium. Compared with nontransgenic mice, HO-1 transgenic HF mice exhibited significantly (PϽ0.05) improved postinfarction survival (94% versus 57%) and less LV dilatation (end-diastolic volume, 46Ϯ8 versus 85Ϯ32 L), mechanical dysfunction (ejection fraction, 65Ϯ9% versus 49Ϯ16%), hypertrophy (LV/tibia length 4.4Ϯ0.4 versus 5.2Ϯ0.6 mg/mm), interstitial fibrosis (11.2Ϯ3.1% versus 18.5Ϯ3.5%), and oxidative stress (3-fold reduction in tissue malondialdehyde). Moreover, myocyte-specific HO-1 overexpression in HF promoted tissue neovascularization and ameliorated myocardial p53 expression (2-fold reduction) and apoptosis. In isolated mitochondria, mitochondrial permeability transition was inhibited by HO-1 in a carbon monoxide (CO)-dependent manner and was recapitulated by the CO donor tricarbonylchloro(glycinato)ruthenium(II) (CORM-3). HO-1-derived CO also prevented H 2 O 2 -induced cardiomyocyte apoptosis and cell death. Finally, in vivo treatment with CORM-3 alleviated postinfarction LV remodeling, p53 expression, and apoptosis. Conclusions-HO-1 induction in the failing heart is an important cardioprotective adaptation that opposes pathological LV remodeling, and this effect is mediated, at least in part, by CO-dependent inhibition of mitochondrial permeability transition and apoptosis. Augmentation of HO-1 or its product, CO, may represent a novel therapeutic strategy for ameliorating HF.

Rationale Endothelial progenitor cells (EPCs) respond to SDF-1 through receptors CXCR7 and CXCR4. Whether SDF-1 receptors involves in diabetes induced EPCs dysfunction remains unknown. Objective To determine the role of SDF-1 receptors in diabetic EPCs dysfunction. Methods and Results CXCR7 expression, but not CXCR4 was reduced in EPCs from db/db mice, which coincided with impaired tube formation. Knockdown of CXCR7 impaired tube formation of EPCs from normal mice, while up-regulation of CXCR7 rescued angiogenic function of EPCs from db/db mice. In normal EPCs treated with oxidized low-density lipoprotein (ox-LDL) or high glucose (HG) also reduced CXCR7 expression, impaired tube formation and increased oxidative stress and apoptosis. The damaging effects of ox-LDL or HG were markedly reduced by SDF-1 pretreatment in EPCs transduced with CXCR7 lentivirus (CXCR7-EPCs) but not in EPCs transduced with control lentivirus (Null-EPCs). Most importantly, CXCR7-EPCs were superior to Null-EPCs for therapy of ischemic limbs in db/db mice. Mechanistic studies demonstrated that ox-LDL or HG inhibited Akt and GSK-3β phosphorylation, nuclear export of Fyn and nuclear localization of Nrf2, blunting Nrf2 downstream target genes HO-1, NQO-1 and catalase, and inducing an increase in EPC oxidative stress. This destructive cascade was blocked by SDF-1 treatment in CXCR7-EPCs. Furthermore, inhibition of PI3K/Akt prevented SDF-1/CXCR7-mediated Nrf2 activation and blocked angiogenic repair. Moreover, Nrf2 knockdown almost completely abolished the protective effects of SDF-1/CXCR7 on EPC function in vitro and in vivo. Conclusions Elevated expression of CXCR7 enhances EPC resistance to diabetes-induced oxidative damage and improves therapeutic efficacy of EPCs in treating diabetic limb ischemia. The benefits of CXCR7 are mediated predominantly by an Akt/GSK-3β/Fyn pathway via increased activity of Nrf2.

␣1-adrenergic receptors (ARs) play a major role in blood pressure regulation. The three ␣1-AR subtypes (A͞C, B, and D) stimulate contraction of isolated arteries, but it is uncertain how different subtypes contribute to blood pressure regulation in the intact animal. We studied the role of the ␣1A͞C subtype by using gene knockout. ␣1A͞C knockout (KO) mice were viable and overtly normal. The LacZ reporter gene replaced ␣1A͞C coding sequence in the KO, and ␤-galactosidase staining was present in resistance arteries and arterioles, but not in the thoracic aorta or its main branches. By tail cuff manometer and arterial catheter in conscious mice, ␣1A͞C KO mice were hypotensive at rest, with an 8 -12% reduction of blood pressure dependent on ␣1A͞C gene copy number. A61603, an ␣1A͞C-selective agonist, caused a pressor response that was lost in the KO and reduced but significant in heterozygous mice with a single copy of the ␣1A͞C. A subtype-nonselective agonist [phenylephrine (PE)] caused a pressor response in KO mice, but the final arterial pressure was only 85% of wild type. The baroreflex was reset in the KO, and heart rate variability was decreased. After baroreflex blockade with atropine, PE increased blood pressure but did not change heart rate. Cardiac and vascular responses to the ␤-AR agonist isoproterenol were unchanged, and the arterial lumen area was not altered. We conclude that the ␣1A͞C-AR subtype is a vasopressor expressed in resistance arteries and is required for normal arterial blood pressure regulation. ␣1A͞C-selective antagonists might be desirable antihypertensive agents.

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