The identification of cardiac progenitor cells in mammals raises the possibility that the human heart contains a population of stem cells capable of generating cardiomyocytes and coronary vessels. The characterization of human cardiac stem cells (hCSCs) would have important clinical implications for the management of the failing heart. We have established the conditions for the isolation and expansion of c-kit-positive hCSCs from small samples of myocardium. Additionally, we have tested whether these cells have the ability to form functionally competent human myocardium after infarction in immunocompromised animals. Here, we report the identification in vitro of a class of human c-kit-positive cardiac cells that possess the fundamental properties of stem cells: they are self-renewing, clonogenic, and multipotent. hCSCs differentiate predominantly into cardiomyocytes and, to a lesser extent, into smooth muscle cells and endothelial cells. When locally injected in the infarcted myocardium of immunodeficient mice and immunosuppressed rats, hCSCs generate a chimeric heart, which contains human myocardium composed of myocytes, coronary resistance arterioles, and capillaries. The human myocardium is structurally and functionally integrated with the rodent myocardium and contributes to the performance of the infarcted heart. Differentiated human cardiac cells possess only one set of human sex chromosomes excluding cell fusion. The lack of cell fusion was confirmed by the Cre-lox strategy. Thus, hCSCs can be isolated and expanded in vitro for subsequent autologous regeneration of dead myocardium in patients affected by heart failure of ischemic and nonischemic origin.generation of human myocardium ͉ progenitor cells ͉ stem cell niches

Abstract-Diabetes leads to a decompensated myopathy, but the etiology of the cardiac disease is poorly understood.Oxidative stress is enhanced with diabetes and oxygen toxicity may alter cardiac progenitor cell (CPC) function resulting in defects in CPC growth and myocyte formation, which may favor premature myocardial aging and heart failure. We report that in a model of insulin-dependent diabetes mellitus, the generation of reactive oxygen species (ROS) leads to telomeric shortening, expression of the senescent associated proteins p53 and p16 INK4a , and apoptosis of CPCs, impairing the growth reserve of the heart. However, ablation of the p66 shc gene prevents these negative adaptations of the CPC compartment, interfering with the acquisition of the heart senescent phenotype and the development of heart failure with diabetes. ROS elicit 3 cellular reactions: low levels activate cell growth, intermediate quantities trigger cell apoptosis, and high amounts initiate cell necrosis. CPC replication predominates in diabetic p66shcϪ/Ϫ , whereas CPC apoptosis and myocyte apoptosis and necrosis prevail in diabetic wild type. Expansion of CPCs and developing myocytes preserves cardiac function in diabetic p66shcϪ/Ϫ , suggesting that intact CPCs can effectively counteract the impact of uncontrolled diabetes on the heart. The recognition that p66shc conditions the destiny of CPCs raises the possibility that diabetic cardiomyopathy is a stem cell disease in which abnormalities in CPCs define the life and death of the heart. Together, these data point to a genetic link between diabetes and ROS, on the one hand, and CPC survival and growth, on the other. Key Words: cardiac stem cells Ⅲ myocyte regeneration Ⅲ replicative senescence Ⅲ telomeric shortening D eath of cardiac cells with chronic loss of myocytes and vascular structures has been proposed as the underlying cause of the anatomical and functional alterations of the diabetic heart. 1 However, myocyte death and defects in the mechanical behavior, regulatory proteins, and Ca 2ϩ cycling of myocytes with diabetes have left unanswered the question of whether these variables play a primary role in the onset of the myopathy or represent secondary events related to the progression of the cardiac disease. Similar abnormalities occur with myocyte hypertrophy associated with ischemic and nonischemic cardiomyopathy, and myocyte death is commonly found in the failing heart. Accumulating evidence supports the notion that the heart possesses a compartment of multipotent progenitor cells (CPCs) that differentiate into myocytes, endothelial cells, and smooth muscle cells in vitro 2-4 and in vivo. 2 The heart constantly renews itself and an imbalance between cell death and regeneration may be present with diabetes and could be mediated by defects in growth and survival of CPCs.Hyperglycemia leads to enzymatic O-glycosylation of proteins, including the transcription factor p53, whose activation upregulates the local renin-angiotensin system and the synthesis of angiotensin II (Ang II)...

Coronary artery disease is the most common cause of cardiac failure in the Western world, and to date there is no alternative to bypass surgery for severe coronary atherosclerosis. We report that c-kitpositive cardiac progenitor cells (CPCs) activated with insulin-like growth factor 1 and hepatocyte growth factor before their injection in proximity of the site of occlusion of the left coronary artery in rats, engrafted within the host myocardium forming temporary niches. Subsequently, CPCs divided and differentiated into endothelial cells and smooth muscle cells and, to a lesser extent, into cardiomyocytes. The acquisition of vascular lineages appeared to be mediated by the up-regulation of hypoxia-inducible factor 1␣, which promoted the synthesis and secretion of stromal-derived factor 1 from hypoxic coronary vessels. Stromal-derived factor 1 was critical in the conversion of CPCs to the vascular fate. CPCs formed conductive and intermediate-sized coronary arteries together with resistance arterioles and capillaries. The new vessels were connected with the primary coronary circulation, and this increase in vascularization more than doubled myocardial blood flow in the infarcted myocardium. This beneficial effect, together with myocardial regeneration attenuated postinfarction dilated myopathy, reduced infarct size and improved function. In conclusion, locally delivered activated CPCs generate de novo coronary vasculature and may be implemented clinically for restoration of blood supply to the ischemic myocardium.coronary blood flow ͉ infarct size ͉ myocardial regeneration ͉ stem cells ͉ vasculogenesis

Marijuana is one of the most commonly used and abused drugs. Δ-9-tetrahydrocannabinol (Δ-9-THC), the primary psychoactive component in marijuana, is FDA-approved to ameliorate AIDS-associated wasting. Because cannabinoid receptors are expressed on cells of the immune system, it is possible that chronic Δ-9-THC use may impact HIV disease progression. Until recently, longitudinal, controlled, systems-approach studies on the effects of cannabinoids on disease progression were lacking. Data from our controlled studies in non-human primates show chronic Δ-9-THC administration prior to and during simian immunodeficiency virus (SIV) infection ameliorates disease progression, attenuates viral load and tissue inflammation, significantly reducing morbidity and mortality of SIV-infected macaques. Identification of possible mechanisms responsible for this modulation of disease progression is complicated due to the multiplicity of cannabinoid-mediated effects, tissue specific responses to the viral infection, multiple cellular mechanisms involved in inflammatory responses, coordinated neuroendocrine and localized responses to infection, and kinetics of viral replication. Emerging results from our studies reveal that the overall mechanisms mediating the protective effects of cannabinoids involve novel epigenomic regulatory mechanisms in need of systematic investigation. Here, we review the evidence supporting an immunomodulatory role for cannabinoids and its impact to disease progression with focus on HIV/SIV infection.

These data suggest negative modulation of insulin signaling coupled with enhanced Ub-proteasome system activity may be central mechanisms underlying chronic binge alcohol-induced accentuation of SIV-associated muscle wasting.

Chronic alcohol abuse is associated with skeletal muscle myopathy. Previously, we demonstrated that chronic binge alcohol (CBA) consumption by rhesus macaques accentuates skeletal muscle wasting at end-stage of simian immunodeficiency virus (SIV) infection. A proinflammatory, prooxidative milieu and enhanced ubiquitin proteasome activity were identified as possible mechanisms leading to loss of skeletal muscle. The possibility that impaired regenerative capacity, as reflected by the ability of myoblasts derived from satellite cell (SCs) to differentiate into myotubes has not been examined. We hypothesized that the inflammation and oxidative stress in skeletal muscle from CBA animals impair the differentiation capacity of myoblasts to form new myofibers in in vitro assays. We isolated primary myoblasts from the quadriceps femoris of rhesus macaques that were administered CBA or isocaloric sucrose (SUC) for 19 mo. Proliferation and differentiation potential of cultured myoblasts were examined in vitro. Myoblasts from the CBA group had significantly reduced PAX7, MYOD1, MYOG, MYF5, and MEF2C expression. This was associated with decreased myotube formation as evidenced by Jenner-Giemsa staining and myonuclei fusion index. No significant difference in the proliferative ability, cell cycle distribution, or autophagy was detected between myoblasts isolated from CBA and SUC groups. Together, these results reflect marked dysregulation of myoblast myogenic gene expression and myotube formation, which we interpret as evidence of impaired skeletal muscle regenerative capacity in CBA-administered macaques. The contribution of this mechanism to alcoholic myopathy warrants further investigation.

Background Chronic binge alcohol (CBA) administration exacerbates skeletal muscle (SKM) wasting at the terminal stage of simian immunodeficiency virus (SIV) infection in rhesus macaques. This is associated with a pro-inflammatory and oxidative milieu which we have previously shown to be associated with a disrupted balance between anabolic and catabolic mechanisms. In this study, we attempted to characterize the SKM gene expression signature in CBA-administered SIV-infected macaques; using the same animals from the previous study. Methods Administration of intragastric alcohol or sucrose to male rhesus macaques began three months prior to SIV infection and continued throughout the duration of study. Gene transcriptomes of SKM excised at necropsy (~10 mo. post-SIV) from healthy naive control (Control), sucrose-administered, SIV-infected (SUC-SIV), and CBA-administered, SIV-infected (CBA-SIV) macaques were evaluated in microarray datasets. The Protein Analysis Through Evolutionary Relationships (PANTHER) classification tool was used to filter differentially regulated genes based on their predicted function into select biological processes relevant to SKM wasting which were: inflammation, extracellular matrix (ECM) remodeling, and metabolism. Results In total, 1124 genes were differentially regulated between SUC-SIV and controls, 2022 genes were differentially expressed between the CBA-SIV and controls and 836 genes were differentially expressed between CBA-SIV and SUC-SIV animals. The relevance of altered gene expression was reflected in the up-regulation of pro-inflammatory CCL-2, CCL-8, CX3CL1, SELE, HP, and TNFRS10A mRNA expression. In addition, ECM remodeling was reflected in the up-regulation of TIMP-1, MMP2 and MMP9 mRNA expression and TGF-β protein expression. In addition, hydroxyproline content and picrosirius staining reflected increased collagen deposition in the CBA-SIV muscle tissue. Conclusions The results of the study demonstrate SKM inflammation as an important underlying mechanism for muscle wasting. In addition, the study provides evidence of SKM fibrotic transformation as a factor in CBA-induced accentuation of SIV-associated muscle wasting.

Persons living with HIV/AIDS (PLWHA) frequently use cannabinoids, either recreationally by smoking marijuana or therapeutically (delta-9-tetrahydrocannabinol; Δ(9)-THC dronabinol). Previously, we demonstrated that chronic Δ(9)-THC administration decreases early mortality in male simian immunodeficiency virus (SIV)-infected macaques. In this study, we sought to examine whether similar protective effects resulted from chronic cannabinoid administration in SIV-infected female rhesus macaques. Clinical and viral parameters were evaluated in eight female rhesus macaques that received either Δ(9)-THC (0.18-0.32 mg/kg, intramuscularly, twice daily) or vehicle (VEH) starting 28 days prior to intravenous inoculation with SIVmac251. SIV disease progression was assessed by changes in body weight, mortality, viral levels in plasma and mucosal sites, and lymphocyte subsets. In contrast to our results in male animals, chronic Δ(9)-THC did not protect SIV-infected female rhesus macaques from early mortality. Markers of SIV disease, including viral load and CD4(+)/CD8(+) ratio, were not altered by Δ(9)-THC compared to control females; however, females that received chronic Δ(9)-THC did not gain as much weight as control animals. In addition, Δ(9)-THC administration increased total CXCR4 expression in both peripheral and duodenal CD4(+) and CD8(+) T lymphocytes prior to SIV inoculation. Although protection from early mortality was not evident, chronic Δ(9)-THC did not affect clinical markers of SIV disease progression. The contrasting effects of chronic Δ(9)-THC in males versus females remain to be explained, but highlight the need for further studies to explore the sex-dependent effects of Δ(9)-THC and other cannabinoids on the HIV disease course and their implications for virus transmission.