Chronic binge alcohol consumption alters myogenic gene expression and reduces in vitro myogenic differentiation potential of myoblasts from rhesus macaques.

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.

Chronic binge alcohol administration accentuates expression of pro-fibrotic and inflammatory genes in the skeletal muscle of simian immunodeficiency virus-infected macaques.

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 3 months prior to SIV infection and continued throughout the duration of study. Gene transcriptomes of SKM excised at necropsy (~10 months post-SIV) from healthy na\xEFve control (Control), sucrose-administered, SIV-infected (SUC-SIV), and CBA-administered, SIV-infected (CBA-SIV) macaques were evaluated in microarray data sets. The Protein Analysis Through Evolutionary Relationships 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, 1,124 genes were differentially regulated between SUC-SIV and Controls, 2,022 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, MMP 2, and MMP 9 mRNA expression and transforming growth factor-beta 1 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.

Dysregulation of myelopoiesis by chronic alcohol administration during early SIV infection of rhesus macaques.

BACKGROUND: Chronic alcohol intoxication suppresses immune function and increases osteoporosis risk suggesting bone-tissue cytotoxicity. Human immunodeficiency virus infection leads to similar impairments. This study investigated the effects of chronic alcohol administration during the early stage of simian immunodeficiency virus (SIV) infection on hematopoietic stem and progenitor cells (HSPCs) and their differentiated progeny in the bone marrow and peripheral blood of rhesus macaques. METHODS: Rhesus macaques were administered alcohol or sucrose daily for a period of 3 months prior to intrarectal inoculation with 250 TCID50 of SIVmac251 . Bone marrow aspirates and blood samples were taken prior to and 2 weeks after SIV infection. Bone marrow cells (BMCs) were assessed using flow cytometric phenotyping for upstream HSPCs and for differentiated cells of the monocyte-granulocyte lineages. Likewise, cells were quantitated in peripheral blood. RESULTS: Of the bone marrow HSPCs, only the common lymphoid progenitor (CLP) was altered by alcohol administration pre-SIV (38 ± 9.4/10(6) BMCs vs. 226 ± 64.1/10(6) BMCs, sucrose vs. alcohol). Post-SIV, the frequency of CLPs in the bone marrow of alcohol-administered macaques decreased compared with the sucrose-administered macaques (107 ± 47.6/10(6) BMCs vs. 43 ± 16.3/10(6) BMCs). However, marrow mature cells of the monocyte lineage, specifically macrophages and osteoclast progenitors, were increased by both chronic alcohol administration and SIV infection (287% and 662%, respectively). As expected, mature cells such as granulocytes (polymorphonuclear cells), B cells, and CD4+ T cells in the peripheral blood were decreased by SIV infection (37 to 62% decline from preinfection), but not affected after 3 months of chronic alcohol administration. CONCLUSIONS: Chronic alcohol administration disrupts myelomonocytic development in the bone marrow during the early period of SIV infection promoting macrophage and osteoclast lineages. We predict this shift in CLP:macrophage/osteoclast balance creates an environment that favors bone resorption and immunosuppression.

Disrupted anabolic and catabolic processes may contribute to alcohol-accentuated SAIDS-associated wasting.

BACKGROUND: Alcohol abuse is a comorbid factor in many human immunodeficiency virus (HIV)-infected patients. Previously, we demonstrated that chronic binge alcohol accentuates loss of body mass at terminal stage of simian immunodeficiency virus (SIV) infection. The purpose of this study was to investigate changes in pathways that may contribute to muscle wasting in chronic binge alcohol-fed SIV-infected macaques. METHODS: The impact of chronic binge alcohol during SIV infection on insulin signaling and the ubiquitin (Ub)-proteasome system-regulators of protein synthesis and degradation-was examined in SIV-infected macaques. RESULTS: SIV infection induced an inflammatory and pro-oxidative milieu in skeletal muscle, which was associated with decreased insulin-stimulated phosphatidylinositol 3-kinase (PI-3k) activity and upregulated gene expression of mTOR and atrogin-1, and protein expression of Ub-proteasome system 19S base. Chronic binge alcohol accentuated the skeletal muscle pro-oxidative milieu and 19S base expression. Additionally, chronic binge alcohol increased skeletal muscle protein expression of protein-tyrosine phosphatase 1B (a negative regulator of insulin signaling) and 19S proteasome regulator non-ATPase (Rpn) 6 subunit and Rpn12, and suppressed PI-3K activity. Animals that were alcohol-fed and SIV-infected for >15 months had increased Ub-proteasome system activity. CONCLUSIONS: 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.

Formation of large coronary arteries by cardiac progenitor cells.

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-kit-positive 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 1alpha, 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.

Lymphocyte apoptosis in murine Pneumocystis pneumonia.

BACKGROUND: Apoptosis of lymphocytes is important in the termination of an immune response to infection but has also been shown to have detrimental effects in animal models of systemic infection and sepsis. We sought to characterize lymphocyte apoptosis in an animal model of pneumonia due to Pneumocystis murina, an infection localized to the lungs. METHODS: Control mice and mice depleted of CD4+ lymphocytes were inoculated with Pneumocystis. Apoptosis of lung and spleen lymphocytes was assayed by flow cytometry and PCR assay of apoptotic proteins. RESULTS: In control mice, apoptosis of lung lymphocytes was maximal just after the infection was cleared from lung tissue and then declined. However, in CD4-depleted mice, apoptosis was also upregulated in recruited lymphocytes in spite of progressive infection. In splenic lymphocytes, apoptosis was observed early at 1 week after inoculation and then declined. Apoptosis of lung lymphocytes in control mice was associated with a decrease in mRNA for Bcl-2 and an increase in mRNA for Bim. In CD4-depleted mice, lavaged CD8+ cells did change intracellular Bcl-2 but showed increased mRNA for Bim. CONCLUSION: Apoptosis of both pulmonary and extrapulmonary lymphocytes is part of the normal host response to Pneumocystis but is also triggered in CD4-deficient animals with progressive infection. In normal mice apoptosis of pulmonary lymphocytes may serve to terminate the immune response in lung tissue. Apoptosis of lung lymphocytes takes place via both the intrinsic and extrinsic apoptotic pathways and is associated with changes in both pro- and anti-apoptotic proteins.

Diabetes promotes cardiac stem cell aging and heart failure, which are prevented by deletion of the p66shc gene.

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 p16INK4a, and apoptosis of CPCs, impairing the growth reserve of the heart. However, ablation of the p66shc 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 p66shc-/-, whereas CPC apoptosis and myocyte apoptosis and necrosis prevail in diabetic wild type. Expansion of CPCs and developing myocytes preserves cardiac function in diabetic p66shc-/-, 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.

Chronic Δ9-tetrahydrocannabinol administration may not attenuate simian immunodeficiency virus disease progression in female rhesus macaques.

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.

Modulation of gut-specific mechanisms by chronic δ(9)-tetrahydrocannabinol administration in male rhesus macaques infected with simian immunodeficiency virus: a systems biology analysis.

Our studies have demonstrated that chronic Δ(9)-tetrahydrocannabinol (THC) administration results in a generalized attenuation of viral load and tissue inflammation in simian immunodeficiency virus (SIV)-infected male rhesus macaques. Gut-associated lymphoid tissue is an important site for HIV replication and inflammation that can impact disease progression. We used a systems approach to examine the duodenal immune environment in 4- to 6-year-old male rhesus monkeys inoculated intravenously with SIVMAC251 after 17 months of chronic THC administration (0.18-0.32 mg/kg, intramuscularly, twice daily). Duodenal tissue samples excised from chronic THC- (N=4) and vehicle (VEH)-treated (N=4) subjects at ∼5 months postinoculation showed lower viral load, increased duodenal integrin beta 7(+)(ß7) CD4(+) and CD8(+) central memory T cells, and a significant preferential increase in Th2 cytokine expression. Gene array analysis identified six genes that were differentially expressed in intestinal samples of the THC/SIV animals when compared to those differentially expressed between VEH/SIV and uninfected controls. These genes were identified as having significant participation in (1) apoptosis, (2) cell survival, proliferation, and morphogenesis, and (3) energy and substrate metabolic processes. Additional analysis comparing the duodenal gene expression in THC/SIV vs. VEH/SIV animals identified 93 differentially expressed genes that participate in processes involved in muscle contraction, protein folding, cytoskeleton remodeling, cell adhesion, and cell signaling. Immunohistochemical staining showed attenuated apoptosis in epithelial crypt cells of THC/SIV subjects. Our results indicate that chronic THC administration modulated duodenal T cell populations, favored a pro-Th2 cytokine balance, and decreased intestinal apoptosis. These findings reveal novel mechanisms that may potentially contribute to cannabinoid-mediated disease modulation.

Cannabinoid neuroimmune modulation of SIV disease.

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 on disease progression with focus on HIV/SIV infection.

Chronic Δ-9-tetrahydrocannabinol administration increases lymphocyte CXCR4 expression in rhesus macaques.

Cannabinoids have been reported to produce various immunomodulatory effects, which could potentially impact the host response to bacterial or viral infection. We have recently demonstrated that chronic Δ-9-tetrahydrocannabinol (THC; 0.32 mg/kg i.m., BID) decreased early mortality in rhesus macaques infected with simian immunodeficiency virus (SIV). However, the possibility that prolonged THC administration affects lymphocyte counts, phenotype, and proliferation indices has not been addressed. We examined expression of proliferative and phenotypic markers in circulating lymphocytes of male young adult rhesus macaques chronically-treated with THC (i.m. twice daily 0.32 mg/kg) for 12 months. Chronic THC administration did not alter lymphocyte subtypes, naïve and memory subsets, proliferation, or apoptosis of T lymphocytes when compared to time-matched vehicle-treated controls. However, chronic THC increased T lymphocyte CXCR4 expression on both CD4+ and CD8+ T lymphocytes compared to control. These results show that chronic THC administration produces changes in T cell phenotype, which can potentially contribute to host immunomodulation to infectious challenges.

Stem cells, phenotypic inversion, and differentiation.

Stem cells possess the potential to cure a myriad of ailments ranging from congenital diseases to illnesses acquired through the physiological process of aging. In the adult, these cells are extremely rare and often difficult to isolate in numbers sufficient to apply to medical treatment. Ex vivo expansion of these cells will be required for most meaningful interventions. The discovery of stem/progenitor cell inversion offers a new avenue for obtaining sufficient numbers of stem cells. Adult progenitor cells are much more common than quiescent stem cells and can be isolated with minimal interventions; therefore, inversion of progenitors to stem cells may become a feasible approach for therapeutic purposes. Stem cells are known to possess few mitochondria, and mitochondrial biogenesis is required for stem cell differentiation. The microtubule cytoskeleton is a major regulator for mitochondrial biogenesis. Investigations in the area of controlling cell differentiation and inducing phenotypic inversion, possibly through manipulation of mitochondrial biogenesis, may contribute to stem cell-based therapies.

Human cardiac stem cells.

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.