Venkata Naga Srikanth Garikipati

Embryonic Stem Cell-Derived Exosomes Promote Endogenous Repair Mechanisms and Enhance Cardiac Function Following Myocardial Infarction

RATIONALE Embryonic stem cells (ESCs) hold great promise for cardiac regeneration but are susceptible to various concerns. Recently, salutary effects of stem cells have been connected to exosome secretion. ESCs have the ability to produce exosomes, however, their effect in the context of the heart is unknown. OBJECTIVE Determine the effect of ESC-derived exosome for the repair of ischemic myocardium and whether c-kit(+) cardiac progenitor cells (CPCs) function can be enhanced with ESC exosomes. METHODS AND RESULTS This study demonstrates that mouse ESC-derived exosomes (mES Ex) possess ability to augment function in infarcted hearts. mES Ex enhanced neovascularization, cardiomyocyte survival, and reduced fibrosis post infarction consistent with resurgence of cardiac proliferative response. Importantly, mES Ex augmented CPC survival, proliferation, and cardiac commitment concurrent with increased c-kit(+) CPCs in vivo 8 weeks after in vivo transfer along with formation of bonafide new cardiomyocytes in the ischemic heart. miRNA array revealed significant enrichment of miR290-295 cluster and particularly miR-294 in ESC exosomes. The underlying basis for the beneficial effect of mES Ex was tied to delivery of ESC specific miR-294 to CPCs promoting increased survival, cell cycle progression, and proliferation. CONCLUSIONS mES Ex provide a novel cell-free system that uses the immense regenerative power of ES cells while avoiding the risks associated with direct ES or ES-derived cell transplantation and risk of teratomas. ESC exosomes possess cardiac regeneration ability and modulate both cardiomyocyte and CPC-based repair programs in the heart.

Negative Regulation of miR‐375 by Interleukin‐10 Enhances Bone Marrow‐Derived Progenitor Cell‐Mediated Myocardial Repair and Function After Myocardial Infarction

Poor survival and function of transplanted cells in ischemic and inflamed myocardium likely compromises the functional benefit of stem cell‐based therapies. We have earlier reported that co‐administration of interleukin (IL)−10 and BMPAC enhances cell survival and improves left ventricular (LV) functions after acute myocardial infarction (MI) in mice. We hypothesized that IL‐10 regulates microRNA‐375 (miR‐375) signaling in BMPACs to enhance their survival and function in ischemic myocardium after MI and attenuates left ventricular dysfunction after MI. miR‐375 expression is significantly upregulated in BMPACs upon exposure to inflammatory/hypoxic stimulus and also after MI. IL‐10 knockout mice display significantly elevated miR‐375 levels. We report that ex vivo miR‐375 knockdown in BMPAC before transplantation in the ischemic myocardium after MI significantly improve the survival and retention of transplanted BMPACs and also BMPAC‐mediated post‐infarct repair, neovascularization, and LV functions. Our in vitro studies revealed that knockdown of miR‐375‐enhanced BMPAC proliferation and tube formation and inhibited apoptosis; over expression of miR‐375 in BMPAC had opposite effects. Mechanistically, miR‐375 negatively regulated 3‐phosphoinositide‐dependent protein kinase‐1 (PDK‐1) expression and PDK‐1‐mediated activation of PI3kinase/AKT signaling. Interestingly, BMPAC isolated from IL‐10‐deficient mice showed elevated basal levels of miR‐375 and exhibited functional deficiencies, which were partly rescued by miR‐375 knockdown, enhancing BMPAC function in vitro and in vivo. Taken together, our studies suggest that miR‐375 is negatively associated with BMPAC function and survival and IL‐10‐mediated repression of miR‐375 enhances BMPAC survival and function. Stem Cells 2015;33:3519–3529

Sirtuin‐6 deficiency exacerbates diabetes‐induced impairment of wound healing

Delayed wound healing is one of the major complications in diabetes and is characterized by chronic proinflammatory response, and abnormalities in angiogenesis and collagen deposition. Sirtuin family proteins regulate numerous pathophysiological processes, including those involved in promotion of longevity, DNA repair, glycolysis and inflammation. However, the role of sirtuin 6 (SIRT6), a NAD+‐dependent nuclear deacetylase, in wound healing specifically under diabetic condition remains unclear. To analyse the role of SIRT6 in cutaneous wound healing, paired 6‐mm stented wound was created in diabetic db/db mice and injected siRNA against SIRT6 in the wound margins (transfection agent alone and nonsense siRNA served as controls). Wound time to closure was assessed by digital planimetry, and wounds were harvested for histology, immunohistochemistry and Western blotting. SIRT6‐siRNA‐treated diabetic wound showed impaired healing, which was associated with reduced capillary density (CD31‐staining vessels) when compared to control treatment. Interestingly, SIRT6 deficiency decreased vascular endothelial growth factor expression and proliferation markers in the wounds. Furthermore, SIRT6 ablation in diabetic wound promotes nuclear factor‐κB (NF‐κB) activation resulting in increased expression of proinflammatory markers (intercellular adhesion molecule‐1, vascular cell adhesion molecule‐1, tumor necrosis factor‐α and interleukin‐1β) and increased oxidative stress. Collectively, our findings demonstrate that loss of SIRT6 in cutaneous wound aggravates proinflammatory response by increasing NF‐κB activation, oxidative stress and decrease in angiogenesis in the diabetic mice. Based on these findings, we speculate that the activation of SIRT6 signalling might be a potential therapeutic approach for promoting wound healing in diabetics.

Mitochondrial dysfunction and its impact on diabetic heart.

Mitochondrial dysfunction and associated oxidative stress are strongly linked to cardiovascular, neurodegenerative, and age associated disorders. More specifically cardiovascular diseases are common in patients with diabetes and significant contributor to the high mortality rates associated with diabetes. Studies have shown that the heart failure risk is increased in diabetic patients even after adjusting for coronary artery disease and hypertension. Although the actual basis of the increased heart failure risk is multifactorial, increasing evidences suggest that imbalances in mitochondrial function and associated oxidative stress play an important role in this process. This review summarizes these abnormalities in mitochondrial function and discusses potential underlying mechanisms. This article is part of a Special Issue entitled: Oxidative Stress and Mitochondrial Quality in Diabetes/Obesity and Critical Illness Spectrum of Diseases - edited by P. Hemachandra Reddy.

Therapeutic inhibition of miR-375 attenuates post-myocardial infarction inflammatory response and left ventricular dysfunction via PDK-1-AKT signalling axis

Aims Increased miR-375 levels has been implicated in rodent models of myocardial infarction (MI) and with patients with heart failure. However, no prior study had established a therapeutic role of miR-375 in ischemic myocardium. Therefore, we assessed whether inhibition of MI-induced miR-375 by LNA anti-miR-375 can improve recovery after acute MI. Methods and results Ten weeks old mice were treated with either control or LNA anti miR-375 after induction of MI by LAD ligation. The inflammatory response, cardiomyocyte apoptosis, capillary density and left ventricular (LV) functional, and structural remodelling changes were evaluated. Anti-miR-375 therapy significantly decreased inflammatory response and reduced cardiomyocyte apoptosis in the ischemic myocardium and significantly improved LV function and neovascularization and reduced infarct size. Repression of miR-375 led to the activation of 3-phosphoinositide-dependent protein kinase 1 (PDK-1) and increased AKT phosphorylation on Thr-308 in experimental hearts. In corroboration with our in vivo findings, our in vitro studies demonstrated that knockdown of miR-375 in macrophages modulated their phenotype, enhanced PDK-1 levels, and reduced pro-inflammatory cytokines expression following LPS challenge. Further, miR-375 levels were elevated in failing human heart tissue. Conclusion Taken together, our studies demonstrate that anti-miR-375 therapy reduced inflammatory response, decreased cardiomyocyte death, improved LV function, and enhanced angiogenesis by targeting multiple cell types mediated at least in part through PDK-1/AKT signalling mechanisms.

Restoration of Hydrogen Sulfide Production in Diabetic Mice Improves Reparative Function of Bone Marrow Cells.

Background: Bone marrow cell (BMC)–based treatment for critical limb ischemia in diabetic patients yielded a modest therapeutic effect resulting from cell dysfunction. Therefore, approaches that improve diabetic stem/progenitor cell functions may provide therapeutic benefits. Here, we tested the hypothesis that restoration of hydrogen sulfide (H2S) production in diabetic BMCs improves their reparative capacities. Methods: Mouse BMCs were isolated by density-gradient centrifugation. Unilateral hind limb ischemia was conducted in 12- to 14-week-old db/+ and db/db mice by ligation of the left femoral artery. The H2S level was measured by either gas chromatography or staining with florescent dye sulfidefluor 7 AM. Results: Both H2S production and cystathionine &ggr;-lyase (CSE), an H2S enzyme, levels were significantly decreased in BMCs from diabetic db/db mice. Administration of H2S donor diallyl trisulfide (DATS) or overexpression of CSE restored H2S production and enhanced cell survival and migratory capacity in high glucose (HG)–treated BMCs. Immediately after hind limb ischemia surgery, the db/+ and db/db mice were administered DATS orally and/or given a local intramuscular injection of green fluorescent protein–labeled BMCs or red fluorescent protein–CSE–overexpressing BMCs (CSE-BMCs). Mice with hind limb ischemia were divided into 6 groups: db/+, db/db, db/db+BMCs, db/db+DATS, db/db+DATS+BMCs, and db/db+CSE-BMCs. DATS and CSE overexpression greatly enhanced diabetic BMC retention in ischemic hind limbs followed by improved blood perfusion, capillary/arteriole density, skeletal muscle architecture, and cell survival and decreased perivascular CD68+ cell infiltration in the ischemic hind limbs of diabetic mice. It is interesting to note that DATS or CSE overexpression rescued high glucose–impaired migration, tube formation, and survival of BMCs or mature human cardiac microvascular endothelial cells. Moreover, DATS restored nitric oxide production and decreased endothelial nitric oxide synthase phosphorylation at threonine 495 levels in human cardiac microvascular endothelial cells and improved BMC angiogenic activity under high glucose condition. Last, silencing CSE by siRNA significantly increased endothelial nitric oxide synthase phosphorylation at threonine 495 levels in human cardiac microvascular endothelial cells. Conclusions: Decreased CSE-mediated H2S bioavailability is an underlying source of BMC dysfunction in diabetes mellitus. Our data indicate that H2S and overexpression of CSE in diabetic BMCs may rescue their dysfunction and open novel avenues for cell-based therapeutics of critical limb ischemia in diabetic patients.

Interleukin-10 inhibits chronic angiotensin II-induced pathological autophagy

BACKGROUND Although autophagy is an essential cellular salvage process to maintain cellular homeostasis, pathological autophagy can lead to cardiac abnormalities and ultimately heart failure. Therefore, a tight regulation of autophagic process would be important to treat chronic heart failure. Previously, we have shown that IL-10 strongly inhibited pressure overload-induced hypertrophy and heart failure, but role of IL-10 in regulation of pathological autophagy is unknown. Here we tested the hypothesis that IL-10 inhibits angiotensin II-induced pathological autophagy and this process, in part, leads to improve cardiac function. METHODS AND RESULTS Chronic Ang II strongly induced mortality, cardiac dysfunction in IL-10 Knockout mice. IL-10 deletion exaggerated pathological autophagy in response to Ang II treatment. In isolated cardiac myocytes, IL-10 attenuated Ang II-induced pathological autophagy and activated Akt/mTORC1 signaling. Pharmacological or molecular inhibition of Akt and mTORC1 signaling attenuated IL-10 effects on Ang II-induced pathological autophagy. Furthermore, lysosomal inhibition in autophagic flux experiments further confirmed that IL-10 inhibits pathological autophagy via mTORC1 signaling. CONCLUSION Our data demonstrate a novel role of IL-10 in regulation of pathological autophagy; thus can act as a potential therapeutic molecule for treatment of chronic heart disease.

IL-10 Accelerates Re-Endothelialization and Inhibits Post-Injury Intimal Hyperplasia following Carotid Artery Denudation

The role of inflammation on atherosclerosis and restenosis is well established. Restenosis is thought to be a complex response to injury, which includes early thrombus formation, acute inflammation and neo-intimal growth. Inflammatory cells are likely contributors in the host response to vascular injury, via cytokines and chemokines secretion, including TNF-alpha (TNF). We have previously shown that IL-10 inhibits TNF and other inflammatory mediators produced in response to cardiovascular injuries. The specific effect of IL-10 on endothelial cell (ECs) biology is not well elucidated. Here we report that in a mouse model of carotid denudation, IL-10 knock-out mice (IL-10KO) displayed significantly delayed Re-endothelialization and enhanced neo-intimal growth compared to their WT counterparts. Exogenous recombinant IL-10 treatment dramatically blunted the neo-intimal thickening while significantly accelerating the recovery of the injured endothelium in WT mice. In vitro, IL-10 inhibited negative effects of TNF on ECs proliferation, ECs cell cycle, ECs-monocyte adhesion and ECs apoptosis. Furthermore, IL-10 treatment attenuated TNF-induced smooth muscle cells proliferation. Our data suggest that IL-10 differentially regulate endothelial and vascular smooth cells proliferation and function and thus inhibits neo-intimal hyperplasia. Thus, these results may provide insights necessary to develop new therapeutic strategies to limit vascular restenosis during percutaneous coronary intervention (PCI) in the clinics.

Interleukin 10 Inhibits Bone Marrow Fibroblast Progenitor Cell-Mediated Cardiac Fibrosis in Pressure Overloaded Myocardium

Background: Activated fibroblasts (myofibroblasts) play a critical role in cardiac fibrosis; however, their origin in the diseased heart remains unclear, warranting further investigation. Recent studies suggest the contribution of bone marrow fibroblast progenitor cells (BM-FPCs) in pressure overload–induced cardiac fibrosis. We have previously shown that interleukin-10 (IL10) suppresses pressure overload–induced cardiac fibrosis; however, the role of IL10 in inhibition of BM-FPC–mediated cardiac fibrosis is not known. We hypothesized that IL10 inhibits pressure overload–induced homing of BM-FPCs to the heart and their transdifferentiation to myofibroblasts and thus attenuates cardiac fibrosis. Methods: Pressure overload was induced in wild-type (WT) and IL10 knockout (IL10KO) mice by transverse aortic constriction. To determine the bone marrow origin, chimeric mice were created with enhanced green fluorescent protein WT mice marrow to the IL10KO mice. For mechanistic studies, FPCs were isolated from mouse bone marrow. Results: Pressure overload enhanced BM-FPC mobilization and homing in IL10KO mice compared with WT mice. Furthermore, WT bone marrow (from enhanced green fluorescent protein mice) transplantation in bone marrow–depleted IL10KO mice (IL10KO chimeric mice) reduced transverse aortic constriction–induced BM-FPC mobilization compared with IL10KO mice. Green fluorescent protein costaining with &agr;-smooth muscle actin or collagen 1&agr; in left ventricular tissue sections of IL10KO chimeric mice suggests that myofibroblasts were derived from bone marrow after transverse aortic constriction. Finally, WT bone marrow transplantation in IL10KO mice inhibited transverse aortic constriction–induced cardiac fibrosis and improved heart function. At the molecular level, IL10 treatment significantly inhibited transforming growth factor-&bgr;–induced transdifferentiation and fibrotic signaling in WT BM-FPCs in vitro. Furthermore, fibrosis-associated microRNA (miRNA) expression was highly upregulated in IL10KO-FPCs compared with WT-FPCs. Polymerase chain reaction–based selective miRNA analysis revealed that transforming growth factor-&bgr;–induced enhanced expression of fibrosis-associated miRNAs (miRNA-21, -145, and -208) was significantly inhibited by IL10. Restoration of miRNA-21 levels suppressed the IL10 effects on transforming growth factor-&bgr;–induced fibrotic signaling in BM-FPCs. Conclusions: Our findings suggest that IL10 inhibits BM-FPC homing and transdifferentiation to myofibroblasts in pressure-overloaded myocardium. Mechanistically, we show for the first time that IL10 suppresses Smad–miRNA-21–mediated activation of BM-FPCs and thus modulates cardiac fibrosis.

Tiny Shuttles for Information Transfer: Exosomes in Cardiac Health and Disease

Intercellular communication mediated by exosomes, nano-sized extracellular vesicles, is crucial for preserving vascular integrity and in the development of cardiovascular and other diseases. As natural carriers of signal molecules, exosomes released from sources such as blood cells, endothelial cells, immune cells, smooth muscle cells, etc., can modify a multitude of cellular bioactivities. They do so by shuttling lipids, proteins, and nucleic acids between donor and recipient cells while circulating in body fluids and in the extracellular space. A recent surge of interest in the field of exosomal biology is in part due to the recognition that the molecules they carry can act as facilitators of both pathogenesis but can also initiate protective and rescue signaling. This mini-review describes current knowledge on exosome function in health and disease including cardiovascular disease.