Haichang Wang

Rosuvastatin enhances the therapeutic efficacy of adipose-derived mesenchymal stem cells for myocardial infarction via PI3K/Akt and MEK/ERK pathways

The poor viability of transplanted stem cells hampers their therapeutic efficacy for treatment of myocardial infarction. The aim of this study was to investigate whether rosuvastatin improved survival of adipose-derived mesenchymal stem cells (AD-MSCs) after transplantation into infarcted hearts. AD-MSCs isolated from Tg(Fluc-egfp) mice which constitutively express both firefly luciferase (Fluc) and enhanced green fluorescent protein were transplanted into infarcted hearts with or without rosuvastatin administration. Longitudinal in vivo bioluminescence imaging and histological staining revealed that rosuvastatin enhanced the survival of engrafted AD-MSCs. Furthermore, combined therapy of AD-MSC and rosuvastatin reduced fibrosis, decreased cardiomyocyte apoptosis, and preserved heart function. AD-MSCs were then subjected to hypoxia and serum deprivation injury in vitro to mimic the ischemic environment. Rosuvastatin (10−6 mmol/L) enhanced the viability and paracrine effect of AD-MSCs, and decreased their apoptotic rate. Western blotting revealed that rosuvastatin supplementation increased Akt and ERK phosphorylation, which resulted in FoxO3a phosphorylation and nuclear export. In addition, rosuvastatin administration decreased the pro-apoptotic proteins Bim and Bax, and increased the anti-apoptotic proteins Bcl-xL and Bcl-2. Furthermore, these effects were abolished by PI3K inhibitor LY294002 and MEK1/2 inhibitor U0126. This study demonstrates that rosuvastatin may improve the survival of engrafted AD-MSCs at least in part through the PI3K/Akt and MEK/ERK1/2 signaling pathways. Combination therapy with rosuvastatin and AD-MSCs has a synergetic effect on improving myocardial function after infarction.

Tanshinone IIA pretreatment protects myocardium against ischaemia/reperfusion injury through the phosphatidylinositol 3-kinase/Akt-dependent pathway in diabetic rats.

Aim: Diabetes Mellitus (DM) is widely acknowledged to increase the risk of cardiovascular death, which warrants the use of aggressive primary prevention strategies. The aim of the present study was to investigate the pretreatment effects of tanshinone IIA (TSN), a traditional Chinese medicine, on myocardial infarct size, apoptosis, inflammation and cardiac functional recovery in diabetic rats subjected to myocardial ischaemia/reperfusion (I/R).

In vivo imaging and evaluation of different biomatrices for improvement of stem cell survival

Therapeutic effects from injection of stem cells are often hampered by acute donor cell death as well as migration away from damaged areas. This is likely due to the fact that injected cells do not have the physical and biochemical cues for ordered engrafment. Here we evaluate 3 common biomatrices (Matrigel, Collagen I, Purmatrix) that has the potential of providing suitable scaffolds needed to enhance stem cell survival. The longitudinal fate of transplanted stem cells was monitored by reporter imaging techniques. Copyright

Effects and mechanisms of ghrelin on cardiac microvascular endothelial cells in rats

Ghrelin is thought to directly exert a protective effect on the cardiovascular system, specifically by promoting vascular endothelial cell function. Our study demonstrates the ability of ghrelin to promote rat CMEC (cardiac microvascular endothelial cell) proliferation, migration and NO (nitric oxide) secretion. CMECs were isolated from left ventricle of adult male Sprague—Dawley rat by enzyme digestion and maintained in endothelial cell medium. Dil‐ac‐LDL (1,1′‐dioctadecyl‐3,3,3′,3′‐ tetramethylindocarbocyanine‐labelled acetylated low‐density lipoprotein) intake assays were used to identify CMECs. Cells were split into five groups and treated with varying concentrations of ghrelin as follows: one control non‐treated group; three ghrelin dosage groups (1×10−9, 1×10−8, 1×10−7 mol/l) and one ghrelin+PI3K inhibitor group (1×10−7 mol/l ghrelin+20 μmol/l LY294002). After 24 h treatment, cell proliferation capability was measured by MTT [3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl‐2H‐tetrazolium bromide] assay and Western blot for PCNA (proliferating cell nuclear antigen) protein expression. Migration of CMECs was detected by transwell assays, and NO secretion of CMECs was measured via nitrate reduction. Protein expression of AKT and phosphorylated AKT in CMECs was measured by Western blot after exposure to various concentrations of ghrelin and the PI3K inhibitor LY294002. Our results indicate that ghrelin significantly enhanced cell growth at concentrations of 10−8 mol/l (0.271±0.041 compared with 0.199±0.021, P=0.03) and 10−7 mol/l (0.296±0.039 compared with 0.199±0.021, P<0.01). However, addition of the PI3K/AKT inhibitor LY294002 inhibited the ghrelin‐mediated enhancement in cell proliferation (0.227±0.042 compared with 0.199±0.021, P=0.15). At a concentration between 10−8 and 10−7 mol/l, ghrelin caused a significant increase in the number of migrated cells compared with the control group (126±9 compared with 98±7, P=0.02; 142±6 compared with 98±7, P<0.01), whereas no such change could be observed in the presence of 20 μmol/l of the PI3K/Akt inhibitor LY294002 (103±7 compared with 98±7, P=0.32). Ghrelin treatment significantly enhanced NO production in a dose‐dependent fashion compared with the untreated control group [(39.93±2.12) μmol/l compared with (30.27±2.71) μmol/l, P=0.02; (56.80±1.98) μmol/l compared with (30.27±2.71) μmol/l, P<0.01]. However, pretreatment with 20 μmol/l LY294002 inhibited the ghrelin‐stimulated increase in NO secretion [(28.97±1.64) μmol/l compared with (30.27±2.71) μmol/l, P=0.37]. In summary, we have found that ghrelin treatment promotes the proliferation, migration and NO secretion of CMECs through activation of PI3K/AKT signalling pathway.

Myocardial Protective Effect of Extracellular Superoxide Dismutase Gene Modified Bone Marrow Mesenchymal Stromal Cells on Infarcted Mice Hearts

Aim: Extracellular superoxide dismutase (ecSOD) is a unique scavenger of superoxide anions and a promising target of gene therapy for ischemia/reperfusion injury (I/R). However, conventional gene therapies have limitation in effectiveness and efficiency. This study aimed to investigate the protective effects of ecSOD gene modified bone marrow mesenchymal stromal cells (BMSCs) on cardiac function improvement in mice infarcted heart. METHODS & RESULTS: BMSCs were isolated from Fluc+ transgenic mice (Tg FVB[Fluc+]) and transfected by adenovirus combined with human ecSOD gene. ELISA was performed to determine ecSOD protein level. Female syngeneic FVB mice were randomized into 5 groups: (1) Sham group (sham); (2) MI group (MI); (3) MI+BMSCs group (BMSC); (4) MI+BMSCs-vector group (BMSC-vector); (5) MI+ BMSCs-ecSOD group (BMSC-ecSOD). MI was accomplished by ligation of the left anterior descending artery. BMSCs (2x106) were injected into the border zone of infarction. In vivo bioluminescence imaging (BLI) was performed to monitor transplanted BMSCs viability. Echocardiography and histological staining revealed that BMSCs-ecSOD significantly reduced myocardial infarction size and improved cardiac function. Lucigenin chemiluminescence, DHE and TUNEL staining demonstrated that BMSCs-ecSOD delivery reduced ROS level and cell apoptosis both in vivo and in vitro. Western blot assay revealed that ecSOD supplementation increased FoxO3a phosphorylation in cardiomyocytes. Moreover, quantitative real-time PCR showed that pro-apoptotic factors (bim and bax) were decreased while the anti-apoptotic factor mir-21 expression was increased after ecSOD intervention. CONCLUSION: Intra-myocardial transplantation of adenovirus-ecSOD transfected BMSCs could exert potential cardiac protection against MI, which may be partly through reduction of oxidative stress and improvement of BMSCs survival.

Percutaneous intramyocardial delivery of mesenchymal stem cells induces superior improvement in regional left ventricular function compared with bone marrow mononuclear cells in porcine myocardial infarcted heart.

Aim: To investigate the efficacy and feasibility of percutaneous intramyocardial injection of bone marrow mesenchymal stem cells (MSC) and autologous bone marrow-derived mononuclear cells (BMMNC) on cardiac functional improvement in porcine myocardial infarcted hearts. Methods and Results: Acute myocardial infarction (AMI) was induced in 22 minipigs by temporary balloon occlusion of the left anterior descending coronary artery for 60min.Two weeks post AMI, BMMNC (n = 7, 245 ± 98×106), MSC (n = 8, 56 ± 17×106), or phosphate buffered saline (PBS; n = 7) were injected intramyocardially. Cardiac function and myocardial perfusion were analyzed by echocardiography and gated single-photon emission computed tomography/computed tomography (SPECT/CT) at 1 week before AMI and 2 and 10 weeks after AMI. Cell engraftment, proliferation, vascular density, and cardiac fibrosis were evaluated by histology analysis. In all groups, the echocardiography revealed no significant change in the left ventricular ejection fraction (LVEF), left ventricular end-systolic volume (LVESV), or left ventricular end-diastolic volume (LVEDV) at 10 weeks after AMI compared with those at 2 weeks after AMI. However, the wall motion score index (WMSI) and left ventricular systolic wall thickening (WT%) were significantly improved at 10 weeks compared with those at 2 weeks after AMI in the MSC group (WMSI 1.55 ± 0.06 vs. 1.87 ± 0.10, WT 33.4 ± 2.3% vs.24.8 ± 2.7%,p < 0.05) but not in the BMMNC group. In addition, myocardial perfusion quantified by SPECT/CT was improved in both the MSC and BMMNC groups, whereas the MSC group showed a superior improvement in vascular density and collagen volume fraction (p < 0.05). Conclusion: This preclinically relevant study suggests that when delivered by percutaneous (transcatheter) intramyocardial injection, MSC might be more effective than BMMNC to improve ischemia and reperfusion after AMI.

Effect of autologous bone marrow mononuclear cells transplantation in diabetic patients with ST-segment elevation myocardial infarction

BACKGROUNDnTo investigate the efficacy and proposed mechanism of bone marrow mononuclear cells (BMMNCs) transplantation for diabetic and non-diabetic patients with ST-segment elevation myocardial infarction (STEMI).nnnMETHODSnOne hundred and sixteen patients with STEMI who had successfully undergone percutaneous coronary intervention (PCI) were divided into a diabetic group (n=51) and non-diabetic group (n=65). All of the patients received intracoronary injection of BMMNCs.nnnRESULTSnDiabetes down-regulated IGF-1, IGFBP-5, VEGF, SDF-1, IL-6, IL-1α and TNF-α expression and affected the expression of Bmi-1, Gfi1, Tel and Hox-B4 which could prevent premature senescence and maintain the self-renewal capacity of stem cells. Event-free survival rates were not statistically different between the diabetic and non-diabetic group (80% vs. 72.5%, p=0.382). LV ejection fraction (LVEF) and wall motion score index (WMSI) were evaluated by echocardiography and found to be significantly improved in the non-diabetic group compared to the diabetic group over the 4-year follow-up period. Improved myocardial perfusion and reduced infarct size in the non-diabetic group compared to the diabetic group was verified using single-photon emission computed tomographic (SPECT) imaging. The non-diabetic group also had reduced anginal symptoms as assessed by changes in their Seattle Angina Questionnaire scores and Canadian Cardiovascular Society (CCS) Functional Angina classification. An improvement of 6-minute walk distance (6MWD) was also noted to be higher in the non-diabetic group during the follow-up period.nnnCONCLUSIONnThis study indicates that the beneficial effect of BMMNCs transplantation for STEMI is less pronounced in diabetic patients. The mechanism is associated with decreased BMMNCs function in diabetic patients.

Multimodality Imaging Evaluation of Functional and Clinical Benefits of Percutaneous Coronary Intervention in Patients with Chronic Total Occlusion Lesion

Aims: To determine the effects of percutaneous coronary intervention (PCI) on cardiac perfusion, cardiac function, and quality of life in patients with chronic total occlusion (CTO) lesion in left anterior descending (LAD) coronary artery. Methods and Results: Patients (n=99) with CTO lesion in the LAD coronary artery who had successfully undergone PCI were divided into three groups based on the SPECT/CTCA fusion imaging: (a) no severe cardiac perfusion defects (n=9); (b) reversible cardiac perfusion defects (n=40); or (c) fixed cardiac perfusion defects (n=50). No statistical difference of perfusion abnormality was observed at 6 months and 1 year after PCI in group (a). In group (b), SPECT/CTCA fusion imaging demonstrated that cardiac perfusion abnormality was significantly decreased 6 month and 1 year after PCI. Left ventricular ejection fraction (LVEF) increased significantly at 6 months and 1 year follow up. Quality of life improved at 6 months and 1 year after PCI procedure. Moreover, patients in group (c) also benefited from PCI therapy: a decrease in cardiac perfusion abnormality, an increase in LVEF, and an improvement in quality of life. PCI of coronary arteries in addition to LAD did not significantly affect cardiac function and quality of life improvement in each group. Conclusions: PCI exerts functional and clinical benefits in patients with CTO lesion in LAD coronary artery, particularly in patients with reversible cardiac perfusion defects. SPECT/CTCA fusion imaging may serve as a useful tool to evaluate the outcomes of patients with CTO lesion in LAD coronary artery.

Can insulin resistance be reversed by insulin therapy

Insulin resistance (IR) is a state of decreased tissue sensitivity to insulin, which commonly exists in patients with metabolic syndrome and diabetes, and leads to compensatory hyperinsulinemia to maintain normoglycemia. It is characterized by pathway-specific inhibition of the PI3K/Akt signaling, which concerns the positive actions of insulin including glucose and lipid metabolism, while other pathways including the Ras/MAPK pathway, which accounts for the negative actions of insulin such as stimulation of smooth muscle proliferation and secretion of endothelin-1, stay unaffected. Thus it was concerned that insulin therapy may exacerbate the negative effects of insulin in IR states. However, treatment of diabetes with insulin in clinical practice showed uniformly beneficial rather than harmful results. So we hypothesize that insulin therapy may itself reverse insulin resistance, thus avoiding magnification of the MAPK pathway-related deleterious effects. The mechanisms may include the recently revealed anti-inflammatory effects of insulin as well as its conventional glucose and free fatty acids lowering effects, and possibly may also include changes in body fat distribution and plasma adiponectin level. Whether there are direct mechanisms that insulin therapy modulates insulin sensitivity remains to be investigated.

GSK-3β inhibitor modulates TLR2/NF-κB signaling following myocardial ischemia-reperfusion

ObjectiveThe present study defines the expression of Toll-like Receptor 2 (TLR2), and the modulatory role of Glycogen synthase kinase (GSK)-3β inhibitor on TLR2/Nuclear Factor-kappa B (NF-κB) signaling following myocardial ischemia-reperfusion (MI-R) injury in rats.MethodsReal-time reverse transcriptase-polymerase chain reaction (RT-PCR) and immunohistochemistry (IHC) were used to analyze the presence and quantity of TLR2 mRNA and protein. Tumor necrosis factor (TNF)-α mRNA and interleukin-6 (IL-6) mRNA were analyzed by RT-PCR. The activation of NF-κB was detected by Western Blot and the myocardial infarct size by Evans blue-TTC staining.ResultsFollowing 30xa0min of myocardial ischemia, a significant up-regulation of TLR2 mRNA was revealed by RT-PCR from 1 to 24xa0h post reperfusion. IHC demonstrated high protein expression levels of TLR2. Administration of the GSK-3β inhibitor 4-benzyl-2-methyl-1, 2, 4-thiadiazolidine-3, 5-dione (TDZD-8) 5xa0min prior to reperfusion following 1xa0h reperfusion down-regulated mRNA levels of TLR2 and downstream proinflammatory cytokines (Pxa0<xa00.05 vs. MI-R), decreased the activity of NF-κB and the size of the myocardial infarct (Pxa0<xa00.05 vs. MI-R).ConclusionOur results demonstrate that TLR2 and its signaling components are activated by MI-R. TDZD-8 administration attenuates TLR2/NF-κB signaling, suggesting a possible mechanism whereby GSK-3β inhibition improves the outcome of MI-R.