Dongjuan Wang

Glucagon-like peptide-1 protects against cardiac microvascular injury in diabetes via a cAMP/PKA/Rho-dependent mechanism.

Impaired cardiac microvascular function contributes to cardiovascular complications in diabetes. Glucagon-like peptide-1 (GLP-1) exhibits potential cardioprotective properties in addition to its glucose-lowering effect. This study was designed to evaluate the impact of GLP-1 on cardiac microvascular injury in diabetes and the underlying mechanism involved. Experimental diabetes was induced using streptozotocin in rats. Cohorts of diabetic rats received a 12-week treatment of vildagliptin (dipeptidyl peptidase-4 inhibitor) or exenatide (GLP-1 analog). Experimental diabetes attenuated cardiac function, glucose uptake, and microvascular barrier function, which were significantly improved by vildagliptin or exenatide treatment. Cardiac microvascular endothelial cells (CMECs) were isolated and cultured in normal or high glucose medium with or without GLP-1. GLP-1 decreased high-glucose–induced reactive oxygen species production and apoptotic index, as well as the levels of NADPH oxidase such as p47phox and gp91phox. Furthermore, cAMP/PKA (cAMP-dependent protein kinase activity) was increased and Rho-expression was decreased in high-glucose–induced CMECs after GLP-1 treatment. In conclusion, GLP-1 could protect the cardiac microvessels against oxidative stress, apoptosis, and the resultant microvascular barrier dysfunction in diabetes, which may contribute to the improvement of cardiac function and cardiac glucose metabolism in diabetes. The protective effects of GLP-1 are dependent on downstream inhibition of Rho through a cAMP/PKA-mediated pathway.

Effects of ghrelin on homocysteine-induced dysfunction and inflammatory response in rat cardiac microvascular endothelial cells.

Ghrelin is a well‐characterized hormone that has protective effects on endothelial cells. Elevated HCY (homocysteine) can be a cardiovascular risk factor, but it is not known whether ghrelin can inhibit HCY‐induced dysfunction and inflammatory response in rat CMECs (cardiac microvascular endothelial cells). We found that HCY treatment for 24 h inhibited proliferation and NO (nitric oxide) secretion, but with increased cell apoptosis and secretion of cytokines in CMECs. In contrast, ghrelin pretreatment significantly improved proliferation and NO secretion, and inhibited cell apoptosis and secretion of cytokines in HCY‐induced CMECs. In addition, Western blot assay showed that NF‐κB (nuclear factor κB) and cleaved‐caspase 3 expression were elevated, and PCNA (proliferating cell nuclear antigen) and eNOS (endothelial nitric oxide synthase) expression were decreased after treatment with HCY, which was significantly reversed by pretreatment with ghrelin. The data suggest that ghrelin inhibits HCY‐induced CMEC dysfunction and inflammatory response, probably mediated by inhibition of NF‐κB activation.

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.

Apelin protects sarcoplasmic reticulum function and cardiac performance in ischaemia-reperfusion by attenuating oxidation of sarcoplasmic reticulum Ca2+-ATPase and ryanodine receptor.

AIMS Apelin, an endogenous cytokine, has a number of biological effects on the cardiovascular system, including a cardioprotective effect and calcium modulation. Because the intracellular calcium abnormality is considered to play an important role in cardiac dysfunction induced by ischaemia-reperfusion (I/R), the aim of this study was to examine the effects of apelin-13 on I/R-induced changes in cardiac performance and sarcoplasmic reticulum (SR) function. METHODS AND RESULTS Isolated rat hearts were subjected to global ischaemia followed by reperfusion in the absence or presence of apelin-13 and inhibitors of some survival kinases. We found that depressed cardiac performance induced by I/R was attenuated by apelin-13. Furthermore, apelin-13 depressed oxidative stress during I/R. SR function depressed during I/R was partly reversed by apelin-13. SR oxidative modification levels were increased in I/R and reversed by apelin. Inhibitors of phosphatidylinositol-3-kinase and protein kinase C abolished the effects of apelin. Apelin-13 maintained the Ca(2+) transient against I/R in cardiomyocytes. CONCLUSION Apelin protects SR function and cardiac performance during I/R by attenuating oxidation of sarco(endo)plasmic reticulum Ca(2+)-ATPase and RyR.

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.

Effects of cannabinoid receptor type 2 on endogenous myocardial regeneration by activating cardiac progenitor cells in mouse infarcted heart.

Cannabinoid receptor type 2 (CB2) activation is recently reported to promote proliferation of some types of resident stem cells (e.g., hematopoietic stem/progenitor cell or neural progenitor cell). Resident cardiac progenitor cell (CPC) activation and proliferation are crucial for endogenous cardiac regeneration and cardiac repair after myocardial infarction (MI). This study aims to explore the role and possible mechanisms of CB2 receptor activation in enhancing myocardial repair. Our results revealed that CB2 receptor agonist AM1241 can significantly increase CPCs by c-kit and Runx1 staining in ischemic myocardium as well as improve cardiomyocyte proliferation. AM1241 also decreased serum levels of MDA, TNF-α and IL-6 after MI. In addition, AM1241 can ameliorate left ventricular ejection fraction and fractional shortening, and reduce fibrosis. Moreover, AM1241 treatment markedly increased p-Akt and HO-1 expression, and promoted Nrf-2 nuclear translocation. However, PI3K inhibitor wortmannin eliminated these cardioprotective roles of AM1241. In conclusion, AM1241 could induce myocardial regeneration and improve cardiac function, which might be associated with PI3K/Akt/Nrf2 signaling pathway activation. Our findings may provide a promising strategy for cardiac endogenous regeneration after MI.

AKAP150 mobilizes cPKC-dependent cardiac glucotoxicity

Activation of conventional PKCs (cPKC) is a key signaling that directs the cardiac toxicity of hyperglycemia. AKAP150, a scaffold protein of the A-kinase anchoring proteins (AKAPs) family, is less defined regarding its capability to anchor and regulate cardiac cPKC signaling. This study was designed to investigate the role of AKAP150 in cPKC-mediated cardiac glucotoxicity. In cardiac tissues from streptozotocin-induced diabetic rats and high-glucose-treated neonatal rat cardiomyocytes, both mRNA and protein levels of AKAP150 increased significantly, and marked elevations were observed in cPKC activity and both expression and phosphorylation levels of p65 NF-κB and p47(phox). AKAP150 knockdown was established via intramyocardial injection in vivo and transfection in vitro of adenovirus carrying AKAP150-targeted shRNA. Downregulation of AKAP150 reversed diabetes-induced diastolic dysfunction as manifested by decreased left ventricular end-diastolic diameter and early/late mitral diastolic wave ratio. AKAP150 inhibition also abrogated high-glucose-induced cardiomyocyte apoptosis (TUNEL staining and annexin V/propidium iodide flow cytometry) and oxidative stress (ROS production, NADPH oxidase activity, and lipid peroxidation). More importantly, reduced AKAP150 expression significantly inhibited high-glucose-induced membrane translocation and activation of cPKC and suppressed the increases in the phosphorylation of p65 NF-κB and p47(phox). Immunofluorescent coexpression and immunoprecipitation indicated enhanced anchoring of AKAP150 with cPKC within the plasma membrane under hyperglycemia, and AKAP150 preferentially colocalized and functionally bound with PKCα and -β isoforms. These results suggest that cardiac AKAP150 positively responds to hyperglycemia and enhances the efficiency of glucotoxicity signaling through a cPKC/p47(phox)/ROS pathway that induces myocardial dysfunction, cardiomyocyte apoptosis, and oxidative stress.

Activation of κ-opioid receptor by U50,488H improves vascular dysfunction in streptozotocin-induced diabetic rats.

BackgroundEvidence suggests that activation of κ-opioid receptor (KOR) by U50,488H exhibits potential cardiovascular protective properties. However, the effects of U50,488H on vascular dysfunction in diabetes mellitus (DM) are still not clear. The present study was designed to investigate the effects of U50,488H on vascular dysfunction in diabetic rats and explore the underlying mechanisms involved.MethodsRats were randomly divided into control, DM, DM + vehicle, DM + U50,488H and DM + nor-binaltorphimine (nor-BNI) groups. Streptozotocin injection was used to induce DM. Weight, blood glucose, blood pressure and plasma insulin for each group were measured. Arterial functions were assessed with isolated vessels mounted for isometric tension recordings. Angiotensin II (ANG II), soluble intercellular adhesion molecule-1 (sICAM-1), interleukin (IL)-6 and IL-8 levels were measured by ELISA, and endothelial nitric oxide synthase (eNOS) phosphorylation and NF-κB p65 translocation were measured by Western blot.ResultsActivation of KOR by U50,488H reduced the enhanced contractility of aortas to KCl and noradrenaline and increased acetylcholine-induced vascular relaxation, which could also protect the aortal ultrastructure in DM. U50,488H treatment resulted in reduction in ANG II, sICAM-1, IL-6 and IL-8 levels and elevation in NO levels, while these effects were abolished by nor-BNI treatment. Further more, eNOS phosphorylation was increased, and NF-κB p65 translocation was decreased after U50,488H treatment.ConclusionsOur study demonstrated that U50,488H may have therapeutic effects on diabetic vascular dysfunction by improving endothelial dysfunction and attenuating chronic inflammation, which may be dependent on phosphorylation of eNOS and downstream inhibition of NF-кB.

PROTECTIVE EFFECTS OF GLUCAGON-LIKE PEPTIDE-1 VIA CAMP/PKA/RHO DEPENDENT PATHWAY ON CARDIAC MICROVESSELS INJURY IN DIABETES MELLITUS

Objectives Glucagon-like peptide-1 (GLP-1) was a hormone predominately synthesised and secreted by intestinal L-cells. Pharmacological modulation of the GLP-1 had emerged as an important treatment target for diabetes mellitus. In addition to its glucose lowering properties, GLP-1 was found to have multiple cardioprotective effects. Impaired cardiac microvascular function is thought to contribute greatly to the diabetes cardiovascular disease. Yet the effects of GLP-1 on cardiac microvessels remained unclear, this study was aim to investigate the protective effects of GLP-1 on cardiac microvessels injury and the underlying regulatory mechanism in diabetes mellitus. Methods Streptozocin (STZ)-induced diabetic rats (n=45) were randomised to 12 weeks of treatment with vehicle, LAF237 (DPP-IV inhibitor, 1 mg/kg/d) or Exenatide (GLP-1 analogue, 1 nmol/kg/d). Before and after treatment, blood glucose levels and weight were assessed. Cardiac function was examined by echocardiographic measurements; cardiac energetics was examined by 18F-FDG PET/CT. Scanning electron microscopy was used to analyse changes in morphology of cardiac microvessels. Transmission electron microscopy was used to assay cardiac microvascular permeability via lanthanum nitrate tracer. Adult rat cardiac microvascular endothelial cells (CMECs) were isolated and cultured in medium alone (control) or medium containing glucose (25 mmol/l), GLP-1 (10−7 mmol/l), high glucose (25 mmol/l) plus GLP-1 (10−7 mmol/l). First, GLP-1 receptor (GLP-1R) was detected by immunofluorescence and western blot. Then lucigenin-enhanced chemiluminescence assay and dihydroethidine (DHE) staining were used to assess oxidative stress. Tunnel staining and caspase-3 expression were used to assess apoptosis of CMECs. H89 was used to inhibit cAMP/PKA pathway; fasudil was used to inhibit Rho/Rho-kinase (ROCK) pathway; Rho siRNA was transfected into CMECs to silence Rho. The protein expression of Rho, ROCK, p22phox, p47phox and rac-1 was examined by western blot analysis. Results After 12 weeks of treatment with LAF237 or Exetinade, the cardiac function and energetics were improved significantly compared with the vehicle treated groups. Cardiac microvascular barrier function was also improved. We demonstrated that GLP-1R was expressed on CMECs. Compared with vehicle treated groups, ROS production (relative light unit, RLU) (4.06±0.36 vs 2.13±0.31, p<0.05) and apoptotic index (33.95%±5.49% vs 24.39%±3.39%, p <0.05) were significantly decreased by GLP-1 treatment in high glucose-induced CMECs. There were also significant reductions in NADPH oxidase such as p22phox, p47phox and rac-1 expression. However, no difference was found in ROS production (RLU) (0.45±0.57 vs 0.53±0.07, p<0.05), apoptotic index (9.15%±1.33% vs 10.87%±1.65%, p<0.05) and NADPH oxidase expression between non-glucose induced groups. Western blot assay showed that the cAMP/PKA activity was increased and the Rho expression was decreased in high glucose induced CMECs after treatment with GLP-1, which reproduced the same effect as PKA inhibitor H89. Fasudil and transfection with Rho siRNA significantly decreased p22phox, p47phox and rac-1 expression in high-glucose induced CMECs Conclusions GLP-1 could protect the cardiac microvessels against oxidative stress injury, apoptosis and the resultant microvascular barrier dysfunction in diabetic rats, which contribute to the improvement of cardiac function and energetics. The protective effects of GLP-1 are dependent on downstream inhibition of Rho, which is through cAMP/PKA pathway, resulting in subsequent decreased expression of NADPH oxidase.

GW24-e2319 CB2 receptor agonist AM1241 promoted the Infarcted murine heart repair by activating endogenuous cardiac stem/progenitor cells

Objectives The aim of this study was to investigate the ability of CB2 agonist AM1241 to promote murine infarcted myocardium repair by activating resident endogenous cardiac stem/progenitor cells (CSC/CPCs) and possible mechanisms. Methods Acute myocardial infarction (AMI) was induced in mice by ligation of left descending artery. Mice were randomised into following groups with n = 10 each: (1) Sham group, (2) MI group, (3) MI + AM1241 group, (4) MI + AM1241 + AM630 (CB2 receptor antagonist). Then, AM1241(10mg/kg) was intraperitoneal injected into mice for seven consecutive days. Three days post-operation, cardiomyocyte survival and apoptosis was determined by Ki67 and TUNEL staining respectively. The levels of LDH, TNF-α and IL-6 were examined with ELISA assay. The express-ion of CSC/CPCs markers c-kit and Runx1 in infarction border zones (IBZs) of myocardium were evaluated with immohistopathology 7 days later. The mice cardiac function evaluation was performed by echocardiography 3, 7, 14 and 28 days postoperation respectively. Myocardium fibrosis was detected with Masson’s trichrome stain 4 weeks later. The expression of ERK, Keap-1, Nrf2 and heme oxygenase (HO-1) and superoxide dismutase (Cu/Zn-SOD) were performed by Western blot analysis. Results In contrast to sham group, AM1241 could significantly increase c-kit and Runx1 positive CSC/CPCs number [Runx 1 positive cells: (3.32 + 0.25)% vs. (1.14 + 0.13)%, p < 0.01], improve cardiomyocytes survival [Ki67 positive cells: (35.32 + 2.03)% vs. (8.94 + 0.79)%, p < 0.01) and inhibit them apoptosis (p < 0.05), and decrease serum levels of LDH as well as the concertration of TNF-α and IL-6 in injury myocardium (p < 0.05). Meanwhile, AM1241 could ameliorate left venricular ejection fraction (LVEF) and fractional shortening (FS) 14 and 28 days post-operation (p < 0.05), and reduce fibrosis (p < 0.01). Moreover, AM1241 treatment also markedly increased p-ERK, HO-1 and Cu/Zn-SOD expression, promoted Nrf-2 nuclear transocation, but decreased Keap-1 expression (p < 0.05). However, AM630 abolished these beneficial roles of AM1241. In contrast to sham group, AM1241 could significantly increase c-kit and Runx1 positive CSC/CPCs number [Runx 1 positive cells: (3.32 + 0.25)% vs. (1.14 + 0.13)%, p < 0.01], improve cardiomyocytes survival [Ki67 positive cells: (35.32 + 2.03)% vs. (8.94 + 0.79)%, p < 0.01) and inhibit them apoptosis (p < 0.05), and decrease serum levels of LDH as well as the concertration of TNF-α and IL-6 in injury myocardium (p < 0.05). Meanwhile, AM1241 could ameliorate left venricular ejection fraction (LVEF) and fractional shortening (FS) 14 and 28 days post-operation (p < 0.05), and reduce fibrosis (p < 0.01). Moreover, AM1241 treatment also markedly increased p-ERK, HO-1 and Cu/Zn-SOD expression, promoted Nrf-2 nuclear transocation, but decreased Keap-1 expression (p < 0.05). However, AM630 abolished these beneficial roles of AM1241. Conclusions AM1241 could better adverse oxidative stress and inflammation milieu after AMI, benefit CSC/CPCs activation, induce myocardial regeneration, and improve cardiac function, which might be associated with ERK/Nrf 2/ARE signaling pathway activation.