BackgroundExcessive autophagy induced by extravagant oxidative stress is the main reason for diabetes-induced vascular endothelial cells dysfunction. Hydrogen sulfide (H2S) has anti-oxidative effects but its regulation on excessive autophagy of vascular endothelial cells is unclear.MethodsIn this study, aorta of db/db mice (28 weeks old) and rat aortic endothelial cells (RAECs) treated with 40 mM glucose and 500 μM palmitate acted as type II diabetic animal and cellular models, respectively, and 100 μMNaHS was used as an exogenous H2S donor. The apoptosis level was measured by terminal deoxynucleotidyl transferase mediated dUTP nick-end labeling (TUNEL) staining and Hoechst 33342/PI staining. The activities of SOD, CAT and respiratory complexes were also measured. The mRNA levels of SOD and CAT were detected by real-time PCR. AMPK-siRNA was used to detect the effect of AMPK on autophagy. Western blotting was used to detected the protein level.ResultsH2S production was decreased (p < 0.05, p < 0.01) both in vitro and in vivo; NaHS treatment rescued this impairment (p < 0.05, p < 0.01). The expression of adhesive proteins was increased (p < 0.05, p < 0.01) both in vitro and in vivo; NaHS attenuated (p < 0.05, p < 0.01) these alterations. NaHS could protect endothelial cells against apoptosis induced by type II diabetes (p < 0.05, p < 0.01). Furthermore, the expressions and activities of SOD and CAT were impaired (p < 0.05, p < 0.01) in endothelial cells of diabetes II; NaHS treatment attenuated (p < 0.05) this impairment. NaHS also increased ATP production (p < 0.05) and activities of respiratory complexes (p < 0.05), and the ratio of p-AMPK to AMPK was also decreased by NaHS (p < 0.01). The level of autophagy in endothelial cells was also decreased (p < 0.05, p < 0.01) by NaHS treatment and AMPK-siRNA treatment. The expression of Nrf2 in the nuclei was increased (p < 0.05) by NaHS treatment.ConclusionExogenous H2S might protect arterial endothelial cells by suppressing excessive autophagy induced by oxidative stress through the Nrf2-ROS-AMPK signaling pathway.Electronic supplementary materialThe online version of this article (doi:10.1186/s13578-016-0099-1) contains supplementary material, which is available to authorized users.

The upregulation of reactive oxygen species (ROS) is a primary cause of cardiomyocyte apoptosis in diabetes cardiomyopathy (DCM). Mitofusin-2 (Mfn-2) is a key protein that bridges the mitochondria and endoplasmic reticulum (ER). Hydrogen sulfide (HS)-mediated cardioprotection is related to antioxidant effects. The present study demonstrated that HS inhibited the interaction between the ER and mitochondrial apoptotic pathway. This study investigated cardiac function, ultrastructural changes in the ER and mitochondria, apoptotic rate using TUNEL, and the expression of ER stress-associated proteins and mitochondrial apoptotic proteins in cardiac tissues in STZ-induced type I diabetic rats treated with or without NaHS (donor of HS). Mitochondria of cardiac tissues were isolated, and MPTP opening and cytochrome (cyt C) and Mfn-2 expression were also detected. Our data showed that hyperglycemia decreased the cardiac function by ultrasound cardiogram, and the administration of exogenous HS ameliorated these changes. We demonstrated that the expression of ER stress sensors and apoptotic rates were elevated in cardiac tissue of DCM and cultured H9C2 cells, but the expression of these proteins was reduced following exogenous HS treatment. The expression of mitochondrial apoptotic proteins, cyt C, and mPTP opening was decreased following treatment with exogenous HS. In our experiment, the expression and immunofluorescence of Mfn-2 were both decreased after transfection with Mfn-2-siRNA. Hyperglycemia stimulated ER interactions and mitochondrial apoptotic pathways, which were inhibited by exogenous HS treatment through the regulation of Mfn-2 expression.

Background: Oxidative stress inducing hyperglycemia and high glucose play an important role in the development of cardiac fibrosis associated with diabetic cardiomyopathy. The endogenous gasotransmitter hydrogen sulfide (H₂S) can act in a cytoprotective manner. However, whether H₂S could inhibit the fibrotic process is unclear. The purpose of our study was to examine the role of H₂S in the development and underlying mechanisms behind diabetic cardiomyopathy. Methods: Diabetic cardiomyopathy was induced in rats by injection of streptozotocin (STZ). Cardiac fibrosis and proliferation of rat neonatal cardiac fibroblasts were induced by hyperglycemia and high glucose. We tested the effects of GYY4137 (a slow-releasing H₂S donor), NaHS (an exogenous H₂S donor) and NADPH oxidase 4 (NOX4) siRNA on reactive oxygen species (ROS) production, MMP-2,9, cystathionine-γ-lyase (CSE), NOX4, and extracellular signal-regulated kinase 1/2 (ERK1/2) to reveal the effects of H₂S on the cardiac fibrosis of diabetic cardiomyopathy. Result: In vivo, NaHS treatment inhibited hyperglycemia-induced expression of type I and III collagen, MMP-2 and MMP-9 in diabetic hearts. Rat neonatal cardiac fibroblast migration and cell survival were inhibited by administration of GYY4137. NOX4 expression was increased by hyperglycemia and high glucose, but was reduced in cardiac fibroblasts treated by NaHS and GYY4137. ROS production, ERK1/2 phosphorylation and MMP-2 and 9 expression were decreased in rat neonatal cardiac fibroblasts treated with GYY4137 and NOX4 siRNA. Conclusion: The present study shows that enhanced NOX4 expression results in cardiac fibrosis through ROS-ERK1/2-MAPkinase-dependent mechanisms in diabetic cardiomyopathy. NOX4 could be an important target for H₂S to regulate redox homeostasis in cardiac fibrosis of diabetic cardiomyopathy.

Aims: Calcium-sensing receptor (CaR) acts as a G protein coupled receptor that mediates the increase of the intracellular Ca²⁺ concentration. The expression of CaR has been confirmed in various cell types, including cardiomyocytes, smooth muscle cells, neurons and vascular endothelial cells. However, whether CaR is expressed and functions in cardiac fibroblasts has remained unknown. The present study investigated whether CaR played a role in cardiac fibroblast proliferation and extracellular matrix (ECM) secretion, both in cultured rat neonatal cardiac fibroblasts and in a model of cardiac hypertrophy induced by isoproterenol (ISO). Methods and Results: Immunofluorescence, immunohistochemistry and Western blot analysis revealed the presence of CaR in cardiac fibroblasts. Calcium and calindol, a specific activator of CaR, elevated the intracellular calcium concentration in cardiac fibroblasts. Pretreatment of cardiac fibroblasts with calhex231, a specific inhibitor of CaR, U73122 and 2-APB attenuated the calindol- and extracellular calcium-induced increase in intracellular calcium ([Ca²⁺]_i). Cardiac fibroblast proliferation and migration were assessed by MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), cell count and the cell scratch assay. ECM production was detected by expression of matrix metalloproteinase-3 and -9 (MMP-3 and -9). Activation of CaR promoted cardiac fibroblast proliferation and migration and ECM secretion. More importantly, calhex231, suppressed cardiac fibroblast proliferation and migration and MMP-3 and -9 expression. To further investigate the effect of CaR on cardiac fibrosis, a model of ISO-induced cardiac hypertrophy was established. Pretreatment with calhex231 prevented cardiac fibrosis and decreased the expression of MMP-3 and -9 expression. Conclusions: Our results are the first report that CaR plays an important role in Ca²⁺ signaling involved in cardiac fibrosis through the phospholipase C- inositol 3,4,5 phosphate (PLC-IP₃) pathway.

AimVascular smooth muscle cell (VSMC) proliferation in response to hyperglycemia is an important process in the development of arterial vessel hyperplasia. The shape change of mitochondria is dynamic and closely related to fission and fusion. Hydrogen sulfide (H2S) was confirmed to have anti-oxidative, anti-inflammatory and anti-proliferative effects. However, little it is known about its effects on mitochondrial morphology induced by hyperglycemia. The aim of the study is to demonstrate that H2S inhibits VSMC proliferation through regulating mitochondrial fission.Methods and resultsWe observe lower H2S levels as well as higher proliferative protein expression levels for proliferative cell nuclear antigen (PCNA) and cyclin D1 and higher mitochondrial fusion–fission protein expression levels for dynamin-related protein 1 (Drp 1) in human kidney arteries and in db/db mouse aorta. Exogenous H2S (100 μM NaHS) inhibits vascular smooth muscle cells of human pulmonary aorta(HPASMC) proliferation and migration in response to high glucose using the BrdU and scratch wound repair assays, decreases proliferative protein (PCNA and cyclin D1) expression, and reduces ROS production in the cytoplasm and mitochondria. When HPASMCs proliferate with a high glucose treatment, the mitochondria become small spheres with a short rod-shaped structure, whereas NaHS, a mitochondrial division inhibitor and siDrp prevent VSMC proliferation and maintain mitochondria as stationary and randomly dispersed with fixed structures.ConclusionExogenous H2S aids in inhibiting mitochondrial fragmentation and affects proliferation in db/db mice and HPASMCs by decreasing Drp 1 expression.Electronic supplementary materialThe online version of this article (doi:10.1186/s13578-016-0102-x) contains supplementary material, which is available to authorized users.