Guangdong Yang

H2S as a Physiologic Vasorelaxant: Hypertension in Mice with Deletion of Cystathionine γ-Lyase

Studies of nitric oxide over the past two decades have highlighted the fundamental importance of gaseous signaling molecules in biology and medicine. The physiological role of other gases such as carbon monoxide and hydrogen sulfide (H2S) is now receiving increasing attention. Here we show that H2S is physiologically generated by cystathionine γ-lyase (CSE) and that genetic deletion of this enzyme in mice markedly reduces H2S levels in the serum, heart, aorta, and other tissues. Mutant mice lacking CSE display pronounced hypertension and diminished endothelium-dependent vasorelaxation. CSE is physiologically activated by calcium-calmodulin, which is a mechanism for H2S formation in response to vascular activation. These findings provide direct evidence that H2S is a physiologic vasodilator and regulator of blood pressure.

Hydrogen sulfide is an endogenous stimulator of angiogenesis

The goal of the current study was to investigate the role of exogenous and endogenous hydrogen sulfide (H2S) on neovascularization and wound healing in vitro and in vivo. Incubation of endothelial cells (ECs) with H2S enhanced their angiogenic potential, evidenced by accelerated cell growth, migration, and capillary morphogenesis on Matrigel. Treatment of chicken chorioallantoic membranes (CAMS) with H2S increased vascular length. Exposure of ECs to H2S resulted in increased phosphorylation of Akt, ERK, and p38. The KATP channel blocker glibenclamide or the p38 inhibitor SB203580 abolished H2S-induced EC motility. Since glibenclamide inhibited H2S-triggered p38 phosphorylation, we propose that KATP channels lay upstream of p38 in this process. When CAMs were treated with H2S biosynthesis inhibitors dl-propylargylglycine or beta-cyano-L-alanine, a reduction in vessel length and branching was observed, indicating that H2S serves as an endogenous stimulator of the angiogenic response. Stimulation of ECs with vascular endothelial growth factor (VEGF) increased H2S release, while pharmacological inhibition of H2S production or KATP channels or silencing of cystathionine gamma-lyase (CSE) attenuated VEGF signaling and migration of ECs. These results implicate endothelial H2S synthesis in the pro-angiogenic action of VEGF. Aortic rings isolated from CSE knockout mice exhibited markedly reduced microvessel formation in response to VEGF when compared to wild-type littermates. Finally, in vivo, topical administration of H2S enhanced wound healing in a rat model, while wound healing was delayed in CSE−/− mice. We conclude that endogenous and exogenous H2S stimulates EC-related angiogenic properties through a KATP channel/MAPK pathway.

Activation of KATP channels by H2S in rat insulin-secreting cells and the underlying mechanisms

H2S is an important gasotransmitter, generated in mammalian cells from l‐cysteine metabolism. As it stimulates KATP channels in vascular smooth muscle cells, H2S may also function as an endogenous opener of KATP channels in INS‐1E cells, an insulin‐secreting cell line. In the present study, KATP channel currents in INS‐1E cells were recorded using the whole‐cell and single‐channel recording configurations of the patch‐clamp technique. KATP channels in INS‐1E cells have a single‐channel conductance of 78 pS. These channels were activated by diazoxide and inhibited by gliclazide. ATP (3 mm) in the pipette solution inhibited KATP channels in INS‐1E cells. Significant amount of H2S was produced from INS‐1E cells in which the expression of cystathinonie gamma‐lyase (CSE) was confirmed. After INS‐1E cells were transfected with CSE‐targeted short interfering RNA (CSE‐siRNA) or treated with dl‐propargylglycine (PPG; 1–5 mm) to inhibit CSE, endogenous production of H2S was abolished. Increase in extracellular glucose concentration significantly decreased endogenous production of H2S in INS‐1E cells, and increased insulin secretion. After transfection of INS‐1E cells with adenovirus containing the CSE gene (Ad‐CSE) to overexpress CSE, high glucose‐stimulated insulin secretion was virtually abolished. Basal KATP channel currents were significantly reduced after incubating INS‐1E cells with a high glucose concentration (16 mm) or lowering endogenous H2S level by CSE‐siRNA transfection. Under these conditions, exogenously applied H2S significantly increased whole‐cell KATP channel currents at concentrations equal to or lower than 100 μm. H2S (100 μm) markedly increased open probability by more than 2‐fold of single KATP channels (inside‐out recording) in native INS‐1E cells (n= 4, P < 0.05). Single‐channel conductance and ATP sensitivity of KATP channels were not changed by H2S. In conclusion, endogenous H2S production from INS‐1E cells varies with in vivo conditions, which significantly affects insulin secretion from INS‐1E cells. H2S stimulates KATP channels in INS‐1E cells, independent of activation of cytosolic second messengers, which may underlie H2S‐inhibited insulin secretion from these cells. Interaction among H2S, glucose and the KATP channel may constitute an important and novel mechanism for the fine control of insulin secretion from pancreatic β‐cells.

Pro-apoptotic effect of endogenous H2S on human aorta smooth muscle cells.

Cystathionine gamma‐lyase (CSE) is a key enzyme in the trans‐sulfuration pathway, which uses L‐cysteine to produce hydrogen sulfide (H2S). The CSE/H2S system has been shown to play an important role in regulating cellular functions in different systems. In the present study, we overexpressed CSE in human aorta smooth muscle cells (HASMCs) using a recombinant defective adenovirus containing CSE gene (Ad‐CSE). Infection of HASMCs with Ad‐CSE resulted in a significant increase in the expression of CSE protein and H2S production. Ad‐CSE transfection inhibited cell growth and stimulated apoptosis, as evidenced by cell viability assay, Hoechst 33258 staining, TUNEL, and caspase 3 activation. CSE‐mediated apoptosis was associated with an increased ERK and p38 MAPK activation, up‐regulation of p21Cip/WAK‐1, and down‐regulation of cyclin D1 expression. After inhibiting endogenous background CSE gene expression, direct administration of H2S at 100 ?M induced apoptosis of HASMCs. The other two endproducts of CSE‐catalyzed enzymatic reaction, ammonium and pyruvate, failed to do so. These results demonstrate that overexpression of CSE stimulates SMC apoptosis due to an increased endogenous production of H2S. Adenovirus‐mediated transfer of CSE gene may provide a novel therapeutic approach in treating vascular diseases linked to abnormal cellular proliferation and vascular remodeling.

Hydrogen sulfide-induced apoptosis of human aorta smooth muscle cells via the activation of mitogen-activated protein kinases and caspase-3

The endogenous production of hydrogen sulfide (H2S) and its physiological functions, including membrane hyperpolarization and smooth muscle cell relaxation, position this gas well in the family of gasotransmitters together with nitric oxide (NO) and carbon monoxide (CO). In this study, we demonstrate that H2S at physiologically relevant concentrations induced apoptosis of human aorta smooth muscle cells (HASMCs). Exposure of HASMCs to H2S did not induce necrosis as verified with Trypan blue exclusion and LDH release analysis. After inhibiting endogenous H2S production, exogenous H2S induced much more significant apoptosis, which was not altered by the presence of albumin or glutathione. H2S treatment increased the activities of ERK and p38 mitogen‐activated protein kinase (MAPK), but not c‐Jun N‐terminal kinase activity. Suppression of extracellular signal‐regulated kinase (ERK) activity, but not of p38 activity, inhibited the H2S‐induced apoptosis of HASMCs. The activation of ERK by H2S in HASMCs was accompanied by increased caspase‐3 activity. Inhibition of caspase‐3 by AC‐DEVD‐CHO attenuated the H2S‐induced cell apoptosis. Inhibition of ERK by U0126 decreased caspase‐3 activity, whereas AC‐DEVD‐CHO did not alter ERK activity. In conclusion, exogenous H2S induces apoptosis of HASMCs, which is significantly affected by the endogenous H2S level. Of the three investigated MAPKs, only ERK played an active role in mediating H2S‐induced apoptosis of HASMCs by activating caspase‐3. These findings may help reveal novel mechanisms for many diseases linked to H2S‐related abnormal cellular proliferation and apoptosis.

Hydrogen Sulfide Protects Against Cellular Senescence via S-Sulfhydration of Keap1 and Activation of Nrf2

AIMS H2S, a third member of gasotransmitter family along with nitric oxide and carbon monoxide, exerts a wide range of cellular and molecular actions in our body. Cystathionine gamma-lyase (CSE) is a major H2S-generating enzyme in our body. Aging at the cellular level, known as cellular senescence, can result from increases in oxidative stress. The aim of this study was to investigate how H2S attenuates oxidative stress and delays cellular senescence. RESULTS Here we showed that mouse embryonic fibroblasts isolated from CSE knockout mice (CSE KO-MEFs) display increased oxidative stress and accelerated cellular senescence in comparison with MEFs from wild-type mice (WT-MEFs). The protein expression of p53 and p21 was significantly increased in KO-MEFs, and knockdown of p53 or p21 reversed CSE deficiency-induced senescence. Incubation of the cells with NaHS (a H2S donor) significantly increased the glutathione (GSH) level and rescued KO-MEFs from senescence. Nrf2 is a master regulator of the antioxidant response, and Keap1 acts as a negative regulator of Nrf2. NaHS S-sulfhydrated Keap1 at cysteine-151, induced Nrf2 dissociation from Keap1, enhanced Nrf2 nuclear translocation, and stimulated mRNA expression of Nrf2-targeted downstream genes, such as glutamate-cysteine ligase and GSH reductase. INNOVATION These results provide a mechanistic insight into how H2S signaling mediates cellular senescence induced by oxidative stress. CONCLUSION H2S protects against cellular aging via S-sulfhydration of Keap1 and Nrf2 activation in association with oxidative stress.

Hydrogen sulfide (H2S) metabolism in mitochondria and its regulatory role in energy production

Although many types of ancient bacteria and archea rely on hydrogen sulfide (H2S) for their energy production, eukaryotes generate ATP in an oxygen-dependent fashion. We hypothesize that endogenous H2S remains a regulator of energy production in mammalian cells under stress conditions, which enables the body to cope with energy demand when oxygen supply is insufficient. Cystathionine γ-lyase (CSE) is a major H2S-producing enzyme in the cardiovascular system that uses cysteine as the main substrate. Here we show that CSE is localized only in the cytosol, not in mitochondria, of vascular smooth-muscle cells (SMCs) under resting conditions, revealed by Western blot analysis and confocal microscopy of SMCs transfected with GFP-tagged CSE plasmid. After SMCs were exposed to A23187, thapsigargin, or tunicamycin, intracellular calcium level was increased, and CSE translocated from the cytosol to mitochondria. CSE was coimmunoprecipitated with translocase of the outer membrane 20 (Tom20) in mitochondrial membrane. Tom20 siRNA significantly inhibited mitochondrial translocation of CSE and mitochondrial H2S production. The cysteine level inside mitochondria is approximately three times that in the cytosol. Translocation of CSE to mitochondria metabolized cysteine, produced H2S inside mitochondria, and increased ATP production. Inhibition of CSE activity reversed A23187-stimulated mitochondrial ATP production. H2S improved mitochondrial ATP production in SMCs with hypoxia, which alone decreased ATP production. These results suggest that translocation of CSE to mitochondria on specific stress stimulations is a unique mechanism to promote H2S production inside mitochondria, which subsequently sustains mitochondrial ATP production under hypoxic conditions.

Decreased Endogenous Production of Hydrogen Sulfide Accelerates Atherosclerosis

Background— Cystathionine &ggr;-lyase (CSE) produces hydrogen sulfide (H2S) in the cardiovascular system. The deficiency of CSE in mice leads to a decreased endogenous H2S level, an age-dependent increase in blood pressure, and impaired endothelium-dependent vasorelaxation. To date, there is no direct evidence for a causative role of altered metabolism of endogenous H2S in atherosclerosis development. Methods and Results— Six-week-old CSE gene knockout and wild-type mice were fed with either a control chow or atherogenic paigen-type diet for 12 weeks. Plasma lipid profile and homocysteine levels, blood pressure, oxidative stress, atherosclerotic lesion size in the aortic roots, cell proliferation, and adhesion molecule expression were then analyzed. CSE-knockout mice fed with atherogenic diet developed early fatty streak lesions in the aortic root, elevated plasma levels of cholesterol and low-density lipoprotein cholesterol, hyperhomocysteinemia, increased lesional oxidative stress and adhesion molecule expression, and enhanced aortic intimal proliferation. Treatment of CSE-knockout mice with NaHS, but not N-acetylcysteine or ezetimibe, inhibited the accelerated atherosclerosis development. Double knockout of CSE and apolipoprotein E gene expression in mice exacerbated atherosclerosis development more than that in the mice with only apolipoprotein E or CSE knockout. Conclusions— Endogenously synthesized H2S protects vascular tissues from atherogenic damage by reducing vessel intimal proliferation and inhibiting adhesion molecule expression. Decreased endogenous H2S production predisposes the animals to vascular remodeling and early development of atherosclerosis. The CSE/H2S pathway is an important therapeutic target for protection against atherosclerosis.

H2S, Endoplasmic Reticulum Stress, and Apoptosis of Insulin-secreting Beta Cells

Cystathionine γ-lyase (CSE) is a key enzyme in the trans-sulfuration pathway, which uses l-cysteine to produce hydrogen sulfide (H2S). Functional changes of pancreatic beta cells induced by endogenous H2S have been reported, but the effect of the CSE/H2S system on pancreatic beta cell survival has not been known. In this study, we demonstrate that H2Sat physiologically relevant concentrations induced apoptosis of INS-1E cells, an insulin-secreting beta cell line. Transfection of INS-1E cells with a recombinant defective adenovirus containing the CSE gene (Ad-CSE) resulted in a significant increase in CSE expression and H2S production. Ad-CSE transfection also stimulated apoptosis. The other two end products of CSE-catalyzed enzymatic reaction, ammonium and pyruvate, had no effects on INS-1E cell apoptosis, indicating that overexpression of CSE may stimulate INS-1E cell apoptosis via increased endogenous production of H2S. Both exogenous H2S (100 μm) and Ad-CSE transfection inhibited ERK1/2 but activated p38 MAPK. Interestingly, BiP and CHOP, two indicators of endoplasmic reticulum (ER) stress, were up-regulated in H2S-and CSE-mediated apoptosis in INS-1E cells. After suppressing CHOP mRNA expression, H2S-induced apoptosis of INS-1E cells was significantly decreased. Inhibition of p38 MAPK, but not of ERK1/2, inhibited the expression of BiP and CHOP and decreased H2S-stimulated apoptosis, suggesting that p38 MAPK activation functions upstream of ER stress to initiate H2S-induced apoptosis. It is concluded that H2S induces apoptosis of insulin-secreting beta cells by enhancing ER stress via p38 MAPK activation. Our findings may help unmask a novel role of CSE/H2S system in regulating pancreatic functions under physiological condition and in diabetes.

Pancreatic islet overproduction of H2S and suppressed insulin release in Zucker diabetic rats.

Hydrogen sulfide (H2S) has been traditionally known for its toxic effects on living organisms. The role of H2S in the homeostatic regulation of pancreatic insulin metabolism has been unclear. The present study is aimed at elucidating the effect of endogenously produced H2S on pancreatic insulin release and its role in diabetes development. Diabetes development in Zucker diabetic fatty (ZDF) rats was evaluated in comparison with Zucker fatty (ZF) and Zucker lean (ZL) rats. Pancreatic H2S production and insulin release were also assayed. It was found that H2S was generated in rat pancreas islets, catalyzed predominantly by cystathionine γ-lyase (CSE). Pancreatic CSE expression and H2S production were greater in ZDF rats than in ZF or ZL rats. ZDF rats exhibited reduced serum insulin level, hyperglycemia, and insulin resistance. Inhibition of pancreatic H2S production in ZDF rats by intraperitoneal injection of DL-propargylglycine (PPG) for 4 weeks increased serum insulin level, lowered hyperglycemia, and reduced hemoglobin A1c level (P<0.05). Although in ZF rats it also reduced pancreatic H2S production and serum H2S level, PPG treatment did not alter serum insulin and glucose level. Finally, H2S significantly increased KATP channel activity in freshly isolated rat pancreatic β-cells. It appears that insulin release is impaired in ZDF because of abnormally high pancreatic production of H2S. New therapeutic approach for diabetes management can be devised based on our observation by inhibiting endogenous H2S production from pancreas.