Vasorelaxation elicited by endogenous and exogenous hydrogen sulfide in mouse mesenteric arteries.

H2S causes vasorelaxation however there is considerable heterogeneity in the reported pharmacological mechanism of this effect. This study examines the contribution of endogenously released H2S in the regulation of vascular tone and the mechanism of H2S-induced vasorelaxation in small resistance-like arteries. Mesenteric arteries from C57 and eNOS-/- mice were mounted in myographs to record isometric force. Vasorelaxation responses to NaHS were examined in the presence of various inhibitors of vasorelaxation pathways. Expression and activity of the H2S-producing enzyme, cystathionine-γ-lyase (CSE), were also examined. CSE was expressed in vascular smooth muscle and perivascular adipose cells from mouse mesenteric artery. The substrate for CSE, L-cysteine, caused a modest vasorelaxation (35%) in arteries from C57 mice and poor vasorelaxation (10%) in arteries from eNOS-/- mice that was sensitive to the CSE inhibitor DL-propargylglycine. The fast H2S donor, NaHS, elicited a full and biphasic vasorelaxation response in mesenteric arteries (EC50 (1) 8.7 µM, EC50 (2) 0.6 mM), which was significantly inhibited in eNOS-/- vessels (P < 0.05), unaffected by endothelial removal, or blockers at any point in the NO via soluble guanylate cyclase and cGMP (NO-sGC-cGMP) vasorelaxation pathway. Vasorelaxation to NaHS was significantly inhibited by blocking K+ channels of the KCa and KV subtypes and the Cl-/HCO3- exchanger (P < 0.05). Further experiments showed that NaHS can significantly inhibit voltage-gated Ca2+ channel function (P < 0.05). The vasorelaxant effect of H2S in small resistance-like arteries is complex, involving eNOS, K+ channels, Cl-/HCO3- exchanger, and voltage-gated Ca2+ channels. CSE is present in the smooth muscle and periadventitial adipose tissue of these resistance-like vessels and can be activated to cause modest vasorelaxation under these in vitro conditions.

Chronic NaHS treatment decreases oxidative stress and improves endothelial function in diabetic mice.

Hydrogen sulphide (H2S) is endogenously produced in vascular tissue and has anti-oxidant and vasoprotective properties. This study investigates whether chronic treatment using the fast H2S donor NaHS could elicit a vasoprotective effect in diabetes. Diabetes was induced in male C57BL6/J mice with streptozotocin (60 mg/kg daily, ip for 2 weeks) and confirmed by elevated blood glucose and glycated haemoglobin levels. Diabetic mice were then treated with NaHS (100 µmol/kg/day) for 4 weeks, and aortae collected for functional and biochemical analyses. In the diabetic group, both endothelium-dependent vasorelaxation and basal nitric oxide (NO•) bioactivity were significantly reduced ( p < 0.05), and maximal vasorelaxation to the NO• donor sodium nitroprusside was impaired ( p < 0.05) in aorta compared to control mice. Vascular superoxide generation via nicotine adenine dinucleotide phosphate (NADPH) oxidase ( p < 0.05) was elevated in aorta from diabetic mice which was associated with increased expression of NOX2 ( p < 0.05). NaHS treatment of diabetic mice restored endothelial function and exogenous NO• efficacy back to control levels. NaHS treatment also reduced the diabetes-induced increase in NADPH oxidase activity, but did not affect NOX2 protein expression. These data show that chronic NaHS treatment reverses diabetes-induced vascular dysfunction by restoring NO• efficacy and reducing superoxide production in the mouse aorta.

Decreased vascular H2S production is associated with vascular oxidative stress in rats fed a high-fat western diet.

A Western-style high-fat diet is known to cause vascular dysfunction and oxidative stress. H2S contributes to the regulation of vascular function and acts as a vasoprotective molecule; however, the effects of high-fat diet on vascular H2S production and function are not known. The aim of this study was to investigate the effects of high-fat diet on vascular function and H2S production. Wistar hooded rats were fed a western diet (WD, 21 % fat) or control rat chow (6 % fat) for 12 weeks. At the end of the experiment, the aorta was collected for assessing vascular function and NO and H2S bioavailability. Superoxide anion production was quantitated by lucigenin-enhanced chemiluminescence. The expression of NADPH oxidase subunit Nox2 and the H2S-producing protein cystathionine-γ-lyase (CSE) were examined by Western blotting. WD rats had significantly higher body weight and body fat than control (p < 0.001). Endothelial function and NO bioavailability were significantly reduced in the WD group (p < 0.05), but vascular smooth muscle cell function was unaffected. Vascular superoxide production and Nox2 expression were significantly increased in the aorta from WD rats. L-Cysteine-induced vasorelaxation was reduced in the WD group (p < 0.05) and insensitive to the inhibition of the CSE. In addition, vascular H2S bioavailability and CSE expression were significantly reduced in the aorta from WD rats (p < 0.01). These data show that fat feeding induces vascular oxidative stress and a reduction in endothelial function. Furthermore, there is a reduced capacity for both basal and stimulated vascular H2S production via CSE in fat fed rats.

Hydrogen sulfide treatment reduces blood pressure and oxidative stress in angiotensin II-induced hypertensive mice.

Hydrogen sulfide (H2S) is increasingly recognized as a gasotransmitter with protective effects in the cardiovascular system. The aim of the study was to examine the effects of chronic NaHS treatment on blood pressure, vascular function and oxidative stress in an in vivo model of hypertension and oxidative stress. Male C57Bl6/J mice were rendered hypertensive with 0.7 mg kg(-1) per day angiotensin II (AngII) for 14 days administered via implanted mini-pumps. The mice were treated with NaHS (10 µmol kg(-1) per day) to deliver H2S or an inhibitor of cystathionine-γ-lyase, DL-propargylglycine (PPG 30 mg kg(-1) per day) via intraperitoneal (i.p.) injection. Systolic blood pressure was measured and vascular function examined by myography. Vascular superoxide production was measured by lucigenin-enhanced chemiluminescence. AngII infusion significantly increased systolic blood pressure (P < 0.001). This increase was significantly attenuated by treatment with NaHS (P < 0.001). Both aortic endothelial function and NO bioavailability were significantly attenuated in the AngII group (P < 0.01) but this attenuation was reversed by NaHS treatment. Similarly, aortic superoxide anion production was significantly enhanced by AngII (P < 0.01), and this was reversed by NaHS treatment, and also exacerbated by PPG treatment (P < 0.001). These data show that in a mouse model of hypertension and oxidative stress induced by AngII, exogenous H2S treatment in vivo reduces blood pressure, endothelial dysfunction and vascular oxidative stress, while inhibiting endogenous H2S production in vivo is deleterious. This furthers the evidence that H2S is a vasoprotective molecule that may be a useful treatment target in cardiovascular disease.

Effect of type 1 diabetes on the production and vasoactivity of hydrogen sulfide in rat middle cerebral arteries.

Hydrogen sulfide (H2S) is produced endogenously in vascular tissue and has both vasoregulation and antioxidant effects. This study examines the effect of diabetes-induced oxidative stress on H2S production and function in rat middle cerebral arteries. Diabetes was induced in rats with streptozotocin (50 mg/kg, i.v.). Middle cerebral artery function was examined using a small vessel myograph and superoxide anion generation measured using nicotinamide adenine dinucleotide phosphate (NADPH)-dependent lucigenin-enhanced chemiluminescence. Cystathionine-γ-lyase (CSE) mRNA expression was measured via RT-PCR. Diabetic rats had elevated blood glucose and significantly reduced cerebral artery endothelial function. Maximum vasorelaxation to the H2S donor NaHS was unaffected in diabetic cerebral arteries and was elicited via a combination of K(+), Cl(-), and Ca(2+) channel modulation, although the contribution of Cl(-) channels was significantly less in the diabetic cerebral arteries. Vasorelaxation to the H2S precursor l-cysteine and CSE mRNA were significantly increased in diabetic cerebral arteries. Cerebral artery superoxide production was significantly increased in diabetes, but this increase was attenuated ex vivo by incubation with the H2S donor NaHS. These data confirm that cerebral artery endothelial dysfunction and oxidative stress occurs in diabetes. Endogenous H2S production and activity is upregulated in cerebral arteries in this model of diabetes. Vasorelaxation responses to exogenous H2S are preserved and exogenous H2S attenuates the enhanced cerebral artery generated superoxide observed in the diabetic group. These data suggest that upregulation of endogenous H2S in diabetes may play an antioxidant and vasoprotective role.

Chronic NaHS Treatment Is Vasoprotective in High-Fat-Fed ApoE(-/-) Mice.

Hydrogen sulfide is emerging as an important mediator of vascular function that has antioxidant and cytoprotective effects. The aim of this study was to investigate the role of endogenous H2S and the effect of chronic exogenous H2S treatment on vascular function during the progression of atherosclerotic disease. ApoE(-/-) mice were fed a high-fat diet for 16 weeks and treated with the H2S donor NaHS or the cystathionine- γ -lyase (CSE) inhibitor D,L-propargylglycine (PPG), to inhibit endogenous H2S production for the final 4 weeks. Fat-fed ApoE(-/-) mice displayed significant aortic atherosclerotic lesions and significantly impaired endothelial function compared to wild-type mice. Importantly, 4 weeks of NaHS treatment significantly reduced vascular dysfunction and inhibited vascular superoxide generation. NaHS treatment significantly reduced the area of aortic atherosclerotic lesions and attenuated systolic blood pressure. Interestingly, inhibiting endogenous, CSE-dependent H2S production with PPG did not exacerbate the deleterious vascular changes seen in the untreated fat-fed ApoE(-/-) mice. The results indicate NaHS can improve vascular function by reducing vascular superoxide generation and impairing atherosclerotic lesion development. Endogenous H2S production via CSE is insufficient to counter the atherogenic effects seen in this model; however exogenous H2S treatment has a significant vasoprotective effect.

3',4'-Dihydroxyflavonol reduces superoxide and improves nitric oxide function in diabetic rat mesenteric arteries.

BACKGROUND: 3',4'-Dihydroxyflavonol (DiOHF) is an effective antioxidant that acutely preserves nitric oxide (NO) activity in the presence of elevated reactive oxygen species (ROS). We hypothesized that DiOHF treatment (7 days, 1 mg/kg per day s.c.) would improve relaxation in mesenteric arteries from diabetic rats where endothelial dysfunction is associated with elevated oxidant stress. METHODOLOGY/PRINCIPAL FINDINGS: In mesenteric arteries from diabetic rats there was an increase in ROS, measured by L-012 and 2',7'-dichlorodihydrofluorescein diacetate fluorescence. NADPH oxidase-derived superoxide levels, assayed by lucigenin chemiluminescence, were also significantly increased in diabetic mesenteric arteries (diabetes, 4892±946 counts/mg versus normal 2486±344 counts/mg, n = 7-10, p<0.01) associated with an increase in Nox2 expression but DiOHF (2094±300 counts/mg, n = 10, p<0.001) reversed that effect. Acetylcholine (ACh)-induced relaxation of mesenteric arteries was assessed using wire myography (pEC(50) = 7.94±0.13 n = 12). Diabetes significantly reduced the sensitivity to ACh and treatment with DiOHF prevented endothelial dysfunction (pEC(50), diabetic 6.86±0.12 versus diabetic+DiOHF, 7.49±0.13, n = 11, p<0.01). The contribution of NO versus endothelium-derived hyperpolarizing factor (EDHF) to ACh-induced relaxation was assessed by evaluating responses in the presence of TRAM-34+apamin+iberiotoxin or N-nitro-L-arginine+ODQ respectively. Diabetes impaired the contribution of both NO (maximum relaxation, R(max) diabetic 24±7 versus normal, 68±10, n = 9-10, p<0.01) and EDHF (pEC(50), diabetic 6.63±0.15 versus normal, 7.14±0.12, n = 10-11, p<0.01) to endothelium-dependent relaxation. DiOHF treatment did not significantly affect the EDHF contribution but enhanced NO-mediated relaxation (R(max) 69±6, n = 11, p<0.01). Western blotting demonstrated that diabetes also decreased expression and increased uncoupling of endothelial NO synthase (eNOS). Treatment of the diabetic rats with DiOHF significantly reduced vascular ROS and restored NO-mediated endothelium-dependent relaxation. Treatment of the diabetic rats with DiOHF also increased eNOS expression, both in total and as a dimer. CONCLUSIONS/SIGNIFICANCE: DiOHF improves NO activity in diabetes by reducing Nox2-dependent superoxide production and preventing eNOS uncoupling to improve endothelial function.

Mechanism of vasorelaxation and role of endogenous hydrogen sulfide production in mouse aorta.

This study aimed to elucidate the molecular mechanism of H(2)S-induced vasorelaxation. Vasorelaxation responses to the H(2)S donor NaHS and the H(2)S precursor L: -cysteine were examined by measuring isometric tone of mouse aortic rings in a small vessel myograph. H(2)S concentrations in Krebs' solution were determined with a polarographic sensor. H(2)S expression was examined by Western blot, and H(2)S production from CSE was assayed using a spectroscopic method. In pre-constricted mouse aorta, NaHS (1 µM-3 mM) elicited vasorelaxation of 95 ± 7%, EC(50) 189 ± 69 µM. This response was unaffected by removal of the endothelium. Maximum vasorelaxation was significantly attenuated by global blockade of K(+) channels (50 mM K(+)) and the K(ATP) channel blocker glibenclamide (10 µM) alone (P < 0.01, ANOVA). Specific inhibition of K(Ca), K(IR), or K(V) channels elicited a significant shift to the right in the concentration-response curve to NaHS (P < 0.01, ANOVA) without affecting maximum relaxation. NaHS-mediated vasorelaxation was inhibited by the Cl(-) channel inhibitor DIDS (1 mM, P < 0.05, t test), and NaHS caused a significant concentration-dependent inhibition of voltage-gated Ca(2+) channels (P < 0.001, two-way ANOVA). The H(2)S-producing enzyme cystathionine-γ-lyase (CSE) was expressed in mouse aorta and had activity of 7 ± 3 µmol H(2)S/g/min. L: -cysteine (1 µM-3 mM) elicited a CSE-dependent vasorelaxation of mouse aorta with intact endothelium (20 ± 7%), but not when the endothelium was removed. CSE inhibitors DL: -propargylglycine (20 mM) and ß-cyanoalanine (1 mM) caused concentration-dependent contraction of mouse aorta. In mouse aorta, H(2)S elicits endothelium-independent vasorelaxation involving several different ion channels and seems to converge at the vascular smooth muscle cell voltage-gated Ca(2+) channel. The L: -cysteine-CSE-H(2)S pathway contributes to vasorelaxation and appears to modulate basal vessel tone.