Tetrahydrobiopterin attenuates homocysteine induced endothelial dysfunction

Homocysteine is an independent risk factor for atherosclerotic vascular disease. It impairs endothelial function via increasing superoxide production and quenching nitric oxide (NO) release. Tetrahydrobiopterin (BH₄) is a critical cofactor that couples nitric oxide synthase and facilitates the production of nitric oxide (vs. superoxide anions). In the first study, the effects of hyperhomocysteinemia (0.1 mM, 3 h) on endothelium-dependent vasorelaxation to ACh and A23187 were examined in isolated segments of rat aortae in the presence or absence of BH₄ (0.1 mM). In the second study, the effects of hyperhomocysteinemia (24 h) on nitric oxide production and superoxide release (using lucigenin chemiluminescence) were studied in human umbilical vein endothelial cells in the absence or presence of BH₄ (10 M). Homocysteine incubation impaired receptor-dependent and -independent endothelial function to ACh and A23187. This effect was attenuated by BH₄. Furthermore, homocysteine exposure increased superoxide production and impaired agonist-stimulated nitric oxide release. These effects were attenuated by BH₄ (p < 0.05). Hyperhomocysteinemia impairs endothelial function, in part due to a diminished bioavailability of BH₄ with resultant uncoupling of nitric oxide synthase. BH₄ may represent an important target for strategies aimed at improving endothelial dysfunction secondary to hyperhomocysteinemia.     

Induction of oxidative stress by homocyst(e)ine impairs endothelial function

Previous studies have demonstrated a relationship between hyperhomocysteinemia and endothelial dysfunction, reduced bioavailability of nitric oxide, elastinolysis and, vascular muscle cell proliferation. In vivo decreased nitric oxide production is associated with increased matrix metalloproteinase (MMP) activity and formation of nitrotyrosine. To test the hypothesis that homocysteine neutralizes vascular endothelial nitric oxide, activates metalloproteinase, causes elastinolysis and vascular hypertrophy, we isolated aortas from normotensive Wistar rats and cultured them in medium containing homocysteine, and calf serum for 14 days. Homocysteine-mediated impairment of endothelial-dependent vasodilatation was reversed by co-incubation of homocysteine with nicotinamide (an inhibitor of peroxinitrite and nitrotyrosine), suggesting a role of homocysteine in redox-mediating endothelial dysfunction and nitrotyrosine formation. The Western blot analysis, using anti-nitrotyrosine antibody, on aortic tissue homogeneates demonstrated decreased nitrotyrosine in hyperhomocysteinemic vessels treated with nicotinamide. Zymographic analysis revealed increased elastinolytic gelatinase A and B (MMP-2, -9) in homocysteine treated vessels and the treatment with nicotinamide decreases the homocysteine-induced MMP activation. Morphometric analyses revealed significant medial hypertrophic thickening (1.4 +/- 0.2-fold of control, P = 0.03) and elastin disruption in homocysteine-treated vessels as compared to control. To determine whether homocysteine causes endothelial cell injury, cross-sections of aortas were analyzed for caspase activity by incubating with Ac-YVAD-AMC (substrate for apoptotic enzyme, caspase). The endothelium of homocysteine treated vessels, and endothelial cells treated with homocysteine, showed marked labeling for caspase. The length-tension relationship of homocysteine treated aortas was shifted to the left as compared to untreated aortas, indicating reduced vascular elastic compliance in homocysteine-treated vessels. Co-incubation of homocysteine and inhibitors of MMP, tissue inhibitor of metalloproteinase-4 (TIMP-4), and caspase, YVAD-CHO, improved vascular function. The results suggest that alteration in vascular elastin/collagen ratio and activation of MMP-2 are associated with decreased NO production in hyperhomocysteinemia.     

Cerebroprotective effects of hydrogen sulfide in homocysteine-induced neurovascular permeability: Involvement of oxidative stress,arginase, and matrix metalloproteinase-9

An elevated level of homocysteine (Hcy) leads to hyperhomocysteinemia (HHcy), which results in vascular dysfunction and pathological conditions identical to stroke symptoms. Hcy increases oxidative stress and leads to increase in blood–brain barrier permeability and leakage. Hydrogen sulfide (H₂S) production during the metabolism of Hcy has a cerebroprotective effect, although its effectiveness in Hcy-induced neurodegeneration and neurovascular permeability is less explored. Therefore, the current study was designed to perceive the neuroprotective effect of exogenous H ₂S against HHcy, a cause of neurodegeneration. To test this hypothesis, we used four groups of mice: control, Hcy, control + sodium hydrosulfide hydrate (NaHS), and Hcy + NaHS, and an HHcy mice model in Swiss albino mice by giving a dose of 1.8 g of dl -Hcy/L in drinking for 8–10 weeks. Mice that have 30 µmol/L Hcy were taken for the study, and a H ₂S supplementation of 20 μmol/L was given for 8 weeks to all groups of mice. HHcy results in the rise of the levels of superoxide and nitrite, although a concomitant decrease in the level of superoxide dismutase, catalase, glutathione peroxidase, reduced glutathione, and arginase in oxidative stress and a concomitant decrease in the endogenous level of H ₂S. Although H ₂S supplementation ameliorated, the effect of HHcy and the levels of H ₂S returned to the average level in HHcy animals supplemented with H ₂S. Interestingly, H ₂S supplementation ameliorated neurovascular remodeling and neurodegeneration. Thus, our study suggested that H ₂S could be a beneficial therapeutic candidate for the treatment of Hcy-associated neurodegeneration, such as stroke and neurovascular disorders.     

Differential Effects of Cystathionine-γ-lyase-Dependent Vasodilatory H(2)S in Periadventitial Vasoregulation of Rat and Mouse Aortas

Background

Hydrogen sulfide (H₂S) is a potent vasodilator. However, the complex mechanisms of vasoregulation by H₂S are not fully understood. We tested the hypotheses that (1) H₂S exerts vasodilatory effects by opening KCNQ-type voltage-dependent (K_v) K⁺ channels and (2) that H₂S-producing cystathionine-γ-lyase (CSE) in perivascular adipose tissue plays a major role in this pathway.

Methodology/Principal Findings

Wire myography of rat and mouse aortas was used. NaHS and 5-(4-hydroxyphenyl)-3H-1,2-dithiole-3-thione (ADTOH) were used as H₂S donors. KCNQ-type K_v channels were blocked by XE991. 4-Propargylglycine (PPG) and ß-cyano-l-alanine (BCA), or 2-(aminooxy)-acetic acid (AOAA) were used as inhibitors of CSE or cystathionine-ß-synthase (CBS), respectively. NaHS and ADTOH produced strong vasorelaxation in rat and mouse aortas, which were abolished by KCNQ channel inhibition with XE991. Perivascular adipose tissue (PVAT) exerted an anticontractile effect in these arteries. CSE inhibition by PPG and BCA reduced this effect in aortas from rats but not from mice. CBS inhibition with AOAA did not inhibit the anticontractile effects of PVAT. XE991, however, almost completely suppressed the anticontractile effects of PVAT in both species. Exogenous l-cysteine, substrate for the endogenous production of H₂S, induced vasorelaxation only at concentrations >5 mmol/l, an effect unchanged by CSE inhibition.

Conclusions/Signficance

Our results demonstrate potent vasorelaxant effects of H₂S donors in large arteries of both rats and mice, in which XE991-sensitive KCNQ-type channel opening play a pivotal role. CSE-H₂S seems to modulate the effect of adipocyte-derived relaxing factor in rat but not in mouse aorta. The present study provides novel insight into the interaction of CSE-H₂S and perivascular adipose tissue. Furthermore, with additional technical advances, a future clinical approach targeting vascular H₂S/KCNQ pathways to influence states of vascular dysfunction may be possible.     

同型半胱氨酸致动脉粥样硬化的作用机制及最新进展

1969年,在研究患有同型半胱氨酸遗传代谢疾病的儿童血管生理时,首次提出了动脉粥样硬化的同型半胱氨酸理论。事实上,自从1969年发现动脉粥样硬化的同型半胱氨酸理论后,许多回顾与展望人类的观察性研究已经将高同型半胱氨酸血症作为动脉粥样硬化的一个独立危险因素。本文主要从以下几个方面探讨同型半胱氨酸致动脉粥样硬化的作用机制:氧化应激,内皮功能障碍和炎症;微生物,脂蛋白和脆性斑块的形成;基质金属蛋白酶和基质金属蛋白酶2。     

cGMP-Dependent Protein Kinase Contributes to Hydrogen Sulfide-Stimulated Vasorelaxation

A growing body of evidence suggests that hydrogen sulfide (H₂S) is a signaling molecule in mammalian cells. In the cardiovascular system, H₂S enhances vasodilation and angiogenesis. H₂S-induced vasodilation is hypothesized to occur through ATP-sensitive potassium channels (K_ATP); however, we recently demonstrated that it also increases cGMP levels in tissues. Herein, we studied the involvement of cGMP-dependent protein kinase-I in H₂S-induced vasorelaxation. The effect of H₂S on vessel tone was studied in phenylephrine-contracted aortic rings with or without endothelium. cGMP levels were determined in cultured cells or isolated vessel by enzyme immunoassay. Pretreatment of aortic rings with sildenafil attenuated NaHS-induced relaxation, confirming previous findings that H₂S is a phosphodiesterase inhibitor. In addition, vascular tissue levels of cGMP in cystathionine gamma lyase knockouts were lower than those in wild-type control mice. Treatment of aortic rings with NaHS, a fast releasing H₂S donor, enhanced phosphorylation of vasodilator-stimulated phosphoprotein in a time-dependent manner, suggesting that cGMP-dependent protein kinase (PKG) is activated after exposure to H₂S. Incubation of aortic rings with a PKG-I inhibitor (DT-2) attenuated NaHS-stimulated relaxation. Interestingly, vasodilatory responses to a slowly releasing H₂S donor (GYY 4137) were unaffected by DT-2, suggesting that this donor dilates mouse aorta through PKG-independent pathways. Dilatory responses to NaHS and L-cysteine (a substrate for H₂S production) were reduced in vessels of PKG-I knockout mice (PKG-I−/−). Moreover, glibenclamide inhibited NaHS-induced vasorelaxation in vessels from wild-type animals, but not PKG-I−/−, suggesting that there is a cross-talk between K_ATP and PKG. Our results confirm the role of cGMP in the vascular responses to NaHS and demonstrate that genetic deletion of PKG-I attenuates NaHS and L-cysteine-stimulated vasodilation.     

Endogenous hydrogen sulfide is involved in the pathogenesis of atherosclerosis

Atherosclerosis is a chronic, complex, and progressive pathological process in large and medium sized arteries. The exact mechanism of this process remains unclear. Hydrogen sulfide (H₂S), a novel gasotransmitter, was confirmed as playing a major role in the pathogenesis of many cardiovascular diseases. It plays a role in vascular smooth muscle cell (VSMC) proliferation and apoptosis, participates in the progress of hyperhomocysteinemia (HHCY), inhibits atherogenic modification of LDL, interferes with vascular calcification, intervenes with platelet function, and there are interactions between H₂S and inflammatory processes. The role of H₂S in atherosclerotic pathogenesis highlights the mysteries of atherosclerosis and inspires the search for innovative therapeutic strategies. Here, we review the studies to date that have considered the role of H₂S in atherosclerosis.     

Hydrogen sulfide attenuates homocysteine-induced osteoblast dysfunction by inhibiting mitochondrial toxicity

Homocysteine (Hcy) is detrimental to bone health in a mouse model of diet-induced hyperhomocysteinemia (HHcy). However, little is known about Hcy-mediated osteoblast dysfunction via mitochondrial oxidative damage. Hydrogen sulfide (H₂S) has potent antioxidant, anti-inflammatory, and antiapoptotic effects. In this study, we hypothesized that the H₂S mediated recovery of osteoblast dysfunction by maintaining mitochondrial biogenesis in Hcy-treated osteoblast cultures in vitro. MC3T3-E1 osteoblastic cells were exposed to Hcy treatment in the presence or absence of an H₂S donor (NaHS). Cell viability, osteogenic differentiation, reactive oxygen species (ROS) production were determined. Mitochondrial DNA copy number, adenosine triphosphate (ATP) production, and oxygen consumption were also measured. Our results demonstrated that administration of Hcy increases the intracellular Hcy level and decreases intracellular H₂S level and expression of the cystathionine β-synthase/Cystathionine γ-lyase system, thereby inhibiting osteogenic differentiation. Pretreatment with NaHS attenuated Hcy-induced mitochondrial toxicity (production of total ROS and mito-ROS, ratio of mitochondrial fission (DRP-1)/fusion (Mfn-2)) and restored ATP production and mitochondrial DNA copy numbers as well as oxygen consumption in the osteoblast as compared with the control, indicating its protective effects against Hcy-induced mitochondrial toxicity. In addition, NaHS also decreased the release of cytochrome c from the mitochondria to the cytosol, which induces cell apoptosis. Finally, flow cytometry confirmed that NaHS can rescue cells from apoptosis induced by Hcy. Our studies strongly suggest that NaHS has beneficial effects on mitochondrial toxicity, and could be developed as a potential therapeutic agent against HHcy-induced mitochondrial dysfunction in cultured osteoblasts in vitro.     

Redox Biochemistry of Hydrogen Sulfide

H₂S, the most recently discovered gasotransmitter, might in fact be the evolutionary matriarch of this family, being both ancient and highly reduced. Disruption of γ-cystathionase in mice leads to cardiovascular dysfunction and marked hypertension, suggesting a key role for this enzyme in H₂S production in the vasculature. However, patients with inherited deficiency in γ-cystathionase apparently do not present vascular pathology. A mitochondrial pathway disposes sulfide and couples it to oxidative phosphorylation while also exposing cytochrome c oxidase to this metabolic poison. This report focuses on the biochemistry of H₂S biogenesis and clearance, on the molecular mechanisms of its action, and on its varied biological effects.     

Relative Contributions of Cystathionine ��-Synthase and ��-Cystathionase to H2S Biogenesis via Alternative Trans-sulfuration Reactions

In mammals, the two enzymes in the trans-sulfuration pathway, cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE), are believed to be chiefly responsible for hydrogen sulfide (H₂S) biogenesis. In this study, we report a detailed kinetic analysis of the human and yeast CBS-catalyzed reactions that result in H₂S generation. CBS from both organisms shows a marked preference for H₂S generation by β-replacement of cysteine by homocysteine. The alternative H₂S-generating reactions, i.e. β-elimination of cysteine to generate serine or condensation of 2 mol of cysteine to generate lanthionine, are quantitatively less significant. The kinetic data were employed to simulate the turnover numbers of the various CBS-catalyzed reactions at physiologically relevant substrate concentrations. At equimolar concentrations of CBS and CSE, the simulations predict that H₂S production by CBS would account for ∼25–70% of the total H₂S generated via the trans-sulfuration pathway depending on the extent of allosteric activation of CBS by S-adenosylmethionine. The relative contribution of CBS to H₂S genesis is expected to decrease under hyperhomocysteinemic conditions. CBS is predicted to be virtually the sole source of lanthionine, and CSE, but not CBS, efficiently cleaves lanthionine. The insensitivity of the CBS-catalyzed H₂S-generating reactions to the grade of hyperhomocysteinemia is in stark contrast to the responsiveness of CSE and suggests a previously unrecognized role for CSE in intracellular homocysteine management. Finally, our studies reveal that the profligacy of the trans-sulfuration pathway results not only in a multiplicity of H₂S-yielding reactions but also yields novel thioether metabolites, thus increasing the complexity of the sulfur metabolome.Hydrogen sulfide (H₂S)² elicits an array of physiological effects, including modulation of blood pressure and reduction of ischemia reperfusion injury (1, 2). Other novel effects of H₂S include induction of a state of suspended animation in mouse by decreasing oxygen consumption and drastically reducing the metabolic rate (3) and synchronizing ultradian metabolic oscillation in yeast (4). Under conditions of metabolic cycling in yeast, H₂S production is catalyzed by sulfite reductase in the sulfur assimilation pathway (4). Inhibition of sulfite reductase reduces H₂S production and in turn perturbs metabolic oscillations. H₂S is a specific and potent inhibitor of cytochrome c oxidase in the electron transport chain (3).Although concentrations of H₂S have been reported to range from 50 to 160 μm in brain (5–7) and 30–50 μm in the peripheral system (8), these appear to be grossly overestimated (9). Significantly lower H₂S concentrations of 17 and 14 nm in liver and brain, respectively, have been reported recently (9). The very significant discrepancy between these and the previous estimates of H₂S levels presumably derives from the earlier use of acidic conditions that led to the release of acid-labile sulfur from iron-sulfur centers.In mammals, the primary catalysts for H₂S generation are reported to be the two pyridoxal phosphate (PLP)-dependent enzymes involved in the trans-sulfuration pathway, cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE) (10, 11). The trans-sulfuration pathway operates in the reverse direction in mammals serving to convert homocysteine to cysteine (Fig. 1), although in yeast and bacteria the pathway is involved in sulfur assimilation from sulfate to cysteine. CBS is widely assumed to be the major contributor to H₂S production in the brain because of its relatively high expression in this organ (10). However, a recent study reported that 3-mercaptopyruvate sulfurtransferase together with cysteine aminotransferase might also generate H₂S in brain (12). The relative contributions of these enzymes and of CSE, which is also present in brain (13, 14), to H₂S production remain to be assessed. Genetic disruption of CSE in mouse leads to cardiac deficits, including pronounced hypertension and reduced endothelium-dependent vasorelaxation, consistent with a major role for CSE in the peripheral system (1). However, brain H₂S levels are reportedly unchanged in CSE^−/− mice.Open in a separate windowFIGURE 1.Diversity of reactions catalyzed by the trans-sulfuration pathway. The turnover numbers (v/[E]) estimated at physiological substrate concentrations, i.e. 10 μm homocysteine, 100 μm cysteine, 560 μm serine, and 5 μm cystathionine, are shown in parentheses for each reaction. The thick arrows highlight reactions that are sensitive to elevated levels of homocysteine. The fold change represents the fold increase in the turnover number of a given reaction under conditions of severe hyperhomocysteinemia (200 μm homocysteine).Despite the growing recognition of the varied physiological effects of H₂S, our understanding of its regulation and mechanism of its biosynthesis is poor. We have recently reported on the complex kinetics of H₂S generation by human CSE (15). The profligacy of the human enzyme affords H₂S generation by a multiplicity of routes involving cysteine and/or homocysteine as substrates. Kinetic simulations predict an increasingly important contribution of homocysteine to H₂S generation with increasing grade of hyperhomocysteinemia, a risk factor for cardiovascular and neurodegenerative diseases (16–18). In addition to H₂S, a variety of products is generated in these reactions, including two novel sulfur metabolites, lanthionine and homolanthionine, which represent the condensation products between 2 mol of cysteine and homocysteine, respectively. Although the steady-state kinetic parameters for H₂S generation from cysteine and homocysteine have been reported for human CBS (hCBS) (19), a comparable detailed kinetic analysis of H₂S generation by CBS by multiple pathways and their sensitivity to the grade of hyperhomocysteinemia is not known. Furthermore, the relative contributions of CBS and CSE to H₂S and lanthionine generation at physiologically relevant concentrations of substrate are not known.Human CBS is a unique heme containing PLP-dependent enzyme (20) that catalyzes the β-replacement of serine by homocysteine to produce cystathionine. The latter is further metabolized by CSE in an α,γ-elimination reaction to produce cysteine. Although yeast and human CBS are highly homologous and catalyze the same chemical reaction with similar kinetic parameters, the yeast enzyme lacks heme and is not allosterically regulated by S-adenosylmethionine (AdoMet) (21).In this study, we have elucidated the kinetics of H₂S biogenesis by yeast and human CBS and used simulations to estimate the relative contributions of CBS and CSE to H₂S production at physiologically relevant concentrations of substrate. We find that CBS and CSE share a common feature, i.e. catalytic promiscuity. However, in contrast to CSE, which is proficient at catalyzing reactions at the β- and γ-carbons of substrates (15), CBS activity is confined to chemical transformations at the β-position. Our studies provide new insights into the existence of alternative trans-sulfuration reactions that can be a source of diverse sulfur metabolites, viz. H₂S, lanthionine, and homolanthionine increasing the diversity of the sulfur metabolome.     

Mitochondrial mechanism of oxidative stress and systemic hypertension in hyperhomocysteinemia

Formation of homocysteine (Hcy) is the constitutive process of gene methylation. Hcy is primarily synthesized by de-methylation of methionine, in which s-adenosyl-methionine (SAM) is converted to s-adenosyl-homocysteine (SAH) by methyltransferase (MT). SAH is then hydrolyzed to Hcy and adenosine by SAH-hydrolase (SAHH). The accumulation of Hcy leads to increased cellular oxidative stress in which mitochondrial thioredoxin, and peroxiredoxin are decreased and NADH oxidase activity is increased. In this process, Ca2+-dependent mitochondrial nitric oxide synthase (mtNOS) and calpain are induced which lead to cytoskeletal de-arrangement and cellular remodeling. This process generates peroxinitrite and nitrotyrosine in contractile proteins which causes vascular dysfunction. Chronic exposure to Hcy instigates endothelial and vascular dysfunction and increases vascular resistance causing systemic hypertension. To compensate, the heart increases its load which creates adverse cardiac remodeling in which the elastin/collagen ratio is reduced, causing cardiac stiffness and diastolic heart failure in hyperhomocysteinemia.     

Vascular dysfunction produced by hyperhomocysteinemia is more severe in the presence of low folate

Earlier we reported that dietary folate depletion causes hyperhomocysteinemia (HHcy) and arterial dysfunction in rats (Symons JD, Mullick AE, Ensunsa JL, Ma AA, and Rutledge JC. Arterioscler Thromb Vasc Biol 22: 772-780, 2002). Both HHcy and low folate (LF) are risk factors for cardiovascular disease. Therefore, the dysfunction we observed could have resulted from HHcy, LF, and/or their combination (HHcy + LF). We tested the hypothesis that HHcy-induced vascular dysfunction is more severe in the presence of LF. Four groups of rats consumed diets for approximately 10 wk that produced plasma homocysteine (microM) and liver folate (microg folate/g liver) concentrations, respectively, of 7 +/- 1 and 15 +/- 1 (Control; Con; n = 16), 17 +/- 2 and 15 +/- 2 (HHcy; n = 17), 10 +/- 1 and 8 +/- 1 (LF; n = 14), and 21 +/- 2 and 8 +/- 1 (HHcy + LF; n = 18). We observed that maximal ACh-evoked vasorelaxation was greatest in aortas and mesenteric arteries from Con rats vs. all groups. While the extent of dysfunction was similar between LF and HHcy animals, it was less severe compared with arteries from HHcy + LF rats. Maximal ACh-evoked vasorelaxation in coronary arteries was not different between Con and LF rats, but both were greater than HHcy + LF animals. In segments of aortas, 1) ACh-evoked vasorelaxation was similar among groups after incubation with the nonenzymatic intracellular O2(-) scavenger Tiron, 2) vascular O2(-) estimated using dihydroethidium staining was greatest in HHcy + LF vs. all groups, and 3) tension development in response to nitric oxide (NO) synthase inhibition was greatest in Con vs. all other groups. We conclude that HHcy + LF evokes greater dysfunction than either HHcy alone (aortas, mesentery) or LF alone (aortas, mesentery, coronary), likely by producing more O2(-) within the vasculature and thereby reducing NO bioavailability.     

Endogenous hydrogen sulfide reduces airway inflammation and remodeling in a rat model of asthma

Endogenous hydrogen sulfide (H₂S) is hypothesized to have an important role in systemic inflammation. We investigated if endogenous H₂S may be a crucial mediator in airway inflammation and airway remodeling in a rat model of asthma and if endogenous H₂S may exert its anti-inflammatory effect by inhibiting inducible nitric oxide synthase (iNOS)/NO pathway. Cystathionine-γ-lyase (CSE; a H₂S-synthesizing enzyme) was mainly expressed in airway and vascular smooth muscle cells in rat lung tissue. Levels of endogenous H₂S was decreased in pulmonary tissue in ovalbumin (OVA)-treated rats. Exogenous administration of NaHS alleviated airway inflammation and airway remodeling: peak expiratory flow (PEF) increased, goblet cell hyperplasia and collagen deposition score decreased, with decreased total cells recovered from bronchoalveolar fluid (BALF) and influx of eosinophils and neutrophils. The H₂S levels of serum and lung tissue were positively correlated with PEF and negatively correlated with the level of eosinophils and neutrophils in BALF, score of lung pathology. NaHS treatment significantly attenuated pulmonary iNOS activation in OVA-treated rats. These results suggest that the CSE/H₂S pathway plays an anti-inflammatory and anti-remodeling part in asthma pathogenesis and could be a novel target in prevention and treatment of asthma.     

Hydrogen Sulphide modulating mitochondrial morphology to promote mitophagy in endothelial cells under high‐glucose and high‐palmitate

Endothelial cell dysfunction is one of the main reasons for type II diabetes vascular complications. Hydrogen sulphide (H₂S) has antioxidative effect, but its regulation on mitochondrial dynamics and mitophagy in aortic endothelial cells under hyperglycaemia and hyperlipidaemia is unclear. Rat aortic endothelial cells (RAECs) were treated with 40 mM glucose and 200 μM palmitate to imitate endothelium under hyperglycaemia and hyperlipidaemia, and 100 μM NaHS was used as an exogenous H₂S donor. Firstly, we demonstrated that high glucose and palmitate decreased H₂S production and CSE expression in RAECs. Then, the antioxidative effect of H₂S was proved in RAECs under high glucose and palmitate to reduce mitochondrial ROS level. We also showed that exogenous H₂S inhibited mitochondrial apoptosis in RAECs under high glucose and palmitate. Using Mito Tracker and transmission electron microscopy assay, we revealed that exogenous H₂S decreased mitochondrial fragments and significantly reduced the expression of p‐Drp‐1/Drp‐1 and Fis1 compared to high‐glucose and high‐palmitate group, whereas it increased mitophagy by transmission electron microscopy assay. We demonstrated that exogenous H₂S facilitated Parkin recruited by PINK1 by immunoprecipitation and immunostaining assays and then ubiquitylated mitofusin 2 (Mfn2), which illuminated the mechanism of exogenous H₂S on mitophagy. Parkin siRNA suppressed the expression of Mfn2, Nix and LC3B, which revealed that it eliminated mitophagy. In summary, exogenous H₂S could protect RAECs against apoptosis under high glucose and palmitate by suppressing oxidative stress, decreasing mitochondrial fragments and promoting mitophagy. Based on these results, we proposed a new mechanism of H₂S on protecting endothelium, which might provide a new strategy for type II diabetes vascular complication.     

Hydrogen sulfide inhibits myocardial injury induced by homocysteine in rats

Hyperhomocysteinemia (HHcy) is a critical independent risk factor for cardiovascular diseases. However, to date, no satisfactory strategies to prevent HHcy exist. Since homocysteine (Hcy) and endogenous H₂S are both metabolites of sulfur-containing amino acids, we aimed to investigate whether a metabolic product of Hcy and H₂S, may antagonize in part the cardiovascular effects of Hcy. In the HHcy rat model injected subcutaneously with Hcy for 3 weeks, H₂S levels and the H₂S-generating enzyme cystathionine γ lyase (CSE) activity in the myocardium were decreased. The intraperitoneal injection of H₂S gas saturation solution significantly reduced plasma total Hcy (tHcy) concentration and decreased lipid peroxidation formation (i.e., lowered manodialdehyde and conjugated diene levels in myocardia and plasma). The activities of myocardial mitochondrial respiratory enzymes succinate dehydrogenase, cytochrome oxidase, and manganese superoxide dismutase, related to reactive oxygen species metabolism, were significantly dysfunctional in HHcy rats. The H₂S administration restored the level of enzyme activities and accelerated the scavenging of H₂O₂ and superoxide anion generated by Hcy in isolated mitochondria. The H₂S treatment also inhibited the expression of glucose-regulated protein 78, a marker of endoplasmic reticulum (ER) stress, induced by Hcy in vivo and in vitro. Thus, HHcy impaired the myocardial CSE/H₂S pathway, and the administration of H₂S protected the myocardium from oxidative and ER stress induced by HHcy, which suggests that an endogenous metabolic balance of sulfur-containing amino acids may be a novel strategy for treatment of HHcy.     

Hyperhomocysteinemia is detrimental to pregnancy in mice and is associated with preterm birth

Elevated levels of homocysteine produce detrimental effects in humans but its role in preterm birth is not known. Here we used a mouse model of hyperhomocysteinemia to examine the relevance of homocysteine to preterm birth. The mouse carries a heterozygous deletion of cystathionine β-synthase (Cbs^+/?). Gestational period was monitored in wild type and Cbs^+/? female mice. Mouse uterine and placental tissues, human primary trophoblast cells, and human myometrial and placental cell lines were used to determine the influence of homocysteine on expression of specific genes in vitro. The activity of BK_Ca channel in the myometrial cell line was monitored using the patch-clamp technique. We found that hyperhomocysteinemia had detrimental effects on pregnancy and induced preterm birth in mice. Homocysteine increased the expression of oxytocin receptor and Cox-2 as well as PGE₂ production in uterus and placenta, and initiated premature uterine contraction. A Cox-2 inhibitor reversed these effects. Gpr109a, a receptor for niacin, induced Cox-2 in uterus. Homocysteine upregulated GPR109A and suppressed BK_Ca channel activity in human myometrial cells. Deletion of Gpr109a in Cbs^+/? mice reversed premature birth. We conclude that hyperhomocysteinemia causes preterm birth in mice through upregulation of the Gpr109a/Cox-2/PGE₂ axis and that pharmacological blockade of Gpr109a may have potential in prevention of preterm birth.     

Tissue-specific downregulation of dimethylarginine dimethylaminohydrolase in hyperhomocysteinemia

Asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide (NO) synthase, has been proposed to be a mediator of vascular dysfunction during hyperhomocysteinemia. Levels of ADMA are regulated by dimethylarginine dimethylaminohydrolase (DDAH). Using both in vitro and in vivo approaches, we tested the hypothesis that hyperhomocysteinemia causes downregulation of the two genes encoding DDAH (Ddah1 and Ddah2). In the MS-1 murine endothelial cell line, the addition of homocysteine decreased NO production but did not elevate ADMA or alter levels of Ddah1 or Ddah2 mRNA. Mice heterozygous for cystathionine beta-synthase (Cbs) and their wild-type littermates were fed either a control diet or a high-methionine/low-folate (HM/LF) diet to produce varying degrees of hyperhomocysteinemia. Maximal relaxation of the carotid artery to the endothelium-dependent dilator acetylcholine was decreased by approximately 50% in Cbs(+/-) mice fed the HM/LF diet compared with Cbs(+/+) mice fed the control diet (P < 0.001). Compared with control mice, hyperhomocysteinemic mice had lower levels of Ddah1 mRNA in the liver (P < 0.001) and lower levels of Ddah2 mRNA in the liver, lung, and kidney (P < 0.05). Downregulation of DDAH expression in hyperhomocysteinemic mice did not result in an increase in plasma ADMA, possibly due to a large decrease in hepatic methylation capacity (S-adenosylmethionine-to-S-adenosylhomocysteine ratio). Our findings demonstrate that hyperhomocysteinemia causes tissue-specific decreases in DDAH expression without altering plasma ADMA levels in mice with endothelial dysfunction.     

Honokiol ameliorates angiotensin II‐induced hypertension and endothelial dysfunction by inhibiting HDAC6‐mediated cystathionine γ‐lyase degradation

Hypertension and endothelial dysfunction are associated with various cardiovascular diseases. Hydrogen sulphide (H₂S) produced by cystathionine γ‐lyase (CSE) promotes vascular relaxation and lowers hypertension. Honokiol (HNK), a natural compound in the Magnolia plant, has been shown to retain multifunctional properties such as anti‐oxidative and anti‐inflammatory activities. However, a potential role of HNK in regulating CSE and hypertension remains largely unknown. Here, we aimed to demonstrate that HNK co‐treatment attenuated the vasoconstriction, hypertension and H₂S reduction caused by angiotensin II (AngII), a well‐established inducer of hypertension. We previously found that histone deacetylase 6 (HDAC6) mediates AngII‐induced deacetylation of CSE, which facilitates its ubiquitination and proteasomal degradation. Our current results indicated that HNK increased endothelial CSE protein levels by enhancing its stability in a sirtuin‐3‐independent manner. Notably, HNK could increase CSE acetylation levels by inhibiting HDAC6 catalytic activity, thereby blocking the AngII‐induced degradative ubiquitination of CSE. CSE acetylation and ubiquitination occurred mainly on the lysine 73 (K73) residue. Conversely, its mutant (K73R) was resistant to both acetylation and ubiquitination, exhibiting higher protein stability than that of wild‐type CSE. Collectively, our findings suggested that HNK treatment protects CSE against HDAC6‐mediated degradation and may constitute an alternative for preventing endothelial dysfunction and hypertensive disorders.