Chaoshu Tang

Role of Hydrogen Sulfide in the Development of Atherosclerotic Lesions in Apolipoprotein E Knockout Mice

Objective—We explored the effect of hydrogen sulfide (H2S) on atherosclerotic progression, particularly on intracellular adhesion molecule-1 (ICAM-1) in apolipoprotein-E knockout (apoE−/−) mice and human umbilical vein endothelial cells (HUVECs). Methods and Results—ApoE−/− mice were treated with sodium hydrosulfide (NaHS) or DL-propargylglycine (PPG); HUVECs were pretreated with NaHS. Compared with control mice, apoE−/− mice showed decreased plasma H2S level and aortic H2S production but increased plasma ICAM-1 and aortic ICAM-1 protein and mRNA. Compared with apoE−/− mice, apoE−/−+NaHS mice showed increased plasma H2S level, but decreased size of atherosclerotic plaque and plasma and aortic ICAM-1 levels, whereas apoE−/−+PPG mice showed decreased plasma H2S level but enlarged plaque size and increased plasma and aortic ICAM-1 levels. NaHS suppressed ICAM-1 expression in tumor necrosis factor (TNF)-&agr;–treated HUVECs. NaHS inhibited I&kgr;B degradation and NF-&kgr;B nuclear translocation in HUVECs treated with TNF-&agr;. Conclusions—The vascular CSE/H2S pathway was disturbed in apoE−/− mice. H2S exerted an antiatherogenic effect and inhibited ICAM-1 expression in apoE−/− mice. H2S inhibited ICAM-1 expression in TNF-&agr;-induced HUVECs via the NF-&kgr;B pathway.

Protective effects of ghrelin on ischemia/reperfusion injury in the isolated rat heart.

Ghrelin, an endogenous ligand of the growth hormone secretagogue receptor, has been reported to have beneficial effects on cardiac function. The authors used the Langendorff model of ischemia/reperfusion (I/R) injury in isolated rat heart to determine whether ghrelin exerts direct cardioprotective effects. Also, the capacity of ghrelin to bind to sarcolemmal membrane fractions before and after ischemia and reperfusion was examined. Compared with vehicle administration, administration of ghrelin (100–10,000 p M) during the reperfusion period resulted in improvement in coronary flow, heart rate, left ventricular systolic pressure, and left ventricular end-diastolic pressure. Ghrelin also enhanced the rates of left ventricular contraction and relaxation after ischemia following reperfusion. Administration of ghrelin during reperfusion reduced myocardial release of lactate dehydrogenase and myoglobin, indicating protection against cardiomyocyte injury. In addition, ghrelin attenuated the depletion of myocardial ATP resulting from ischemia and reperfusion. A receptor-binding assay demonstrated that maximum binding capacity of ghrelin to sarcolemmal membranes was significantly increased after ischemia and was further increased after I/R. However, Scatchard analysis showed that the affinity of ghrelin for its receptor was not altered. The authors have concluded that administration of ghrelin during reperfusion protects against myocardial I/R injury. The cardioprotective effects are independent of growth hormone release and likely involve binding to cardiovascular receptors, a process that is upregulated during I/R.

The possible role of hydrogen sulfide as a smooth muscle cell proliferation inhibitor in rat cultured cells

Hydrogen sulfide (H2S) was recently suggested to be a possible endogenous gasotransmitter in physiological concentration. For the purpose of understanding its possible role in the regulation of the cardiovascular system, we explored the potential effect of H2S on the proliferation of cultured aortic vascular smooth muscle cells (VSMCs) of rats and mitrogen-activated protein kinase (MAPK) as a signaling transduction pathway. Vascular smooth muscle cells were cultured in vitro and the cells were divided into six groups: (1) control group, (2) serum group, (3) endothelin group, (4) NaHS group, (5) serum + NaHS group, and (6) endothelin + NaHS group. VSMC proliferation was measured by [3H]thymidine ([3H]TdR) incorporation and MAPK activity in the VSMCs was determined by radioactivity assay. The results showed that endothelin-1 increased VSMC [3H]TdR incorporation 2.39-fold (P ≪ 0.01) and MAPK activity 1.62-fold (P ≪ 0.01), as compared with controls. Hydrogen sulfide at 5 × 10−5 mol/l, 1 × 10−4 mol/l, and 5 × 10−4 mol/l decreased VSMC [3H]TdR incorporation by 16.8%, 26.60%, and 37.40%, respectively, and reduced MAPK activity by 7.37% (P ≫ 0.05), 23.39%, and 33.57%, respectively (P ≪ 0.01). The results demonstrated that H2S could dose-dependently suppress the proliferation of VSMCs through the MAPK pathway.

Angiotensin II up-regulates soluble epoxide hydrolase in vascular endothelium in vitro and in vivo

Epoxyeicosatrienoic acids (EETs), as metabolites of arachidonic acid, may function as antihypertensive and antiatherosclerotic mediators for vasculature. EETs are degraded by soluble epoxide hydrolase (sEH). Pharmacological inhibition and genetic ablation of sEH have been shown to increase the level of EETs, and treating angiotensin II (Ang II)-infused hypertension rats with sEH-selective inhibitors increased the levels of EETs, with attendant decrease in systolic blood pressure. To elucidate the mechanisms by which Ang II regulates sEH expression, we treated human umbilical vein endothelial cells (ECs) and bovine aortic ECs with Ang II and found increased sEH expression at both the mRNA and protein levels. Transient transfection assays showed that the activity of the human sEH promoter was increased in ECs in response to Ang II. Further analysis of the promoter region of the sEH gene demonstrated that treatment with Ang II, like overexpression of c-Jun/c-Fos, activates the sEH promoter through an AP-1-binding motif. The binding of c-Jun to the AP-1 site of the sEH promoter was confirmed by chromatin immunoprecipitation assays. In contrast, adenovirus overexpression of the dominant-negative mutant of c-Jun significantly attenuated the effects of Ang II on sEH induction. An elevated level of sEH was found in the aortic intima of both spontaneously hypertensive rats and Ang II-infused Wistar rats. Blocking Ang II binding to Ang II receptor 1 by losartan abolished the sEH induction. Thus, AP-1 activation is involved in the transcriptional up-regulation of sEH by Ang II in ECs, which may contribute to Ang II-induced hypertension.

Therapeutic effects of ghrelin on endotoxic shock in rats.

We investigated the effects of ghrelin in a rat endotoxic shock model, and also observed the direct role of endotoxin on ghrelin generation in gastric mucosa. About 55% (11/20) of rats treated with lipopolysaccharide (5 mg/kg i.v.) alone died within 24 h of endotoxin injection. However, administration of ghrelin either at the same time as lipopolysaccharide injection (early treatment) or 12 h after lipopolysaccharide injection (late treatment) significantly decreased the mortality rate and ameliorated the hypotension seen in rats with endotoxic shock. Early and late treatment with ghrelin increased markedly the plasma glucose concentration and decreased the plasma lactate concentration. Early treatment with ghrelin attenuated significantly the deficiency in myocardial ATP content, but late treatment with ghrelin had no effect on myocardial ATP content. The plasma ghrelin level was significantly increased in the rats with endotoxin shock, and it increased further after ghrelin administration. Exposure of rat gastric mucosa in vitro to lipopolysaccharide (1.0 to 100 microg/ml) triggered the release of ghrelin from mucosa tissue in a dose- and time-dependent manner, meaning that lipopolysaccharide stimulated directly gastric mucosa to synthesize and secrete ghrelin. The results suggest that ghrelin could have therapeutic value for endotoxic shock.

Hydrogen sulfide protects rat lung from ischemia–reperfusion injury

Recent studies have indicated that hydrogen sulfide (H(2)S) is capable of modulating many physiological processes, which prompted us to investigate the potential of H(2)S as a lung protective agent. To explore changes in the generation of endogenous H(2)S and the role of H(2)S in the pathogenesis of pulmonary ischemia-reperfusion (I/R) injury in rats, we built an isolated rat lung I/R model. Lungs were subjected to 45 min ischemia followed by reperfusion (45 min) and were pretreated with H(2)S (50 micromol/l or 100 micromol/l) or an irreversible inhibitor of cystathionine-gamma-lyase (CSE), propargylglycine (PPG; 2 mmol/l). We examined indices of lung injury: lung histological change, perfusion flow rate, ratio of lung wet weight to dry weight (w/d), and lung compliance. H(2)S content and CSE protein expression in lung tissues were measured. Malondialdehyde (MDA) content, activities of superoxide dismutase (SOD) and catalase (CAT), and restraint of superoxide anion (O(2)(-)) production in lung tissues were measured to reflect oxidative stress. In the current study, we demonstrated that H(2)S content and CSE activity in lungs after I/R were significantly higher than those in the control group. Preperfusion with H(2)S attenuated the lung I/R injury while preperfusion with PPG aggravated the lung I/R injury. H(2)S preperfusion reduced I/R-induced MDA production and potentiated SOD and CAT activities and the restraint of O(2)(-) production in the lungs under I/R, which attenuated lung oxidative injury. These findings suggest that endogenous CSE/H(2)S pathway might be involved in the pathogenesis of lung I/R injury and that administration of H(2)S might be of clinical benefit in lung I/R injury.

Changes in arterial hydrogen sulfide (H2S) content during septic shock and endotoxin shock in rats

OBJECTIVES To explore the changes of hydrogen sulfide (H(2)S) in vascular tissues of rats with septic shock and endotoxin shock and its possible pathophysiological implication. METHODS Rat models of septic shock induced by cecal ligation and puncture and of endotoxic shock induced by injection of endotoxin were used in this study. The authors measured hymodynamic variations, metabolic data, H(2)S and nitric oxide (NO) contents of different arteries in rats with septic shock and endotoxic shock. RESULTS The results showed that hemodynamic parameters including the heart rate (HR), the mean arterial pressure (BP), and the +dP/dt max decreased markedly, while the left ventricular end-diastolic pressure (LVEDP) increased significantly and the rats developed hypoglycemia and lactic acidosis. Arterial H(2)S contents were significantly increased (P<0.01) in both septic and endotoxic shock (P<0.01). Endogenous H(2)S and NO contents all negatively correlated with BP, cardiac function and the degree of hypoglycemia (P<0.01). CONCLUSIONS The results of our study demonstrated that endogenous vascular H(2)S increased in rats with septic shock and endotoxic shock. It was suggested that endogenous H(2)S was involved in physiological and pathophysiological process during shock.

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

Endogenous hydrogen sulfide (H(2)S) is hypothesized to have an important role in systemic inflammation. We investigated if endogenous H(2)S may be a crucial mediator in airway inflammation and airway remodeling in a rat model of asthma and if endogenous H(2)S may exert its anti-inflammatory effect by inhibiting inducible nitric oxide synthase (iNOS)/NO pathway. Cystathionine-gamma-lyase (CSE; a H(2)S-synthesizing enzyme) was mainly expressed in airway and vascular smooth muscle cells in rat lung tissue. Levels of endogenous H(2)S 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 H(2)S 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/H(2)S 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 sulfide derived from periadventitial adipose tissue is a vasodilator

Background Recently, periadventitial adipose tissue (PAT) was found to secrete bioactive factors playing an important role in the regulation of vascular tension. Hydrogen sulfide (H2S), a novel cardiovascular gasotransmitter, relaxes vessels through the KATP channel in a calcium-dependent and endothelial-independent manner. Methods We first identified the endogenous H2S-generated key enzyme cystathionase (CSE) expressed in adipocytes of PAT and H2S released from PAT in rats. Results The CSE inhibitors DL-propargylglycine and/or beta-cyano-L-alanine largely blocked the vasorelaxing effects on aorta rings (removed adventitia), induced by PAT, the culture medium of PAT or isolated adipocytes from PAT. Phenylephrine, serotonin and angiotensin II inhibited endogenous H2S production from the aortic medium but increased its release from PAT. Endogenous H2S generated in the aorta and PAT was decreased but the level of CSE protein was increased with the aging of rats. In rats with hypertension induced by abdominal aortic banding, H2S generation and CSE protein expression were significantly increased in PAT but not aortic tissues. Transplanting PAT into periadventitia of stenotic aortas ameliorated the elevated arterial blood pressure and decreased angiotensin II level in aorta. Conclusion These results suggested PAT could endogenously generate H2S, which might act as an adipocyte-derived relaxing factor and contribute to the pathogenesis of hypertension.

Apelin protects myocardial injury induced by isoproterenol in rats

We aimed to explore the change in level of apelin and its receptor APJ during myocardial injury and the therapeutic effects of apelin in myocardial injury. Rat myocardial injury was induced by subcutaneous injection of a high dose of isoproterenol (ISO); apelin and APJ mRNA levels were determined by RT-PCR; APJ protein was determined by Western blot; EIA and RIA were used to measure the apelin content and receptor binding, respectively. Plasma lactate dehydrogenase (LDH) activity and myocardial and plasma malondialdehyde (MDA) contents were higher in ISO-treated hearts than that in controls. ISO-treated rats showed lower +/-LV dp/dt(max) values and higher LVEDP value (all P<0.01), which suggested severe heart failure. As well, the apelin content in plasma, atrial and ventricular myocardium was decreased by 27%, 30% and 25% (P<0.01), respectively. The mRNA levels of apelin and APJ in myocardia were also markedly reduced; but the APJ protein level in myocardia was increased. However, administration of apelin significantly ameliorated myocardial injury and ISO-induced heart failure. Compared with the ISO-alone group, the group given low-dosage apelin (5 nmol/kg/day) had 39% and 66% higher +LV dp/dt(max) and -LV dp/dt(max) values, and 40.7% lower LVEDP value (P<0.01), and the leakage of myocardial LDH and increased MDA content were attenuated (all P<0.01). Interestingly, bolus injections of apelin (10 nmol/kg/day) resulted in potent inotropic effects in ISO-treated rats. ISO-induced myocardial injury resulted in hypoexpression of apelin and its receptor APJ, and the administration of exogenous apelin ameliorated heart failure and myocardial injury. Apelin could have a cardioprotective effect, and the apelin-APJ system may be a new therapeutic target in myocardial injury and heart failure.