| Hydrogen sulfide (H2S) is well known as a toxic gas with the characteristic smell of rotten eggs. But it is becoming increasingly clear that mammalian cells also produce H2S. The H2S concentration in rat serum is about 46μmol/L. Endogenous H2S is synthesized naturally in the body from L-cysteine mainly by the activity of two enzymes, cystathionineβ-synthase (CBS) and cystathionineγ-lyase (CSE). CBS seems to be main H2S-forming enzyme in the brain and nervous system, whereas CSE is the main H2S-synthesizing enzyme in the cardiovascular system. It is reported that physiological concentrations of H2S specifically potentiate the activity of the N-methyl-D-aspartate (NMDA) receptor, facilitate the induction of hippocampal long-term potentiation. In cardiovascular system, H2S can relax smooth muscle cells by activating ATP-dependent K+ channels (KATP) and decrease the blood pressure. The deficit of H2S/CSE system may be responsible for the development of spontaneous hypertension. The administration of exogenous H2S can effectively prevent myocardial ischemia injury. The endogenous metabolism and physiological functions of H2S position this gas well in the novel family of gasotransmitters together with nitric oxide (NO) and carbon monoxide (CO).Angiogenesis, the formation of new blood vessels from pre-existing vessels, plays a fundamental role in a variety of physiological and pathological processes. Transient, highly regulated growth of new capillaries occurs during embryonic development, and in reparative processes. Persistent and uncontrolled angiogenesis is observed most dramatically in tumour development. So regulation of angiogenesis is important for medical therapy. NO and CO have been implicated in angiogenesis by many reports and they can interact with H2S. That brings about the hypothesis of the present study, i.e. H2S may regulate the process of angiogenesis. However, most studies concentrate on regulation of blood pressure and effect on smooth muscle cells and there is no line about the potential role of H2S in angiogenesis yet. The purpose of the present study is to assess the physiological role of H2S in the regulation of angiogenesis.First, the expression of CSE and CBS in endothelial cells was investigated. Three kinds of endothelial cells of different species were studied in our experiment. They are primary cultured rat cardiac microvascular endothelial cells (CMEC), RF/6A and bEnd.3. Primers for CSE,CBS and housekeeping genes were designed for each kind of cells. Using RT-PCR, the fragments of CSE, but not CBS, were detected in all three kinds of endothelial cells, which suggested that endothelial cells express CSE mRNA. Furthermore, immunofluorescence staining of CMEC for antibodies against CSE and CBS showed that endothelial cells expressed CSE but not CBS. It was the first time that expression of CSE was found in endothelial cells, which implied that H2S may exert some physiological effects on endothelial cells.Then the effect of H2S on endothelial cells and angiogenesis in vitro was explored. Treatment with high concentrations (500 - 1000μmol/L) of NaHS (H2S donor) resulted in a significant reduction in cell viability and relatively low concentrations (1 - 200μmol/L) didn't affect cell viability as assessed using the MTT method, which indicates high concentrations of H2S may have cellular toxicity. Next cell growth was examined with BrdU incorporation assay and we found that NaHS (10 and 20μmol/L) stimulated endothelial cell growth. The effects of H2S on cell adhesion, cell migration and tube formation were also studied. Similarly, H2S at relatively low concentrations promoted adhesion, migration and tube formation ability of endothelial cells. These data suggested H2S can promote angiogenesis in vitro.We further investigated the mechanism of angiogenic effect of H2S. NaHS treatment induced a dose and time dependent increase in Akt phosphorylation in endothelial cells, suggesting a role for Akt in H2S-induced effects. Both the PI3K inhibitors LY 294002 and wortmannin prevented H2S-induced Akt phosphorylation. Moreover, H2S-induced endothelial cell migration and tube formation were also blocked by either LY 294002 or transfection with the dominant-negative Akt mutant. These data suggest that H2S stimulates angiogenesis by activating Akt. As for ERK and p38, neither of them was activated by H2S. Survivin has been reported to be a downstream effector of the PI3K/Akt pathway and promotes angiogenesis. We found here that survivin level was also increased in response to H2S, suggesting activation of the PI3K/Akt/survivin pathway. Also, integrin levels in RF/6A endothelial cells treated with H2S were measured. NaHS treatment significantly increased integrin a2 andβ1 levels. Whereas expression of integrinα1,αv,β3 andβ5 were not changed significantly in our experiment.The effect of H2S on angiogenesis in vivo was then explored. Matrigel plug assay in C57 mice was used in this study. Matrigel plugs were paraffin embedded for histological examination. Sections were stained with hematoxylin-eosin or Masson's trichrome. There was a significant increase in cellular infiltration and neovascularization in Matrigel following administration of H2S. Neovascularization was further quantified by measuring hemoglobin content in the Matrigel plugs. Compared with vehicle treatment, hemoglobin contents were significantly increased in H2S-administrating group. These data suggest H2S can promote angiogenesis in vivo.Furthermore, we constructed constitutive activation and dominant-negative survivin plasmids for further investigation.In summary, the present study provides the first evidence of expression of CSE in endothelial cells. H2S can promote angiogenesis both in vitro and in vivo, which probably relates to the activation of PI3k/Akt. |
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