eNOS介导外源性硫化氢促进损伤血管再内皮化的机制研究

Early reendothelialization is the key for prevention of coronary stent thrombosis. As a novel gasotransmitter, hydrogen sulfide(H2S)has been proved to possess important physiological and pathophysiologic functions, and exerts regulatory effects on the pathogenesis of various cardiovascular diseases. Our previous data showed that exogenous H2S could accelerate reendothelialization in vivo and enhanced the migration, adhesion capacity of bone marrow derived endothelial progenitor cells(EPCs) in vitro. Recently, accumulating evidence have demonstrated that eNOS is the crucial regulator of EPCs-induced reendothelialization. However, whether eNOS-dependent EPCs mobilization could be involved in H2S-induced the repair process of injured endothelium is unclear. Thus, we propose a hypothesis that eNOS-dependent mobilization bone marrow-derived EPCs by H2S contribute to reendothelialization after vascular injury. To test this hypotheses, the effects of H2S on neointima formation, reendothelialization capacity and EPCs mobilization will be observed in WT and eNOS-deficient mice firstly. Then, we will build WT/Tie-2-GFP and eNOS-/-/Tie-2-GFP chimeric mice with bone marrow transplantation, to investigate the recruitment of EPCs on injured vascular wall, and evaluate the role of eNOS regulation on H2S-induced reendothelialization. Finally, in order to clarify the mechanism of eNOS-mediated EPCs mobilization, the relative signal protein expression and phosphyorylation level of EPCs exposed to NaHS as well as pharmacological inhibitors will also be examined. This study may provide theoretical basis of the mechanism in early reendothelialization after vascular injury , and also provide evidence of effective therapeutic strategy for preventing coronary stent thrombosis .

早期血管再内皮化是防止冠脉支架内血栓的重要策略。我们前期研究发现外源性硫化氢能促进动脉损伤后血管内皮修复，并增强小鼠EPC的黏附和迁移,有关机制尚未明确。研究证实eNOS是骨髓来源EPC参与动脉损伤后再内皮化的关键分子。本项目拟首先观察硫化氢对WT、eNOS基因敲除小鼠颈动脉损伤后EPC动员、血管再内皮化和血管内膜增殖的作用；然后通过构建WT/Tie-2-GFP、 eNOS基因敲除/Tie-2-GFP嵌合鼠，观察硫化氢对动脉损伤后EPC募集的影响，以阐明硫化氢动员EPC，促进损伤血管再内皮化过程中eNOS的关键作用。离体实验部分将使用小鼠骨髓EPC，观察硫化氢对EPC黏附、迁移的影响，检测EPC中相关信号通路蛋白和磷酸化水平，并通过阻断相关分子信号通路，进一步阐明硫化氢经eNOS介导动员EPC的分子机制。研究成果将为深入探讨早期血管再内皮化的相关机制，寻找有效治疗手段奠定坚实的理论基础。

目前，加快血管的再内皮化进程是防止冠脉支架内血栓的重要手段。根据我们前期研究发现外源性硫化氢（H2S）能促进动脉损伤后血管内皮的修复、增强小鼠内皮祖细胞（EPCs）的黏附和迁移。通过本课题，我们证实了eNOS 是骨髓来源的内皮祖细胞（EPCs）参与动脉损伤后再内皮化的关键分子。首先，成功构建WT、eNOS 基因敲除小鼠的颈动脉损伤模型和两者的Tie-2-GFP嵌合鼠模型。使用外源性H2S干预后，WT小鼠的损伤血管段蓝染区的面积明显减少，而eNOS-/-小鼠的H2S处理组和空白组比较未见明显的减少。同时，H2S能以浓度依赖性方式增强EPCs增殖能力、经SDF-1诱导下EPCs的迁移能力和经TNF-a预刺激的内皮细胞黏附能力。而对eNOS-/-小鼠的骨髓源性EPCs的增殖、迁移和黏附的能力，H2S处理组与空白对照组相比无明显差异。使用不同浓度的H2S干预EPCs后检测PI3K/Akt通路后发现，H2S能以浓度依赖性方式上调P-Akt蛋白、P-eNOS蛋白的表达水平和一氧化氮（NO）的水平。在阻断WT小鼠EPCs的PI3K/Akt信号通路后，给予外源性H2S，P-eNOS蛋白的表达水平和NO生成与空白对照组对比无明显差异。并予eNOS的可逆性抑制剂L-NAME刺激后，H2S处理组的EPCs增殖、迁移和黏附能力与空白对照组对比也无明显差异。符合预期计划的设想，在eNOS的介导下，外源性H2S 能加速动脉损伤后再内皮化，而且外源性H2S 能动员EPCs 参与损伤血管内皮修复。外源性H2S 通过激活EPCs 中PI3K/Akt 信号通路，增强eNOS的磷酸化活性，诱导NO合成增加，从而增强EPCs的迁移、黏附及增殖能力。本次课题为外源性H2S 治疗血管损伤性疾病乃至预防晚期支架内血栓形成奠定了坚实的理论基础。