Regulation of gene expression of inducible nitric oxide synthase (iNOS) in cardiovascular system and its molecular mechanism of action

INOS过量产生NO参与内毒素休克和动脉粥样硬化病变的发生发展。为了阐明诱导型一氧化氮合酶在血管内皮细胞中的作用，我们研究了诱导型一氧化氮合酶基因在培养的大鼠血管平滑肌细胞中表达及其作用的分子机制。脂质体转染法将iNOS基因导入VSMC，可导致大量NO生成，并伴有细胞凋亡和DNA合成抑制，提示NO作为促凋亡因子在动脉粥样硬化中起到了预防内膜增厚的作用。NO供体可引起培养的大鼠内皮细胞大量凋亡，其作用可被内皮素1(ET-1)阻断，提示NO作为促凋亡因子发挥作用，而ET-1则作为抗凋亡因子发挥抗凋亡作用，从而相互作用以维持内皮细胞的完整性。炎性细胞因子IL-1和肿瘤坏死因子-α通过激活核因子-kB和降解IkBα而激活核因子-kB，从而导致iNOS基因启动子的反式激活。然而，NO供体通过阻断细胞因子诱导的诱导型一氧化氮合酶α的磷酸化和随后的降解来抑制诱导型一氧化氮合酶基因的表达，这表明诱导型一氧化氮合酶产生的内源性一氧化氮以一种自我反馈的方式抑制诱导型一氧化氮合酶的过度表达。糖皮质激素还通过阻断IkBα的磷酸化和降解来抑制细胞因子诱导的诱导型一氧化氮合酶基因的表达。相反，非甾体抗炎药(NSAID)，如阿司匹林和水杨酸钠，通过阻断(翻译后)水平抑制细胞因子刺激的NO产生，而不影响NF-kB激活或iNOS基因转录。这些数据表明，糖皮质激素和非甾体抗炎药通过通过不同的作用部位阻止NO的产生而发挥抗炎作用。

Excessive production of NO by iNOS is implicated in the development of endotoxic shock and atherosclerotic lesion. To elucidate the role of iNOS-derived NO, we have studied the molecular mechanisms of iNOS gene expression and its actions in cultured rat vascular smooth muscle cells (VSMC). Transfection of iNOS cDNA construct into VSMC by lipofection caused a massive generation of NO accompanied by apoptosis and inhibition of DNA synthesis, suggesting the role of NO as proapoptotic factor to prevent intimal thickening in atherosclerosis. Addition of NO donors caused massive apoptosis of cultured rat endothelial cells (EC), whose effect was blocked by endothelin-1 (ET-1), suggesting that NO functions as a proapoptotic factor, whereas ET-1 function as an anti-apoptotic factor, thereby conteracting with each other to maintain endothelial integrity.Inflammatory cytokines, such as interleukin (IL)-1 and tumor necrosis factor (TNF)-α, activate nuclear factor (NF)-kB by phosphorylation and degradation of IkBα, thereby leading to transactivation of iNOS gene promoter. However, NO donors inhibited cytokines-induced iNOS gene expression by blocking phosphorylation and subsequent degradation of IkBα, suggesting that endogenous NO generated from iNOS inhibits overexpression of iNOS in an autofeedback fashion. Glucocorticoid also inhibited cytokines-induced iNOS gene expression by blockade of IkBα phosphorylation and degradation. In contrast, nonsteroidal anti-inlflammatory drugs (NSAID), such as aspirin and sodium salicylate, inhibited cytokines-stimulated NO production by blocking (post) translational level without affecting NF-kB activation or iNOS gene transcription. These data suggest that glucocorticoid and NSAID exert their anti-inflammatory effects by blocking NO production via different sites of action.