平滑肌细胞腺苷激酶对血管重塑的作用及机理

Vascular smooth muscle cells (VSMC) play a pivotal role during vascular remodeling. However, it is not clear whether and how intracellular adenosine regulated by adenosine kinase (ADK) is critical for vascular remodeling. Our previous studies demonstrate that endothelial ADK knockout elevate endogenous adenosine level resulting in inhibiting reendothelialization, vascular inflammation and enhanced angiogenesis. VSMC ADK knockout can impair cell growth, the neointima formation after carotid artery ligation, decrease methylation and enhance KLF4 expression, a key marker gene for smooth muscle cell phenotypic switch. We hypothesis that SMC intracellular adenosine regulated by ADK is essential for vascular remodeling. Pathological factors can induce ADK expression, and then decrease intracellular adenosine, subsequence induced KLF4 methylation controled by DMNT3a, resulting in enhanced proliferation regulated by KLF4 during vascular remodeling. The goals of this proposal is designed to use tissue-specific ADK-deficient mice, a mouse model of a guide wire-induced transluminal injury of carotid artery, bisulfite sequencing, luciferase activity assay, as well as CHIP and CO-IP to investigate whether intracellular adenosine regulated by ADK can suppress SMC proliferation during vascular remodeling and the underlying epigenetic mechanism. Completion of this project will advance a new ADK-based paradigm for suppression neointimal hyperplasia and may lead to a more effective therapy of arterial disease.

平滑肌细胞(VSMC)是血管重塑关键执行细胞。但其细胞内腺苷激酶(ADK)调节的腺苷代谢系统对血管重塑的作用及机理并不清楚。我们前期研究发现：内皮细胞敲除ADK提升内源性腺苷能促进内皮修复，抑制炎症，促进血管再生。平滑肌ADK敲除抑制细胞增殖；抑制小鼠动脉结扎诱发的血管内膜增生；抑制甲基化，上调平滑肌细胞表型转换重要基因—KLF4。我们提出假设：平滑肌细胞ADK调节的腺苷代谢系统对血管重塑有重要调节作用。病理因素促使ADK表达，降低胞内腺苷浓度，增强DNMT3a调节KLF4甲基化，抑制KLF4基因表达，促进增殖表型，调节血管重塑。本项目拟采用VSMC特异性ADK敲除小鼠、内皮损伤模型、亚硫酸氢钠测序法、启动子活性、CHIP、CO-IP等技术研究ADK调节的腺苷代谢系统对血管重塑作用及机理。该项目从腺苷代谢角度揭示血管重塑平滑肌增殖新机制，为防治血管重塑性疾病提供新的靶点—ADK。

血管重塑是多种原因诱发的血管重新分布、血管结构与功能的改变。血管重塑对心血管疾病发生和发展具有重要作用。但针对机体内源性维持血管稳态的保护性系统研究不多。本项目将采用平滑肌ADK特异性敲除小鼠，研究ADK介导的内源性腺苷代谢通路对血管重塑的作用及机理。..围绕科学假设：平滑肌细胞ADK调节的腺苷代谢系统对血管重塑具有重要调节作用。病理因素促使平滑肌ADK表达增强，导致细胞内腺苷浓度降低；腺苷对DNMT3a的抑制作用减弱，增强了DNMT3a调节的KLF4基因甲基化修饰，抑制KLF4基因表达，促进血管平滑肌细胞的增殖表型，最终调节血管重塑。.本项目重要研究了内容：.1.平滑肌细胞内ADK调节的腺苷代谢系统是否调节血管重塑；.2.ADK调节的腺苷代谢系统是否调节基因组DNA甲基化修饰；.3.ADK调节的腺苷代谢系统如何调节下游转录因子KLF4启动子区域甲基化修饰以及该甲基化修饰作用是否调节血管重塑；.4.靶向干预ADK能否调节内源性腺苷对血管重塑的抑制作用。..研究结果：.1.ADK对病理性血管重塑有重要调节作用，平滑肌特异性ADK 敲除（Myh11-Cre/ERT2/Adkf/f）抑制颈总动脉结扎、guide wire injury诱导的血管内膜层增生；.2.DNA甲基化与血管内膜层增生有密切联系，抑制平滑肌DNA甲基化抑制颈总动脉结扎诱导的血管内膜层增生；.3.ADK对平滑肌DNA甲基化具有重要调节作用，调节KLF4启动子区域甲基化而调节KLF4的表达，抑制平滑肌细胞从收缩表型向合成表型转化，最终抑制病理因素诱导的血管重塑。..本项目科学意义：.阐明ADK调节的腺苷代谢通路与血管重塑中平滑肌细胞增殖表型的关系，在理论价值和临床运用均有一定科学价值。理论上能阐明：⑴ 在血管重塑过程中，机体存在内源性维持血管稳态的保护系统—腺苷代谢调节系统。⑵ 内源性腺苷代谢调节系统维持血管稳态的分子机制—DNA甲基化修饰。临床运用：ADK靶向干扰有望用于血管再狭窄、动脉粥样硬化等心血管疾病的预防和控制。