基质金属蛋白酶在分化抑制因子-1调控易损斑块内血管新生中的作用及其分子机制

Atherosclerotic vulnerable plaque rupture is the major cause of acute cardiovascular disease. Angiogenesis is a pivotal event resulting in plaque rupture. However, the underlying mechanism is poorly understood. Our previous studies have found that inhibitor of differentiation-1 (Id1) could enhance angiogenesis within rupture-prone plaque. On the basis of the fact that activation of matrix metalloproteinase members (MMPs) appears to facilitate angiogenesis, plaque destabilization and be accompanied with abnormal modification of DNA-methylation, therefore, we hypothesized that angiogenesis in rupture-prone plaque is mediated via DNA-methylation modification of MMPs regulated by Id1. To prove the hypothesis, ApoE-/- mouse model, cell transfection, immunohistochemistry and vascular lumen formation test were employed in this study to clarify how MMPs signal take part in regulating neovascularization in instable plaque and obtain MMPs which are specifically regulated by Id1 in a screening fashion. Moreover, based on DNA-Methylation regulation of MMPs ,both in vitro culture systems and in vivo animal model were adopted and combined with bioinformatics, siRNA, CRISPR/cas9 and confocal live image technology to explore the molecular mechanism of regulation of angiogenesis in rupture-prone plaque by MMPs in response to Id1. This study will significantly enhance our understanding of angiogenesis related-mechanism associated with the atherosclerotic progression, and also may provide a new therapeutic target for clinical treatment of atherosclerotic rupture-prone plaques.

动脉粥样硬化（AS）易损斑块的破裂是急性心脑血管疾病发生的主要原因。易损斑块内的血管新生是导致斑块破裂的核心事件，但其分子机制尚未阐明。本实验室前期研究发现：分化抑制因子-1(Id1)促进易损斑块内血管新生。基于基质金属蛋白酶（MMPs）的激活会增强血管新生、促进易损斑块的形成，并伴随MMPs 的DNA甲基化修饰异常，本项目提出如下假设：Id1通过调控MMPs的DNA甲基化修饰介导易损斑块内血管新生。为证实该假设，项目拟通过建立ApoE-/-小鼠动物模型，运用血管管腔形成检验等方法明确MMPs参与调控易损斑块血管新生过程；再以MMPs的DNA甲基化修饰为切入点，采用体外培养系统以及动物模型，结合生物信息学及CRISPR/cas9技术等，探讨MMPs在Id1调控易损斑块血管新生中的作用及其分子机理。本研究不仅有助于阐明易损斑块内血管新生发生的分子机制，并且将为易损斑块的临床治疗提供新的靶点。

易损斑块的破裂是急性心脑血管疾病发生的主要原因。因此，深入研究易损斑块发生机制对降低AS疾病的致残致死率有至关重要的意义。易损板块内的血管新生是导致斑块破裂的核心事件，但是其发生的分子机制一直没能阐明。本研究在ApoE-/-小鼠中成功构建颈总动脉套环狭窄模型和颈动脉扰动切应力模型以模拟高，低和扰动切应力，通过超声检测其血流状态后进行高脂饲料喂养以构建动AS易损斑块模型。通过对斑块形态学、内皮完整性以及血管新生能力的检测，明确了易损斑块内血管新生与切应力的关系：高切应力和扰动切应力都会促进斑块内血管新生。通过在体及体外实验筛选出MT1-MMP、MT4-MMP以及MMP9是参与切应力介导的斑块内血管新生的重要蛋白，高切应力主要影响膜型MT1-MMP和MT4-MMP的表达，而MMP9则主要对扰动切应力产生响应。接着我们在细胞和分子水平上阐述了切应力对力学响应蛋白Id1的调控和Id1调控MT1-MMP、MT4-MMP以及MMP9在血管新生中的力学生物学机制。研究结果显示Id1能调控MT1-MMP、MT4-MMP和MMP9降解胞外基质来增强细胞迁移、侵袭能力，进而调控血管新生过程。此外，本研究还通过甲基化酶抑制剂作用，初步发现MMP9可能是通过甲基化水平改变调节其表达水平，进而调节血管新生过程。综上所述，本研究的所有结果阐明Id1参与高切应力和扰动切应力介导易损斑块形成的分子机制，这将会为深入了解AS易损斑块的发生机制提供一定的理论依据，并且为易损斑块的临床治疗提供新的靶点。