DDR1-p53调控途径对血管衰老过程中平滑肌细胞DNA甲基化的影响机制

Dysregulation of DNA methylation in smooth muscle cells has been shown to play a critical role in vascular aging, however, it remains to be elucidated that how the aging-associated vascular microenvironment results in insufficient DNA methylation. Aging-resulted remodeling of the vascular wall includes increased vascular stiffness, which is mainly attributable to increased stiffness of the extracellular matrix. We have previously reported that extracellular matrix stiffening downregulates the expression DNMT1, an enzyme responsible for catalyzing DNA methylation, to cause insufficient DNA methylation in vascular smooth muscle cells. Recently, we found a negative correlation between DNMT1 expression and discoidin domain receptor (DDR1) phosphorylation in age- or chronic renal failure-induced vascular stiffening; DNMT1 was downregulated whereas DDR1 phosphorylation was elevated in smooth muscle cells in vascular stiffening. In cells cultured on substrates with different stiffness, we found that DNMT1 expression was repressed by increase in substrate stiffness and that this effect could be eliminated when the cells were pretreated with a DDR1 inhibitor, DDR1-IN-1, probably via a p53-dependent mechanism. We hypothesized that the aging-associated mechanical microenvironment downregulates DNMT1 expression in vascular smooth muscle cells through DDR1-mediated mechanotransduction and signaling to result in changes of smooth muscle cell phenotype from contractile to senescence associated secretory phenotype (SASP). In the proposed study, the pathophysiological mechanisms of regulation on DNMT1 expression in smooth muscle cells with SASP will be explored in depth. Verification of the function of DDR1 as a novel mechanosensor will be conducted. The contribution of DDR1-p53-DNMT1 pathway to vascular aging and aging-associated vascular disorders will be ascertained. Our study will further reveal the mechanisms underlying aging-related vascular disorders from a mechanobiological review and might be helpful for the pursuit of the current achievement in the area of DNA methylation toward clinical translational studies.

平滑肌细胞DNA甲基化不足是血管衰老的重要机制，但是衰老血管微环境如何调控DNA甲基化有待探讨。衰老血管特征表现之一为血管硬化尤其是管壁细胞外基质硬化。我们已报道硬化的基质可下调平滑肌细胞中DNA甲基转移酶1（DNMT1）的表达而导致DNA甲基化不足。近期我们发现增龄和肾衰所致硬化血管中DNMT1表达下降，而盘状结构域受体1（DDR1）磷酸化升高；在培养于不同刚度基底表面的平滑肌细胞中，抑制DDR1的磷酸化可能通过减弱基质硬化诱导的p53活化而逆转基质硬化对DNMT1表达的下调。推测衰老血管基质通过DDR1介导的机械力信号转导抑制DNMT1表达，使平滑肌细胞出现衰老样表型。本项目旨在确认机械力信号感受器DDR1通过调节p53活性影响DNMT1表达，明确DDR1-p53-DNMT1调控轴对平滑肌细胞功能的影响及其在衰老相关血管病理改变中的作用，从力学生物学角度解析血管衰老的表观遗传机理。

血管平滑肌细胞具有高度可塑性，其表型可以响应多种微环境因素刺激而发生变化。其中，细胞外基质固有的物理信号（如基质刚度、孔隙、几何与拓扑等）可能与生物化学因素单独或协同作用，参与调控平滑肌细胞的表型与功能。在多种常见的增龄相关的慢性血管炎性疾病中，如动脉粥样硬化，动脉壁刚度显著增加；病变血管中的平滑肌细胞呈现“合成”和“促炎”表型，从而分泌趋化因子和细胞因子，调控单核/巨噬细胞浸润，加重血管炎症。理解平滑肌细胞如何感知微环境中的机械信号，从而调控下游基因表达与活性、影响细胞功能，对动脉粥样硬化相关血管疾病的病理机制理解和防治非常重要。在本项目中，申请人发现高基质刚度激活了平滑肌细胞中的酪氨酸激酶受体DDR1，且机械刺激引起的DDR1活化不依赖于其经典配体胶原与受体的结合；该工作还揭示了由DDR1介导的细胞内力学信号转导通路，证明高基质刚度引起的DDR1激活通过ERK1/2-p53信号轴抑制下游靶基因DNMT1表达；利用平滑肌细胞特异性DNMT1缺陷小鼠和急、慢性血管硬化和衰老的小鼠模型，发现DDR1-DNMT1信号轴在血管平滑肌细胞中调控细胞收缩功能和促炎因子的合成；结合二维微图案模型和三维人工血管移植模型，发现了基质几何形状所传递的物理信号在血管平滑肌细胞中诱导了DNMT1的亚细胞重新分布和mtDNA甲基化水平的变化、DNMT1 与 mtDNA 的结合、mtDNA 基因转录、线粒体功能和能量代谢改变，提示基质几何形状是血管平滑肌细胞功能的关键决定因素。该研究的科学价值在于对机械信号传感器的发现和动脉粥样硬化的力学调控过程的阐明；应用价值在于，设计具有可变刚度、可控微/纳米结构的工程血管移植物可能是优化其性能的有效策略。