周期性张应力经Integrinβ1/OCT4正反馈环路触发血管平滑肌细胞表型转化致胸主动脉夹层发生的作用机制

The phenotype transition of vascular smooth muscle cells (VSMC) as response to injury factors is the pivotal issue during the pathogenesis of thoracic aortic dissection (TAD). We have previously reported that OCT4 would promote such VSMC phenotype transition. In recent years, the exploration in the field of clarifying the effect and mechanisms of mechanical factors on triggering the initiation of TAD pathogenesis process and finally the occurrence of it confirmed that, pathologically enhanced cyclic strain (CS) of aortic wall during the cardiac cycle would also induce the phenotype transition of VSMC. Our preliminary experimental results have showed that enhanced CS would upregulate OCT4 activity in VSMC, overexpression of OCT4 would upregulate the expression level of the mechanosensing molecule integrinβ1, and stimulating integrinβ1 with enhanced CS would activate OCT4 via AKT pathway and thus promote VSMC phenotype transition. Based on these results, we proposed the hypothesis that CS would trigger VSMC phenotype transition via integrinβ1/OCT4 positive feedback loop. In order to verify this hypothesis, we would first evaluate the mechanisms of OCT4 regulating the expression of integrinβ1 as well as the mechanisms of integrinβ1 upregulating the biological activity OCT4 via AKT pathway. Then we would verify the existence of integrinβ1/OCT4 positive feedback loop regulation mode under high CS stimulation. Finally, we would clarify the effect of integrinβ1/OCT4 positive feedback loop in high CS promoted VSMC phenotype transition and the development of TAD. Results of this program would provide theoretical basis for exploring potential molecule target for drug therapy of TAD.

血管平滑肌细胞（VSMC）损伤应答性表型转化是胸主动脉夹层（TAD）致病中轴环节，我们已报道OCT4可促进上述转化。近年对力学因素触发TAD的探索证实，与心搏周期一致的主动脉壁周期性张应力（CS）亢进亦可诱发VSMC表型转化。前期已证实高CS可上调VSMC的OCT4活性，OCT4可上调力学感应器Integrinβ1表达，高CS刺激Integrinβ1经AKT活化OCT4并促VSMC表型转化。据此提出假说：CS经Integrinβ1/OCT4正反馈环路触发VSMC表型转化。为其验证，拟先明确OCT4调控Integrinβ1表达及Integrinβ1经AKT上调OCT4活性的机制，验证Integrinβ1/OCT4在高CS下的正反馈环路调控模式，再经体内外实验阐明Integrinβ1/OCT4环路在高CS触发VSMC表型转化及TAD发病中的作用，结果有望为寻找药物干预TAD的靶点提供理论依据。

血管平滑肌细胞（VSMC）去分化性表型转化是胸主动脉夹层（TAD）这一致死性胸部大血管疾病的发病中轴环节。本项目负责人在既往主持并已结题的国家自然科学基金青年项目（81300233）资助下已公开报道在TAD临床标本中表达升高的OCT4通过调控下游关键分子元件促进VSMC发生上述表型转化。血流机械力学因素异常升高则是TAD发病的始动触发因子，现已明确心搏射血对主动脉壁的周期性张应力（CS）亢进可促VSMC发生上述表型转化，结合前期实验显示高CS可促VSMC中OCT4磷酸化激活并入核等结果，本项目提出并明确了在TAD“CS经Integrinβ1/OCT4正反馈环路触发VSMC表型转化”这一尚无文献报道的科学假说内容。.本项目通过细胞水平过表达和敲低实验明确了VSMC中OCT4与Integrinβ1相互调控作用。OCT4正向调控Integrinβ1转录表达，而高CS作用于VSMC通过Integrinβ1上调了OCT4磷酸化水平并促其进入胞核。报告基因系统和ChIP-PCR明确了OCT4与Integrinβ1启动子序列结合的直接转录上调作用。通过VSMC中AKT活性的诱导和抑制，明确了Integrinβ1在高CS刺激VSMC后对OCT4活化及入核的促进作用是通过AKT介导的。在此基础上通过细胞功能实验证实了高CS在 VSMC通过Integrin β1/AKT/OCT4 正反馈环路调控机制促VSMC发生去分化性表型转化的作用，分别干预该环路中的关键节点分子Integrinβ1、AKT、OCT4，当降低其表达或活性时均可阻断该正反馈交互调控效应并抑制VSMC表型去分化。此外我们发现另一主动脉扩张性疾病胸主动脉瘤（TAA）临床标本中Integrin β1表达升高，TAA小鼠模型中Integrin α1/β1表达也较正常主动脉显著增加，抑制Integrin α1/β1可显著缓解TAA进展。在细胞水平阻断Integrin α1/β1可降低VSMC增殖、迁移能力、MMP2、胶原I/III含量，细胞和动物实验显示Integrin α1/β1通过AKT和FAK通路实现对VSMC表型去分化的促进作用。综上，项目研究结果显示Integrin β1/OCT4 正反馈环路是高血流应力作用下触发VSMC表型转化及TAD/TAA的重要分子机制，对其干预或可为临床延缓TAD破裂和TAA进展提供潜在方法。