整合素连接激酶调控低氧诱导肺动脉平滑肌细胞表型转换的机制

The transition from the contractile phenotype of normal pulmonary artery smooth muscle (PASMC) to proliferative and synthetic phenotype is a key pathologically responsible for hypoxia-induced pulmonary arterial remodeling. Serum response factor (SRF), regulates most SMC marker genes expression by binding to CArG elements. The discovery of SRF cofactor myocardin was the most important advance in our understanding of the transcription mechanisms that regulate SMC-specific gene expression. However, it is not known how the functions of myocardin affected by hypoxia during the phenotypic modulation of PASMC. Our previously studies have found that hypoxia decreased the integrin-linked kinase (ILK) activity and expression. In addition, ILK can repress the expression of myocardin for target. We used constructing recombinant adenovirus vectors for gene overexpression and RNA interference, protein and chromatin immunoprecipitation and luciferase reporter analysis and so on, from the aspects of molecular, cell, tissue and animal model, in order to verify the hypothesis that ILK regulate hypoxia- induced PASMC phenotypic modulation by regulating the function of myocardin, so as to provide a new idea for the prevention and treatment of hypoxia pulmonary hypertension.

肺动脉平滑肌细胞(PASMC)从收缩表型转向合成表型，在低氧性肺血管重构中起着关键作用。血清效应因子（SRF）只有与CArG 盒子DNA 结合，才能够激活参与SMC收缩和合成表型基因的转录表达。目前已知Myocardin 是SRF 最有效的辅激活因子，在低氧调节PASMC表型调控中发挥重要作用。但低氧究竟通过何种信号机制影响Myocardin的表达及功能尚未可知。整合素连接激酶(ILK)被认为是一种早期低氧反应性因子。申请者的初步结果表明低氧可抑制ILK激酶活性并抑制其表达，ILK可靶向下调Myocardin表达。本项目拟通过基因沉默及过表达技术、蛋白/染色质免疫共沉淀分析、荧光素酶报告基因分析等方法，从分子、细胞、组织和整体水平进一步论证“ILK通过下调Myocardin活性参与低氧诱导PASMC表型转换的机理”这一假说，为认识低氧性肺动脉高压形成的病理机制提供重要的科学依据。

低氧性肺动脉高压是一组发病机制复杂、死亡率高、预后差的进行性临床综合征。目前，除肺移植外尚缺乏有效的治疗手段，故探明低氧性肺动脉高压的发病机制为后期临床上提供有效的治疗方法至关重要。低氧条件下肺动脉平滑肌细胞发生表型转换进行引起增殖、迁移增加是低氧性肺动脉高压的重要病理过程，但其具体机制尚不完全清楚。我们前期研究发现，血管平滑肌细胞发生表型转换的同时伴随整合素连接激酶（ILK）表达下调。我们推测ILK可能在低氧诱导肺动脉平滑肌细胞发生表型转换中发挥重要作用。在本研究中，我们发现急性低氧可引起ILK的激酶活性降低，进而引起心肌素与血清效应因子及收缩基因启动子结合降低。这提示ILK在基因水平即可参与肺动脉平滑肌细胞发生表型转换。我们进一步研究发现，慢性低氧可明显降低ILK及心肌素的表达，采用SiRNA技术敲减ILK表达可靶向降低心肌素的表达，并促使肺动脉平滑肌细胞发生表型转换。并且采用腺病毒过表达ILK可明显逆转低氧引起的心肌素表达降低及表型转换。提示在低氧条件下，ILK可通过作用于其下游因子心肌素参与肺动脉平滑肌细胞表型转换的发生。这可能是ILK参与低氧性肺动脉高压发生发展的重要机制。这可能会为未来低氧性肺动脉高压的防治提供新靶点。