SENP1调控血管平滑肌祖细胞分化的分子机制及其作为血管重构防治靶点的研究

Vascular remodeling is a feature of vascular injury with impact on much vascular pathology or injury, such as pulmonary arterial hypertension and restenosis after percutaneous coronary intervention (PCI). Vascular smooth muscle cells (VSMCs) proliferation is a key event for these vascular diseases. Recent studies have shown that various progenitor cell subsets could be a source of VSMCs which include SMC progenitors. Despite the different sources of SMC progenitor cells are implicated, there are few therapies that target this process and molecular mechanisms underlying this process remain incompletely understood. Thus, a better understanding of this process and identification of novel therapeutic targets are necessary. Maintenance of SMC progenitors requires transcriptional silencing of Serum Response Factor (SRF)-dependent SMC differentiation marker genes within these different environments. SRF can be a SUMO1 target on its lysine residue 147, which is located in the MADS box crucial for DNA binding. Defective SRF SUMOylation alters transcription activity. Thus, the SUMO1 acceptor site on SRF and its coactivators, such as myocardin，Msx1 and Foxo4, are critical for full activation of target genes by SRF and is promoter-dependent. Preliminary evidence in our lab demonstrates that SENP1 deletion in VSMC augmented neointima formation after carotid injury. Furthermore, in an in vitro iPSC-based VSMC differentiation system SENP1 deletion induces an increased expression of SPC markers (KLF4, KLF5, MYH10) and a decreased expression of SMC markers (MYH11, ACTA2, MYOCD). We hypothesize that SENP1 coordinates with SRF and its coactivators to regulate SPC gene expression following vascular injury. Our study is to elucidate the function and mechanism by which SENP1 mediates SPC differentiation during vascular injury and provide new therapeutic targets for the treatment of vascular diseases.

SMC是构成血管中膜的主要细胞成分，近年来许多学者发现血管损伤内膜增厚部位的SMC更有可能或至少有一部分来源于其前体细胞-SPCs。提示我们SPC的分化动员有可能成为这类血管疾病的新治疗靶点，但是该过程具体的发生机制还有待深入研究。血清应答因子SRF是SMC分化成熟的关键转录因子，能被苏素蛋白SUMO1修饰，SENP1是SUMO1特异性的蛋白酶，能够将目标蛋白去苏素化。我们的前期结果发现SENP1平滑肌特异性敲除后加剧颈动脉结扎引起的血管内膜增生，并且在SPC向SMC分化过程中SENP1敲低可导致SMC标志基因表达降低，而SPC标志基因表达增加。我们猜想SENP1是否能够通过将SUMO1-SRF去苏素化，增加deSUMOylation-SRF与Myocardin等辅助活化因子的相互作用，进而提高deSUMOylation-SRF对SMC标志基因的转录活性，促进SPC向SMC分化。

血管平滑肌细胞（VSMC）可分为分化（收缩）型与去分化（合成）型，去分化引起的VSMC异常增殖与迁移是导致冠状动脉介入（PCI）术后再狭窄的重要原因之一。苏素化（SUMOylation，SUMO）是一种蛋白质翻译后修饰方式，可影响蛋白质的生理功能。SENP1是肽键内切酶中的一种，可使苏素化的蛋白质去苏素化（deSUMOylation），已有研究表明SENP1可使多种蛋白去苏素化，进而影响细胞的生理功能。但SENP1对VSMC表型转换的调节作用机制还有待研究。本研究结果发现，血管平滑肌细胞条件性敲除SENP1后，在颈动脉结扎诱导的血管损伤模型中，颈动脉结扎可引起血管狭窄及内膜增厚，与野生型小鼠相比，在血管平滑肌细胞条件性敲除SENP1小鼠中，颈动脉结扎后血管狭窄加剧，血管内膜进一步增厚。提示我们SENP1在血管损伤引起的内膜增生中具有重要的作用。另外，我们还发现SENP1-SMKO后可导致VSMC中代表VSMC分化的特异性标志基因MYH11, MYOCD, ACTA2等表达下降，提示我们SENP1在VSMC表型转换中具有关键作用。研究表明血清应答因子（SRF）与其顺式元件（CArG-box）之间的相互作用对于平滑肌标志基因的转录是非常重要的，SRF对平滑肌特异性基因表达的调节活性涉及SRF的表达、核转位以及翻译后修饰等，其中包括苏素化。因此，我们进一步探讨SENP1是否通过调控SRF的苏素化水平进而调节VSMC表型转换。免疫共沉淀实验结果表明在小鼠主动脉平滑肌细胞中，SENP1敲低可引起SRF苏素化水平升高，反之，过表达SENP1可导致SRF苏素化水平降低。进一步的实验发现SRF-SUMO1活性突变体转染可引起SMC标志基因表达降低，去分化型标志基因表达升高，提示我们SRF苏素化修饰抑制SMC标志基因的转录，促进SMC由分化表型向去分化表型转换，并且这一作用是由于SRF苏素化修饰影响了其与辅助转录因子myocardin的结合介导的。本课题研究揭示了SENP1的表达下调对颈动脉结扎缺血导致的血管损伤模型中内膜增生的作用，SENP1通过调节血清应答因子SRF的苏素化水平，影响SRF与其辅助转录因子Myocardin三元复合物的形成，进一步调控血管平滑肌细胞分化表型标志基因的转录水平，最终调控血管平滑肌细胞的表型转换。