TSP-4调控血管平滑肌细胞STAT4/circANRIL/miR-30b-3p信号轴参与下肢动脉再狭窄发生的机制研究

Arterial restenosis of lower extremity after endovascular therapy is an important clinical issue with urgent need to resolve currently. The over proliferation and migration of vascular smooth muscle cell is key factor to induce the arterial restenosis. In our previous studies, we have found that TSP-4 and STAT4 is closely associated with arterial restenosis of lower extremity. The level of circANRIL significantly increased in the artery with intimal hyperplasia. During arterial restenosis of lower extremity, circANRIL interacts with miR-30b-3p. However, the specific molecular mechanism is unknown. Based on the findings, we hypothesize that TSP-4 promotes the phosphorylation and nuclear translocation of STAT4, which stimulates the formation of circANRIL together with another coordinated transcription factor. The circANRIL acts as a binding sponge of miR-30b-3p and up-regulate the level of α-SMA. In addition, the miR-30b-3p can modulate the formation of circANRIL by SP3. This interaction between them becomes a feedback loop that promotes the proliferation and migration of vascular smooth muscle cell, and finally induces the arterial restenosis of lower extremity. The aim of this study is to clarify the specific mechanism of TSP-4 regulating STAT4/circANRIL/miR-30b-3p signaling axis in vascular smooth muscle cells during arterial restenosis of lower extremity, which may provide new clues for the clinical treatment of patients with arterial restenosis of lower extremity.

下肢动脉腔内治疗后再狭窄是当前亟需解决的临床问题，血管平滑肌细胞（VSMC）过度增殖和迁移是导致再狭窄的重要因素。我们已发表研究证实TSP-4和STAT4与下肢动脉再狭窄密切相关，预实验又首次发现动脉增生内膜中circANRIL显著增加，调控miR-30b-3p参与再狭窄发生，但具体机制不详。本课题据此提出假说：TSP-4促进VSMC内STAT4磷酸化后进入核内，与协同转录因子共同促进circANRIL形成，circANRIL“海绵样吸附”miR-30b-3p，上调α-SMA表达，miR-30b-3p又通过转录因子SP3调控circANRIL，形成反馈环路，促进VSMC增殖和迁移，导致再狭窄发生。我们将从细胞实验、动物实验和临床标本三个层次展开研究，阐明TSP-4调控STAT4/circANRIL/miR-30b-3p信号轴参与下肢动脉再狭窄发生的具体机制，为其临床治疗提供新思路和新靶点。

下肢动脉硬化闭塞症是老年人常见的周围血管疾病，可造成肢体疼痛甚至缺血坏死，严重威胁患者的生存质量。近年来，以血管内球囊扩张、支架植入为代表的血管腔内治疗因其创伤小、恢复快等优势逐步取代放开性搭桥手术，成为ASO的一线治疗方式。长期随访研究发现下肢动脉腔内治疗后再狭窄（restenosis）发生率较高，严重影响靶血管的长期通畅率，成为临床亟待解。课题组围绕下肢动脉腔内治疗后再狭窄发生的具体分子机制开展系列研究，首先完善了体外动脉血管平滑肌细胞的分离、培养、鉴定体系，同时使用导丝损伤血管的方式构建了下肢动脉腔内治疗后再狭窄的小鼠模型。使用基因敲除、免疫荧光染色、双荧光素酶报告基因分析、EMSA、ChIP、Western Blot、q-PCR等实验方法，课题组成员通过一系列体内外实验证实TSP-4能够在血管平滑肌细胞内调控STAT4/circANRIL/miR-30b-3p信号轴，在下肢动脉再狭窄模型中验证了STAT4/circANRIL/miR-30b-3p信号轴激活情况，初步探索了干预该信号轴对下肢动脉再狭窄发生的影响。同时课题组依托本课题基础实验的方法和整体研究思路，围绕下肢动脉再狭窄诊治这一关键词展开系列拓展研究。我们通过一系列体内外实验发现双硫仑能够通过NLRP3/Caspase-1/GSDMD通路阻断中性粒细胞陷阱网形成从而加速糖尿病足愈合速度。课题组成员依托本课题还发现中性粒细胞陷阱网能够通过Hippo通路诱导平滑肌细胞表型转化促进腹主动脉瘤的形成。课题组成员还发现磷酸烯醇丙酮酸羧酸激酶1（PCK1）在组织内血管新生过程中发挥重要作用。