HIF-1α在静脉高压下介导IL-17A调节SMC表型转化的分子机制

The major pathological change of varicose veins (VVs)was the dysfunction of smooth muscle cells (SMCs), which reflected in the phenotypic switching from contract type to synthetic type. Venous hypertension was recognized as the main pathophysiological factor of VV, which demonstrated a higher expression of HIF-1α within the venous wall during hypoxic environment. However, the relationship between the venous hypertension and the phenotypic switching of SMC is still unclear. In preliminary experiment, we identified in patients with VV the differentially expressed proteins of IL-17A, and that combined with its receptor IL-17RA to induce the phenotypic switching of SMC. As reported before, HIF-1α was the major up-regulative factor for IL-17A, suggesting that the IL-17A induced phenotypic switching of SMC might be involved in the pathogenesis of VV by the modulation of HIF-1α. On these results, this project is to explore HIF-1α and IL-17A on the pathogenesis of remodeling dilation of venous wall, including: (1) in vitro: to study the expression and distribution of HIF-1α, IL-17A and IL-17RA on the wall of venous tissues and SMCs from VV patients and control cases respectively, and also to study the function and mechanism of HIF-1α and IL-17A on the phenotypic switching of SMCs. (2) in vivo of mouse models: to clarify the effect and mechanism of HIF-1α and IL-17A on the remodeling dilation of venous wall in the development of VV. In this project, we attempt to provide new research points for the development of VV and to find new targets of medicine treatment for VV.

静脉曲张(Varicose Vein,VV)病理学基础是平滑肌细胞(Smooth Muscle Cell,SMC)功能障碍，即表型从收缩型转化为合成型。静脉高压是VV血流动力学因素，其作用于静脉壁引起SMC功能障碍是VV发病基础，但分子机制尚不明确。有研究显示静脉壁在静脉高压下高表达HIF-1α，我们预实验发现VV患者高表达IL-17A，且结合其受体IL-17RA诱导SMC表型转化，而HIF-1α是IL-17A主要上调因子，提示HIF-1α上调IL-17A诱导SMC表型转化可能参与了VV发病过程。本项目在此基础上，通过免疫组化等技术观察HIF-1α和IL-17A及其受体在静脉壁分布特点；采用WB等方法分析IL-17A对SMC表型转化及其相关联信号通路下游蛋白的影响，明确IL-17A调节SMC表型转化分子机制；建立动物模型，阐明上述机制在VV发病中的作用，为VV开辟新的治疗方案提供理论依据。

静脉曲张是血管外科最常见病症，静脉高压是其发病主要病理生理学基础，但静脉高压通过何种机制引起静脉壁扩张性重塑并不明确。本研究试图从静脉高压这一低氧环境下高表达HIF-1α的现象入手，以平滑肌细胞SMC为靶细胞，以IL-17A为中间体，以SMC表型转变为病理性病变结果，阐明HIF-1α/IL-17A/SMC轴对静脉壁扩张性重塑的影响。首先体外分析HIF-1α和IL-17A作用下静脉壁MMP2/9的表达，结果显示IL-17A显著提高SMC细胞中MMP2/9的表达，HIF-1α只有在Th-17细胞联合作用下才能提高MM2/9的表达，验证了HIF-1α通过诱导Th-17细胞分泌IL-17A作用于SMC，引起SMC从收缩型向合成型转变；其次，体外分析在HIF-1α和IL-17A作用下对SMC凋亡的影响，显示HIF-1α对SMC凋亡的影响并不显著，但IL-17A对SMC凋亡有直接的促进作用，提示HIF-1α可以通过IL-17A抑制SMC的凋亡；最后，体内实验研究HIF-1α对小鼠后肢静脉壁扩张性重塑的影响，成功构建后肢静脉高压从10-20mmHg升高至90-110mmHg的模型，发现在静脉高压的状态下，HIF-1α短期内升高不明显(提示静脉曲张是一个缓慢发病过程，在反复静脉高压的刺激下发生，与静脉转流桥和人工动静脉透析通路是急性静脉高压状态不同)，但注射HIF-1α后待血液中浓度升高，管壁中SMCMMP2/9显著升高，提示在静脉高压下HIF-1α升高是一个慢性过程，而外源性HIF-1α是诱导静脉壁SMC合成型分泌蛋白OPN和PCNA/β显著升高，且收缩性蛋白α-SMA和SM22α/β水平降低，即SMC在HIF-1α发生表型转换。上述研究结果初步证实了在静脉高压这一慢性缺氧模型中HIF-1α升高，刺激Th17细胞活化释放IL-17A，后者与SMC表面IL-17RA结合启动SMC细胞内表型转化信号传导通路，进而诱导SMC由收缩型向合成型转化，合成型SMC分泌MMP2/9引起静脉壁发生扩张性重塑，是静脉壁张力降低和弹性顺应性下降的重要原因。同时，在本实验中，检测相关信号传导通路，试图寻找HIF-1α/IL-17A/SMC轴的分子生物学通路，结果发现，SMC细胞中ERK和AKT信号通路均未受到明显影响，后续将进一步验证其他信号通路在这一过程中所起着的作用。