CaSR-TRPC信号通路在低氧性肺动脉血管重塑中的作用及机制

Pulmonary vasculature remodeling contributes to hypoxic pulmonary arterial hypertension (HPAH). The elevation of intracellular Ca2+ concentration ([Ca2+]i) in pulmonary arterial smooth muscle cells (PASMC) plays an important role in myocyte proliferation and vasculature remodeling. It has been shown that hypoxic upregulation of calcium sensing receptor (CaSR) in pulmonary arterial smooth muscle involved in the pathogenesis of HPAH. We have previously demonstrated that the upregulation of canonical transient receptor potential proteins (TRPC) was associated with the increase of [Ca2+]i in pulmonary vascular smooth muscle and pulmonary vasculature remodeling induced by hypoxia. Our preliminary data showed that knockdown of CaSR in PASMC with siRNA attenuated hypoxic upregulation of TRPC and inhibited PASMC proliferation in hypoxia. We hypothesize that the Ca2+ influx attributable to the activation of CaSR-TRPC signaling pathway and Ca2+ channels is the mechanism underlying the increase of [Ca2+]i in hypoxic PASMC, PASMC proliferation and HPAH. We will develop the PASMC and animal models and measure the regulation relationship between CaSR and TRPC in hypoxia and the effects of activation of CaSR-TRPC signaling pathway on [Ca2+]i in PASMC, PASMC proliferation and vasculature remodeling.If our hypothesis is correct,our study may reveal a novel pathogenic mechanism and provide new medical targets for effective control of HPAH.

肺血管重塑是低氧性肺动脉高压（HPAH）发病的重要环节，细胞内钙是血管重塑关键因素。最新报道HPAH大鼠肺动脉平滑肌钙敏感受体（CaSR）表达上调，但其参与HPAH发病的具体机制尚未阐明。我们前期发现低氧时肺血管平滑肌经典瞬时受体电位蛋白（TRPC）表达上调与平滑肌细胞内钙升高及肺血管重塑密切相关。预实验观察到用siCaSR处理肺动脉平滑肌细胞可抑制低氧时肺动脉平滑肌细胞TRPC表达上调及细胞增殖，我们推测CaSR经TRPC及其相关钙通道介导参与HPAH发病，机制涉及CaSR-TRPC通路相关钙通道介导的钙内流、细胞增殖及血管重塑。为证实该假说，我们将构建细胞及动物模型，采用分子生物学等技术，观察低氧时CaSR与TRPC的调控关系、CaSR-TRPC通路激活对肺动脉平滑肌细胞内钙、细胞增殖、血管重塑的影响及涉及的钙内流通路。本课题将从新的视角阐明HPAH发病机制，为HPAH防治提供新靶点。

肺血管重塑是低氧肺动脉高压发病的重要环节，细胞内钙是血管重塑关键因素。钙敏感受体(CaSR)参与低氧性肺血管重塑，但其具体机制尚不清楚。本课题通过原代培养大鼠远端肺动脉平滑肌细胞并建立慢性低氧性肺动脉高压大鼠模型，利用分子生物学、细胞生理学、免疫学及病理学等方法，研究发现胶原酶消化法原代培养的大鼠远端肺动脉平滑肌细胞具有典型的平滑肌细胞形态学、免疫学及生理学特征，可以广泛用于肺动脉高压发病机制的相关研究；慢性低氧(4% O2 60 h)促进大鼠肺动脉平滑肌细胞肌浆网中Ca2+释放，并增强钙池操纵性钙通道（SOC）活性，使经由SOC内流的钙池操纵性钙内流（SOCE）升高，从而导致肺动脉平滑肌细胞的 [Ca2+]i升高，进而促进大鼠肺动脉平滑肌细胞增殖；慢性低氧显著促进肺动脉平滑肌细胞增殖，并上调CaSR、经典瞬时受体电位蛋白（TRPC）家族成员TRPC1、TRPC6表达，CaSR激动剂（精胺、R568）可进一步促进肺动脉平滑肌细胞增殖，并上调CaSR、TRPC1、TRPC6表达，而CaSR阻滞剂（NPS2143、NPS2390）作用却相反；siCaSR不仅下调肺动脉平滑肌细胞的TRPC1和TRPC6表达，且显著抑制低氧诱导的肺动脉平滑肌细胞增殖；尽管siTRPC1和siTRPC6也显著抑制低氧诱导的肺动脉平滑肌细胞增殖，但对肺动脉平滑肌细胞的CaSR表达水平却无影响；慢性低氧(10% O2 21 d)可建立慢性低氧性肺动脉高压大鼠模型，导致大鼠右心室收缩压（RVSP）、右心室肥厚指数（RVHI）升高、肺动脉发生血管重塑；而CaSR阻滞剂NPS2143可显著抑制慢性低氧引起的RVSP、RVHI升高及肺血管重塑。慢性低氧使大鼠肺动脉平滑肌CaSR、TRPC1、TRPC6表达升高，CaSR阻滞剂NPS2143可以显著抑制慢性低氧引起的CaSR、TRPC1、TRPC6表达水平升高。结论：慢性低氧经CaSR-TRPC1/6通路导致大鼠肺动脉平滑肌细胞[Ca2+]i升高和细胞增殖，进而发生血管重塑、血管管腔狭窄，导致肺动脉压力升高和右心室肥大，从而参与低氧肺动脉高压发病。