Capacitative Ca2+ Entry and TRP Channels in Thromboembolic Pulmonary Hypertension

描述（由申请人提供）：在慢性血栓栓塞性肺动脉高压（CTEPH）患者中，肺血管阻力（PVR）升高是由血栓栓塞阻塞肺动脉近端和远端，以及肺血管平滑肌（PASMC）和内皮细胞（PAEC）过度迁移和增殖引起的肺血管重塑引起的。附着在肺血管壁上的血栓栓子含有大量凝血因子（如凝血酶、纤维蛋白原），可触发血管重构。凝血酶诱导PAEC细胞质[Ca2+]升高，增加内皮通透性，使有丝分裂因子渗透到血管介质中，引起内侧肥大。PASMC中细胞质[Ca2+]的增加通过上调Ca2+敏感信号蛋白和转录因子来刺激细胞增殖。通过激活PASMC中的受体，凝血因子（如凝血酶和纤维蛋白原）也通过瞬态受体电位（TRP）通道促进Ca2+内流而引起胞浆[Ca2+]的增加。我们最近证明：a)正常PASMC在增殖过程中TRP表达增加，Ca2+通过TRP通道进入增加；b)用siRNA抑制TRP表达可减弱PASMC的增殖；c)特发性肺动脉高压患者PASMC中TRP mRNA和蛋白表达（如TRPC3/6）上调。基于这些数据，我们假设肺血管中TRP通道的基因表达异常增强是CTEPH患者血栓栓塞介导的肺血管重构的易感性。为了验证这一假设，我们提出了三个具体目标：1)在CTEPH患者分离的PASMC中，TRP通道是否以及哪些转录上调，以及TRP编码的储存和受体操作的阳离子通道是否在功能上增强；2)检查凝血因子（凝血酶和纤维蛋白原）是否通过激活PASMC中的TRP通道而增加胞浆[Ca2+]，如果是，哪些TRP通道亚基受到凝血酶和纤维蛋白原的功能调节；3)研究凝血酶和纤维蛋白原的有丝分裂作用是否依赖于正常PASMC中Ca2+通过TRP通道进入引起的胞浆[Ca2+]的增加，并在CTEPH患者的PASMC中增强。

DESCRIPTION (provided by applicant): In patients with chronic thromboembolic pulmonary hypertension (CTEPH), elevated pulmonary vascular resistance (PVR) is caused by obstruction of proximal and distal pulmonary arteries with thromboemboli, and by pulmonary vascular remodeling due to excessive migration and proliferation of pulmonary vascular smooth muscle (PASMC) and endothelial (PAEC) cells. The thromboemboli attached to the pulmonary vascular wall include a large amount of blood coagulation factors (e.g., thrombin, fibrinogen), which may serve as triggers for vascular remodeling. A rise in cytosolic [Ca2+] in PAEC induced by thrombin increases endothelial permeability, which allows mitogenic factors to penetrate into the vascular media and cause medial hypertrophy. A rise in cytosolic [Ca2+] in PASMC stimulates cell proliferation by upregulating Ca2+- sensitive signaling proteins and transcription factors. By activating receptors in PASMC, the coagulation factors (e.g., thrombin and fibrinogen) also cause increases in cytosolic [Ca2+] by promoting Ca2+ influx through transient receptor potential (TRP) channels. We have recently demonstrated that a) TRP expression is increased and Ca2+ entry through TRP channels is augmented in normal PASMC during proliferation, b) inhibition of TRP expression with siRNA attenuates PASMC proliferation, c) mRNA and protein expression of TRP (e.g., TRPC3/6) is upregulated in PASMC from patients with idiopathic pulmonary arterial hypertension. Based on these data, we hypothesize that abnormally enhanced gene expression of TRP channels in the pulmonary vasculature serves as a predisposition for thromboemboli-mediated pulmonary vascular remodeling in CTEPH patients. Three specific aims are proposed to test the hypothesis: 1) To examine whether and which TRP channels are transcriptionally upregulated, and whether TRP-encoded store- and receptor-operated cation channels are functionally enhanced, in PASMC isolated from CTEPH patients; 2) To examine whether coagulation factors (thrombin nd fibrinogen) increase cytosolic [Ca2+] by activating TRP channels in PASMC and, if so, which TRP channel subunits are functionally regulated by thrombin and fibrinogen; and 3) To examine whether the mitogenic effect of thrombin and fibrinogen depends on increases in cytosolic [Ca2+] due to Ca2+ entry through TRP channels in normal PASMC and is enhanced in PASMC from CTEPH patients.