Capacitative Ca2+ Entry and TRP Channels in Thromboembolic Pulmonary Hypertension

描述(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)升高是由于血栓栓子阻塞近端和远端肺动脉，以及由于肺血管平滑肌(PASMC)和内皮(PAEC)细胞过度迁移和增殖而导致肺血管重构。附着在肺血管壁上的血栓栓子包括大量的凝血因子(如凝血酶、纤维蛋白原)，它们可能是血管重塑的触发因素。凝血酶诱导的血管内皮细胞胞浆[Ca~(2+)]升高增加内皮通透性，使促有丝分裂因子穿透血管中层，引起中膜肥厚。PASMC胞浆[Ca~(2+)]升高通过上调Ca~(2+)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)也通过瞬时受体电位(TRP)通道促进钙内流，导致胞浆内[Ca~(2+)]升高。我们最近证实：a)在正常的PASMC增殖过程中，Trp的表达增加，通过Trp通道的钙内流增加；b)siRNA抑制Trp的表达可抑制PASMC的增殖；c)特发性肺动脉高压患者的PASMC中Trp(如TRPC3/6)的mRNA和蛋白表达上调。基于这些数据，我们假设肺血管中Trp通道基因表达异常增强是血栓栓子介导的CTEPH患者肺血管重塑的易感因素。为了验证这一假说，提出了三个特定的目标：1)检测从CTEPH患者分离的PASMC中是否以及哪些Trp通道转录上调，以及Trp编码的存储和受体操控的阳离子通道是否在功能上得到增强；2)检测凝血因子(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，则哪些Trp通道亚单位在功能上受到凝血酶和纤维蛋白原的调节；以及3)研究凝血酶和纤维蛋白原的促有丝分裂作用是否依赖于正常PASMC中由于通过Trp通道钙离子进入而导致的胞浆[Ca2+]增加以及在CTEPH患者的PASMC中是否被增强。

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.