Data_Sheet_1_Upregulation of Calcium Homeostasis Modulators in Contractile-To-Proliferative Phenotypical Transition of Pulmonary Arterial Smooth Muscle Cells.PDF

Excessive pulmonary artery (PA) smooth muscle cell (PASMC) proliferation and migration are implicated in the development of pathogenic pulmonary vascular remodeling characterized by concentric arterial wall thickening and arteriole muscularization in patients with pulmonary arterial hypertension (PAH). Pulmonary artery smooth muscle cell contractile-to-proliferative phenotypical transition is a process that promotes pulmonary vascular remodeling. A rise in cytosolic Ca2+ concentration [(Ca2+)cyt] in PASMCs is a trigger for pulmonary vasoconstriction and a stimulus for pulmonary vascular remodeling. Here, we report that the calcium homeostasis modulator (CALHM), a Ca2+ (and ATP) channel that is allosterically regulated by voltage and extracellular Ca2+, is upregulated during the PASMC contractile-to-proliferative phenotypical transition. Protein expression of CALHM1/2 in primary cultured PASMCs in media containing serum and growth factors (proliferative PASMC) was significantly greater than in freshly isolated PA (contractile PASMC) from the same rat. Upregulated CALHM1/2 in proliferative PASMCs were associated with an increased ratio of pAKT/AKT and pmTOR/mTOR and an increased expression of the cell proliferation marker PCNA, whereas serum starvation and rapamycin significantly downregulated CALHM1/2. Furthermore, CALHM1/2 were upregulated in freshly isolated PA from rats with monocrotaline (MCT)-induced PH and in primary cultured PASMC from patients with PAH in comparison to normal controls. Intraperitoneal injection of CGP 37157 (0.6 mg/kg, q8H), a non-selective blocker of CALHM channels, partially reversed established experimental PH. These data suggest that CALHM upregulation is involved in PASMC contractile-to-proliferative phenotypical transition. Ca2+ influx through upregulated CALHM1/2 may play an important role in the transition of sustained vasoconstriction to excessive vascular remodeling in PAH or precapillary PH. Calcium homeostasis modulator could potentially be a target to develop novel therapies for PAH.

肺动脉（pulmonary artery, PA）平滑肌细胞（smooth muscle cell, PASMC）的过度增殖与迁移，参与了以同心性动脉壁增厚和小动脉肌化为特征的病理性肺血管重构的发生发展，该过程可见于肺动脉高压（pulmonary arterial hypertension, PAH）患者中。肺动脉平滑肌细胞收缩型向增殖型表型转化是促进肺血管重构的关键过程。肺动脉平滑肌细胞内胞质游离钙浓度[(Ca²+)cyt]升高，既是肺血管收缩的触发因素，也是肺血管重构的刺激源。本研究发现，钙稳态调节蛋白（calcium homeostasis modulator, CALHM）——一种受电压与细胞外钙变构调控的钙（及ATP）通道——在肺动脉平滑肌细胞收缩型向增殖型表型转化过程中表达上调。原代培养的、于含血清与生长因子的培养基中增殖的肺动脉平滑肌细胞（proliferative PASMC）中，CALHM1/2的蛋白表达水平显著高于同一只大鼠新鲜分离的收缩型肺动脉平滑肌细胞（contractile PASMC）。增殖型肺动脉平滑肌细胞中上调的CALHM1/2，与pAKT/AKT及pmTOR/mTOR比值升高、细胞增殖标志物增殖细胞核抗原（proliferating cell nuclear antigen, PCNA）表达增加呈正相关；而血清饥饿与雷帕霉素可显著下调CALHM1/2的表达。此外，与正常对照组相比，野百合碱（monocrotaline, MCT）诱导的肺动脉高压模型大鼠的新鲜分离肺动脉组织，以及肺动脉高压患者的原代培养肺动脉平滑肌细胞中，CALHM1/2的表达均出现上调。对已造模的实验性肺动脉高压大鼠，腹腔注射CALHM通道非选择性阻断剂CGP 37157（0.6 mg/kg，每8小时一次）可部分逆转病情。上述结果表明，CALHM表达上调参与了肺动脉平滑肌细胞收缩型向增殖型的表型转化过程。经上调的CALHM1/2介导的钙内流，可能在肺动脉高压或毛细血管前肺动脉高压患者中，参与了持续性肺血管收缩向过度血管重构的转化过程。钙稳态调节蛋白有望成为开发肺动脉高压新型治疗手段的潜在靶点。