1 Keenan Research Centre for Biomedical Science, St. Michael's Hospital, Toronto, Ontario, Canada.
2 Ludwig Boltzmann Institute for Lung Vascular Research, Graz, Austria.
Am J Respir Crit Care Med. 2018 Jan 15;197(2):244-260. doi: 10.1164/rccm.201702-0386OC.
Vascular remodeling in pulmonary arterial hypertension (PAH) results from smooth muscle cell hypertrophy and proliferation of vascular cells. Loss of BMPR-II (bone morphogenetic protein receptor 2) signaling and increased signaling via TGF-β (transforming growth factor β) and its downstream mediators SMAD (small body size [a C. elegans protein] mothers against decapentaplegic [a Drosophila protein family])-2/3 has been proposed to drive lung vascular remodeling; yet, proteomic analyses indicate a loss of SMAD3 in PAH.
We proposed that SMAD3 may be dysregulated in PAH and that loss of SMAD3 may present a pathophysiological master switch by disinhibiting its interaction partner, MRTF (myocardin-related transcription factor), which drives muscle protein expression.
SMAD3 levels were measured in lungs from PAH patients, rats treated either with Sugen/hypoxia or monocrotaline (MCT), and in mice carrying a BMPR2 mutation. In vitro, effects of SMAD3 or BMPR2 silencing or SMAD3 overexpression on cell proliferation or smooth muscle hypertrophy were assessed. In vivo, the therapeutic and prophylactic potential of CCG1423, an inhibitor of MRTF, was investigated in Sugen/hypoxia rats.
SMAD3 was downregulated in lungs of patients with PAH and in pulmonary arteries of three independent PAH animal models. TGF-β treatment replicated the loss of SMAD3 in human pulmonary artery smooth muscle cells (huPASMCs) and human pulmonary artery endothelial cells. SMAD3 silencing increased proliferation and migration in huPASMCs and human pulmonary artery endothelial cells. Coimmunoprecipitation revealed reduced interaction of MRTF with SMAD3 in TGF-β-treated huPASMCs and pulmonary arteries of PAH animal models. In huPASMCs, loss of SMAD3 or BMPR-II increased smooth muscle actin expression, which was attenuated by MRTF inhibition. Conversely, SMAD3 overexpression prevented TGF-β-induced activation of an MRTF reporter and reduced actin stress fibers in BMPR2-silenced huPASMCs. MRTF inhibition attenuated PAH and lung vascular remodeling in Sugen/hypoxia rats.
Loss of SMAD3 presents a novel pathomechanism in PAH that promotes vascular cell proliferation and-via MRTF disinhibition-hypertrophy of huPASMCs, thereby reconciling the parallel induction of a synthetic and contractile huPASMC phenotype.
肺动脉高压（PAH）中的血管重构是由平滑肌细胞肥大和血管细胞增殖引起的。骨形态发生蛋白受体 2（BMPR-II）信号的丧失和 TGF-β（转化生长因子β）及其下游介质 SMAD（小体大小[秀丽隐杆线虫蛋白]对抗 decapentaplegic[果蝇蛋白家族]）-2/3 的信号增加被认为是导致肺血管重构的原因；然而，蛋白质组学分析表明 PAH 中 SMAD3 的丢失。
我们提出 SMAD3 在 PAH 中可能失调，并且 SMAD3 的丢失可能通过解除其相互作用伙伴 MRTF（肌球蛋白相关转录因子）的抑制作用，从而驱动肌肉蛋白表达，呈现出病理生理学主开关。
测量了 PAH 患者、苏根/低氧或单克隆抗体（MCT）治疗的大鼠以及携带 BMPR2 突变的小鼠肺中的 SMAD3 水平。在体外，评估了 SMAD3 或 BMPR2 沉默或 SMAD3 过表达对细胞增殖或平滑肌肥大的影响。体内，研究了 MRTF 抑制剂 CCG1423 在苏根/低氧大鼠中的治疗和预防潜力。
PAH 患者肺和三种独立的 PAH 动物模型的肺动脉中 SMAD3 下调。TGF-β 处理复制了人肺动脉平滑肌细胞（huPASMCs）和人肺动脉内皮细胞中 SMAD3 的丢失。SMAD3 沉默增加了 huPASMCs 和人肺动脉内皮细胞的增殖和迁移。共免疫沉淀显示，在 TGF-β 处理的 huPASMCs 和 PAH 动物模型的肺动脉中，MRTF 与 SMAD3 的相互作用减少。在 huPASMCs 中，SMAD3 或 BMPR-II 的丢失增加了平滑肌肌动蛋白的表达，而 MRTF 抑制则减弱了这种表达。相反，SMAD3 过表达可防止 BMPR2 沉默的 huPASMCs 中 TGF-β 诱导的 MRTF 报告基因的激活，并减少肌动蛋白应力纤维。MRTF 抑制减轻了苏根/低氧大鼠的 PAH 和肺血管重构。
SMAD3 的丢失代表了 PAH 中的一种新的病理机制，它促进了血管细胞的增殖，并通过 MRTF 抑制促进 huPASMCs 的肥大，从而调和 huPASMCs 中合成和收缩表型的平行诱导。
