Mechanobiology of Vascular Remodeling in Pulmonary Arterial Hypertension

描述（由申请人提供）：肺动脉高压（PAH）是一种严重的疾病，其特征是抗凋亡肺动脉内皮细胞（PAEC）和平滑肌细胞（PASMC）过度增殖，进行性肺动脉（PA）硬化，最终导致右心衰和死亡。最近的研究表明，PA僵硬度的增加显著增加了PAH患者的右心室后负荷，并与死亡率的增加有关，然而，PA僵硬度在PAH发病机制中的作用尚未完全阐明。我们使用原子力显微镜（AFM）微压痕在实验PAH中前所未有的微尺度水平上机械表征肺动脉的刚度。我们的初步研究结果表明，在SU5416/缺氧和单氯胆碱（MCT）诱导的PAH大鼠模型中，肺动脉远端基质硬度显著增加3倍以上。此外，在聚丙烯酰胺基质上生长的人PASMC和PAEC具有重塑肺动脉的刚度，表现出惊人的超增殖表型，环氧化酶(COX)-2表达降低，前列腺素I2合成减少，内皮素-1分泌增加。综上所述，我们的研究结果表明，通过之前未被认识到的基质硬化效应，PA壁的基质重塑从根本上使细胞行为倾向于进行性血管重塑。我们假设PA刚度的增加不仅仅是血管壁病理改变的结果，而是基质刚度的增加引发了“重塑表型”，其特征是细胞增殖和肺动脉基质沉积增强，促进血管重塑的机械-生物反馈放大。为了验证我们的假设，我们提出了三个具体目标。在具体目标1中，我们将研究实验性PAH期间PA刚度的时空增加和力学变化的可逆性。我们将利用AFM微凹痕在微米空间尺度上表征SU5416/缺氧和mct诱导的PAH大鼠模型的肺动脉远端局部力学环境。在特异性目标2中，我们将确定基质刚度的增加是否会触发人类PASMC和PAEC中的“重塑表型”，并研究COX-2在协调这些刚度依赖的细胞改变中的作用。我们将研究刚度调节COX-2表达的分子机制，并测试COX-2衍生的前列腺素的刚度依赖性调节是否驱动血管重构的反馈放大。在Specific Aim 3中，我们将阐明刚度如何调节基因表达，并确定人类PASMC和PAEC中涉及刚度依赖基因调控的关键转录因子。我们将使用转录谱分析和生物信息学方法，以及一种新的动态硬化水凝胶系统，在刚性驱动的超增殖细胞表型出现期间，对时间基因表达进行无偏分析。

DESCRIPTION (provided by applicant): Pulmonary arterial hypertension (PAH) is a severe disease characterized by excessive proliferation of apoptosis-resistant pulmonary artery endothelial cells (PAEC) and smooth muscle cells (PASMC), progressive pulmonary arterial (PA) stiffening, and ultimately right heart failure and death. Recent studies suggest that increased PA stiffness contributes significantly to increased right ventricular after-load and is associated with increased mortality in PAH patients, however the role of PA stiffening in the pathogenesis of PAH has not yet been fully elucidated. We have used atomic force microscopy (AFM) micro-indentation to mechanically characterize the stiffness of pulmonary arteries at an unprecedented micro-scale level in experimental PAH. Our preliminary findings demonstrate that distal pulmonary arteries develop significant increases in matrix stiffness by more than three-fold in the rat models of SU5416/hypoxia and monocrotaline (MCT)-induced PAH. Furthermore, human PASMC and PAEC grown on polyacrylamide substrates with the stiffness of remodeled pulmonary arteries develop a striking hyper-proliferative phenotype, decreased expression of cyclooxygenase (COX)-2, reduced prostaglandin I2 synthesis, and increased secretion of endothelin-1. Taken together, our findings suggest that matrix remodeling in the PA wall fundamentally biases cellular behavior towards progressive vascular remodeling via previously unrecognized effects of matrix stiffening. We hypothesize that increases in PA stiffness are not merely a consequence of pathological alterations in the vessel wall, but rather that increases in matrix stiffness trigger a "remodeling phenotype" characterized by enhanced cellular proliferation and matrix deposition in pulmonary arteries, promoting mechano-biological feedback amplification of vascular remodeling. To test our hypothesis, we propose three specific aims. In Specific Aim 1, we will investigate the temporal and spatial increases in PA stiffness and reversibility of mechanical changes during experimental PAH. We will utilize AFM micro-indentation to characterize the local mechanical environment of distal pulmonary arteries at the micron spatial scale in the rat models of SU5416/hypoxia and MCT-induced PAH. In Specific Aim 2, we will determine whether increases in matrix stiffness trigger a "remodeling phenotype" in human PASMC and PAEC and investigate the role of COX-2 in orchestrating these stiffness- dependent cellular alterations. We will investigate the molecular mechanisms by which stiffness modulates COX-2 expression and test whether stiffness-dependent regulation of COX-2-derived prostanoids drives feedback amplification of vascular remodeling. In Specific Aim 3, we will elucidate how stiffness modulates gene expression and identify key transcription factors involved in stiffness-dependent gene regulation in human PASMC and PAEC. We will use transcriptional profiling and bioinformatic approaches, along with a novel dynamic stiffening hydrogel system, to perform an unbiased analysis of temporal gene expression during the stiffness-driven emergence of the hyper-proliferative cellular phenotype.