Mechanobiology of Vascular Remodeling in Pulmonary Arterial Hypertension

描述(申请人提供)：肺动脉高压(PAH)是一种严重的疾病，其特征是抗凋亡的肺动脉内皮细胞(PAEC)和平滑肌细胞(PASMC)过度增殖，进行性肺动脉(PA)硬化，最终导致右心衰竭和死亡。最近的研究表明，PA僵硬的增加显著增加了PAH患者的右室后负荷，并与增加的死亡率有关，然而PA僵硬在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)微压痕技术，在前所未有的微尺度水平上对实验性PAH中的肺动脉僵硬进行了机械表征。我们的初步发现表明，在SU5416/低氧和野百合碱(MCT)诱导的PAH大鼠模型中，远端肺动脉的基质硬度显着增加三倍以上。此外，生长在聚丙烯酰胺基质上的人PASMC和PAEC表现出显著的高增殖表型，环氧合酶(COX)-2的表达减少，前列腺素I2的合成减少，内皮素-1的分泌增加。综上所述，我们的发现表明，PA壁中的基质重塑通过先前未知的基质硬化效应，从根本上偏向于进行性血管重塑的细胞行为。我们假设，PA硬度的增加不仅是血管壁病理改变的结果，而且基质硬度的增加还会触发一种“重塑表型”，其特征是促进肺动脉中细胞的增殖和基质的沉积，从而促进血管重塑的机械生物反馈放大。为了检验我们的假设，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硬度的时间和空间增加以及力学变化的可逆性。我们将利用AFM微压痕技术在微米空间尺度上表征SU5416/低氧和MCT诱导的PAH模型大鼠远端肺动脉的局部力学环境。在特定的目标2中，我们将确定基质硬度的增加是否会触发人PASMC和PAEC的“重塑表型”，并研究COX-2在协调这些硬度依赖的细胞变化中的作用。我们将研究僵硬调节COX-2表达的分子机制，并测试是否僵硬依赖的COX-2衍生前列腺素调节驱动血管重塑的反馈放大。在具体目标3中，我们将阐明刚性如何调节基因表达，并确定参与人PASMC和PAEC刚性依赖基因调控的关键转录因子。我们将使用转录图谱和生物信息学方法，以及一种新的动态硬化水凝胶系统，对在僵硬驱动的高增殖细胞表型出现期间的时间基因表达进行无偏见的分析。

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.