Mechanobiology of Vascular Remodeling in Pulmonary Arterial Hypertension

描述（由申请方提供）：肺动脉高压（PAH）是一种严重疾病，其特征为肺动脉硬化抗性肺动脉内皮细胞（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僵硬度增加显著增加了右心室后负荷，并与PAH患者的死亡率增加相关，但PA僵硬在PAH发病机制中的作用尚未完全阐明。我们已经使用原子力显微镜（AFM）微压痕机械表征肺动脉的硬度在一个前所未有的微观尺度水平在实验PAH。我们的初步研究结果表明，远端肺动脉开发显着增加基质硬度超过三倍，在大鼠模型的SU 5416/缺氧和野百合碱（MCT）诱导的PAH。此外，在聚丙烯酰胺基质上生长的人PASMC和PAEC具有重塑的肺动脉的硬度，发展出显著的过度增殖表型，降低了环氧合酶（考克斯）-2的表达，减少了前列腺素I2的合成，并增加了内皮素-1的分泌。综上所述，我们的研究结果表明，PA壁中的基质重塑从根本上使细胞行为偏向于通过先前未认识到的基质硬化效应进行性血管重塑。我们假设PA硬度的增加不仅仅是血管壁病理改变的结果，而是基质硬度的增加触发了以肺动脉中增强的细胞增殖和基质沉积为特征的“重塑表型”，促进了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刚度的时间和空间增加以及机械变化的可逆性。我们将利用AFM微压痕在微米空间尺度上表征SU 5416/缺氧和MCT诱导的PAH大鼠模型中远端肺动脉的局部力学环境。在具体目标2中，我们将确定基质硬度的增加是否触发人PASMC和PAEC中的“重塑表型”，并研究考克斯-2在协调这些硬度依赖性细胞改变中的作用。我们将研究硬度调节考克斯-2表达的分子机制，并测试是否硬度依赖性调节考克斯-2衍生的前列腺素驱动血管重塑的反馈放大。在具体目标3中，我们将阐明刚度如何调节基因表达，并确定在人PASMC和PAEC中参与刚度依赖性基因调控的关键转录因子。我们将使用转录分析和生物信息学方法，沿着一种新的动态硬化水凝胶系统，对过度增殖细胞表型的僵硬驱动出现期间的时间基因表达进行无偏分析。 公共卫生相关性：肺动脉高压（PAH）是一种严重的疾病，其特征在于肺动脉壁内细胞的异常生长，这可能导致不可逆的血管重塑、肺动脉硬化、右心衰竭和死亡。我们假设肺动脉硬度的增加触发血管壁细胞中的“重塑表型”，从而促进进一步的血管重塑。我们提出的研究的目标是进行严格的分析刚度依赖性的“开关”，导致肺动脉细胞的行为异常，并导致肺血管重构。我们的研究有可能发现靶向治疗的新途径，以预防PAH患者的不可逆血管重塑，右心衰竭和死亡。

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. PUBLIC HEALTH RELEVANCE: Pulmonary arterial hypertension (PAH) is a severe disease characterized by abnormal growth of cells within the pulmonary artery wall, which may lead to irreversible vascular remodeling, stiffening of the pulmonary arteries, failure of the right side o the heart, and death. We hypothesize that increases in pulmonary artery stiffness trigger a "remodeling phenotype" in the cells of the vessel wall which promotes further vascular remodeling. The goal of our proposed studies is to perform a rigorous analysis of the stiffness-dependent "switch" which causes pulmonary artery cells to behave abnormally and lead to pulmonary vascular remodeling. Our research has the potential to discover new pathways for targeted therapy to prevent irreversible vascular remodeling, right heart failure, and death in patients with PAH.