High Shear Stress Alters Gene Regulation in Pulmonary Arterial Hypertension

肺动脉高压（PAH）是一种使人衰弱的疾病，其中肺动脉周围动脉闭塞， 肺引起肺血管阻力升高，最终导致右心衰竭。在这一提议中， 我们将PAH的进展性与高切应力（HSS）对肺动脉（PA）的影响联系起来 内皮细胞（EC）和平滑肌细胞（SMC）的基因调控和功能，我们可能能够 利用这些机械信息来逆转已建立的疾病。针对先前关于“一体化”的RFA， 组学的研究中，我们将PAEC增强子启动子相互作用与不同表达的基因（DEG）联系起来。 层流剪切应力（LSS）的生理条件。LSS导致不同的染色质可及性， 通过ATAC Seq评估，在转录因子KLF 4在H3 K27 ac增强子位点结合DNA的位点， 通过ChIP Seq.然而，我们只能将LSS增强子与三分之一的DEG联系起来， “最近的基因”。通过结合HiChIP和活动联系（ABC）算法，我们能够将 80% DEG的远端增强子，包括跳跃基因和PAH相关基因，如BMPR 2。HSS是 当存在使血管腔变窄的既定血管疾病时，或如果PAH由 肺血流量和压力过高的先天性心脏病在我们的初步研究中，我们表明， HSS对PAEC稳态基因如BMPR 2、JAG 1、ERG和ELN的表达具有重要影响; 此外，内皮间充质转化（EndMT）基因的表达增加，如 SNAI 1，并增加PAEC通透性和单核细胞粘附。我们能够构建PA EC-SMC双层， 纤维蛋白凝胶使我们能够研究LSS和HSS下的血管细胞相互作用。我们的首要假设是 响应于HSS的PAEC和PAEC-SMC相互作用的变化对增强子景观产生不利影响， 基因调控和功能，并可以逆转，以防止进行性PAH病理。为了调查这个， 我们提出三个具体目标。在特定目标1中，我们描述了增强子-启动子中的扰动， 在HSS控制和PAH PAEC下，我们将这些联系起来， 功能异常的渗透性，炎症和EndMT，以及基因和 PAH患者PA组织中的蛋白质。在具体目标2中，我们用PAEC内衬生物制造管状结构 EC周围的管腔和SMC，以确定细胞-细胞相互作用如何影响HSS与LSS介导的 基因调控和功能，包括SMC增殖和弹性蛋白纤维形成。在具体目标3中，我们 重点关注对照组和PAH PAEC中HSS介导的ERG显著降低，以确定该特征是否 是HSS介导的PAEC基因表达和功能异常的必要和充分条件。我们确定 HSS介导的miR-96升高和ERG降低是否可被miR-96逆转 在PAH细胞和ERG缺陷的转基因小鼠中，这些研究将提供新的 通过颠覆HSS介导的进行性PAH的根本原因来逆转疾病的干预途径。

Pulmonary arterial hypertension (PAH) is a debilitating disease in which occlusion of the peripheral arteries of the lung causes elevation in pulmonary vascular resistance that culminates in right heart failure. In this proposal, we relate the progressive nature of PAH to the impact of high shear stress (HSS) on pulmonary arterial (PA) endothelial (EC) and smooth muscle cell (SMC) gene regulation and function with the view that we might be able to use this mechanistic information to reverse established disease. In response to a previous RFA on ‘integrative omics’, we related PAEC enhancer promoter interactions to differentially expressed genes (DEGs) under physiologic conditions of laminar shear stress (LSS). LSS resulted in differential chromatin accessibility, assessed by ATAC Seq, at sites where the transcription factor KLF4 bound DNA at H3K27ac enhancer sites, as assessed by ChIP Seq. However, we could only relate the LSS enhancers to one third of DEGs on the basis of ‘nearest gene’. By incorporating HiChIP and the Activity by Contact (ABC) algorithm, we were able to relate distal enhancers to 80% of DEGs, including skipped genes and those related to PAH, such as BMPR2. HSS is prevalent when there is established vascular disease narrowing the vascular lumen, or if PAH is initiated by the high pulmonary blood flow and pressure of a congenital heart defect. In our Preliminary Studies, we show that HSS has a major impact on expression of PAEC homeostatic genes such as BMPR2, JAG1, ERG, and ELN; moreover, there is heightened expression of endothelial mesenchymal transition (EndMT) genes, such as SNAI1, and increased PAEC permeability and monocyte adhesion. Our ability to build PA EC-SMC bilayers in fibrin gels allows us to study vascular cell interactions under LSS and HSS. Our overarching hypothesis is that changes in PAEC and PAEC-SMC interaction in response to HSS adversely impact the enhancer landscape, gene regulation and function, and can be reversed to prevent progressive PAH pathology. To investigate this, we propose three Specific Aims. In Specific Aim 1, we characterize perturbations in the enhancer-promoter landscape that account for aberrant gene regulation under HSS in control and PAH PAEC, and we link these features to functional abnormalities in permeability, inflammation and EndMT, and to changes in genes and proteins in the PA tissue of PAH patients. In Specific Aim 2, we biofabricate tubular structures with PAEC lining the lumen and SMC surrounding the EC, to determine how cell-cell interactions impact HSS vs LSS mediated gene regulation and function, including SMC proliferation and elastin fiber formation. In Specific Aim 3, we focus on the pronounced HSS mediated reduction in ERG in control and PAH PAEC, to determine if this feature is necessary and sufficient for the HSS-mediated abnormal PAEC gene expression and function. We determine whether the HSS mediated elevation in miR-96 and the reduction in ERG can be subverted by the miR-96 antagomir, both in PAH cells and in a transgenic mouse with deficient ERG. These studies should provide new avenues for intervention to reverse disease by subverting the root cause of HSS mediated-progressive PAH.