High Shear Stress Alters Gene Regulation in Pulmonary Arterial Hypertension

高剪切应力改变肺动脉高压的基因调控

基本信息

批准号：
10557807
负责人：
Marlene Rabinovitch
金额：
$ 73.97万
依托单位：
STANFORD UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-01-28 至 2024-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10557807
关键词：
ATAC-seq Address Adhesions Affect Algorithms Arteries BMPR2 gene Biological Assay Biomanufacturing Blood Pressure Blood Vessels Blood flow Cell Communication Cell Proliferation Cell physiology Cells ChIP-seq Chromatin Collaborations Congenital Heart Defects DNA DNA Binding Data Deposition Disease Distal Elastin Elastin Fiber Endothelial Cells Endothelium Enhancers Exposure to Family Fibrin Functional disorder Funding Gel Gene Expression Gene Expression Regulation Genes Genomics Heterozygote Homeostasis Impairment Inflammation Intervention Knock-out Left Link Lung MADH3 gene Maps Mediating Mesenchymal Metabolism Mus Muscle Cells NF-kappa B Natural regeneration Nature Obstruction Pathology Patients Peripheral Permeability Physiological Proteins Pulmonary Hypertension Pulmonary Vascular Resistance Pulmonary artery structure Reporting Research Personnel SWI/SNF Family Complex Shunt Device Site Smooth Muscle Myocytes Structure Technology Tissues Transgenic Mice Tube Tubular formation Untranslated RNA Vascular Diseases Veno-Occlusive Disease bioprinting chromatin remodeling derepression differential expression gene function genetic variant member monocyte mortality neutrophil novel prevent promoter pulmonary arterial hypertension pulmonary artery endothelial cell recruit response right ventricular failure shear stress transcription factor transcriptome sequencing

项目摘要

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.

肺动脉高压（PAH）是一种使人衰弱的疾病，其周围动脉闭塞肺部导致肺血管阻力升高，最终导致右心衰竭。我们将 PAH 的进展性质与高剪切应力 (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) 基因调控和功能，我们或许能够利用这一机制信息来逆转已确定的疾病，以回应之前关于“综合”的 RFA。组学中，我们将 PAEC 增强子启动子相互作用与差异表达基因 (DEG) 联系起来层流剪切应力（LSS）的生理条件导致染色质可及性的差异，通过 ATAC Seq 评估转录因子 KLF4 在 H3K27ac 增强子位点结合 DNA 的位点，如然而，我们只能根据 ChIP Seq 评估将 LSS 增强子与三分之一的 DEG 相关联。通过结合 HiChIP 和接触活动 (ABC) 算法，我们能够将“最近的基因”关联起来。 80% DEG 的远端增强子，包括跳过的基因和与 PAH 相关的基因，例如 BMPR2。当存在使血管腔变窄的已确定的血管疾病时，或者如果 PAH 是由先天性心脏病的高肺血流量和压力在我们的初步研究中表明。 HSS 对 PAEC 稳态基因（如 BMPR2、JAG1、ERG 和 ELN）的表达有重大影响；此外，内皮间质转化（EndMT）基因的表达令人惊叹，例如 SNAI1，并增加 PAEC 通透性和单核细胞粘附力，我们能够在其中构建 PA EC-SMC 双层。纤维蛋白凝胶使我们能够研究 LSS 和 HSS 下血管细胞的相互作用。我们的首要假设是： PAEC 和 PAEC-SMC 相互作用的变化响应 HSS 对增强剂景观产生不利影响，基因调控和功能，并且可以逆转以预防进行性 PAH 病理学。我们提出了三个具体目标，在具体目标 1 中，我们描述了增强子-启动子中的扰动。解释 HSS 控制下的异常基因调控和 PAH PAEC 的景观，我们将这些联系起来通透性、炎症和 EndMT 功能异常以及基因和在具体目标 2 中，我们生物制造了带有 PAEC 衬里的管状结构。 EC 周围的管腔和 SMC，以确定细胞间相互作用如何影响 HSS 与 LSS 介导的在具体目标 3 中，我们研究了基因调控和功能，包括 SMC 增殖和弹性蛋白纤维形成。重点关注对照和 PAH PAEC 中 HSS 介导的 ERG 显着降低，以确定该特征是否我们确定，对于 HSS 介导的异常 PAEC 基因表达和功能是必要且充分的。 HSS 介导的 miR-96 升高和 ERG 降低是否可以被 miR-96 颠覆 antagomir，无论是在 PAH 细胞还是在 ERG 缺陷的转基因小鼠中，这些研究应该提供新的结果。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。