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.

Public Health Relevance

We study the impact of high shear stress on the aberrant regulation of genes that leads to functional abnormalities causing occlusion of pulmonary arteries in pulmonary arterial hypertension. We build vascular tubular structures to study endothelial interaction with smooth muscle cells under high shear stress, and pursue the genomic and functional sequelae resulting from a reduction in a critical transcription factor, ERG, and how these abnormalities can be corrected.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Research Project (R01)
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Respiratory Integrative Biology and Translational Research Study Section (RIBT)
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Xiao, Lei
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Stanford University
Schools of Medicine
United States
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