Project 3: Endothelial and smooth muscle cell programs for pulmonary arterial hypertension

Stanford University, Stanford CA

Investigators

Abstract

PROJECT SUMMARY: Project 3 Pulmonary arterial hypertension (PAH) is caused by progressive loss and occlusion of distal arteries, owing to endothelial cell (EC) dysfunction, leading to smooth muscle cell (SMC) proliferation and a chronic inflammatory response. Current FDA-approved agents improve survival and quality of life in PAH patients, but there is no cure for PAH short of lung transplantation. BMPR2 is the most frequently mutated gene in PAH and 11 other causal mutations have been identified, about half in the BMPR2 pathway. However, for >70% of PAH patients a genetic cause has not been found. Project 3 will use in vivo Perturb-seq to address the hypothesis that genes that predispose to or protect against PAH impact branches of the BMPR2 signaling pathway in ECs, and that the functions of these genes depend on genetic interactions, environmental stimuli, and tissue-specific wiring of ECs and SMCs. In Aim 1, we will utilize a library of 250 guides shared with Project 1 but delivered by a vector that selectively targets lung ECs in mice. The library contains guides to known PAH causal genes as well as activators and regulators of those genes, and others related to EC function and coronary artery disease (CAD). By relating gene expression in singe cells to each guide, we will identify novel gene programs that indicate predisposition to or protection against PAH. In collaboration with Project 1, we will determine whether gene programs in response to the same perturbation in lung and aortic ECs explain different propensities to PAH and CAD. In Aim 2, we will select 50 genes identified in Aim 1 that are predicted to either cause PAH when there is a genetic predisposition such as Bmpr2 deficiency or reverse PAH when Bmpr2 deficiency and oxidant stress are combined. Analysis of the resulting gene programs will allow us to select genes for individual perturbation to test for their ability to worsen or reverse PAH including those with a different gene program in aortic ECs. Genes in novel programs with the strongest potential link to PAH from Aims 1 and 2 will be prioritized for evaluation in human pulmonary artery (PA)ECs and human tissue samples from PAH patients including those with a BMPR2 mutation. In Aim 3, we will collaborate with Project 2 to study EC-SMC interactions in mice with Smad3 deleted in SMCs, as loss of SMC-Smad3 increases CAD but has been shown to protect against PAH. This will determine how EC programs are altered by cross-talk with SMCs, and whether there is a site-specific difference in EC and SMC gene expression and cross-talk in the lung and the aorta. In this aim, we will also use perfused vascular tubes populated by human PAECs and PASMCs to assess changes in gene expression and cellular responses when SMAD3 is deficient in PASMCs, or when BMPR2 is mutant in both cell types, compared to those identified in mice. The goal of Project 3, enabled by the technical and analytical expertise of the Cores and in collaboration with Projects 1 and 2, is to uncover new features of gene dysregulation that modify propensity to PAH and could be targeted to reverse PAH and not predispose to CAD.