Project 1: Endothelial cell programs for coronary artery disease

Stanford University, Stanford CA

Investigators

Abstract

PROJECT SUMMARY: Project 1 Coronary artery disease (CAD) remains the leading cause of death throughout the world despite therapies targeting leading risk factors, calling for the rapid development of new treatments. One approach is to use human genetic association studies to identify medically targetable pathways. However, finding disease-causing genes has been a slow endeavor because it requires testing one gene at a time and there are over 300 CAD loci. Our lab recently demonstrated that a new technology called Perturb-seq can dramatically increase this discovery pipeline because it can functionally characterize all 300 CAD loci in one experiment by identifying convergence of risk pathways. When we performed this assay in primary human endothelial cells (ECs), we discovered that 41 CAD risk loci converge onto a small number of common pathways that could promote disease risk. The major disease-associated pathway we identified contained several genes previously associated with Cerebral Cavernous Malformations (CCM), a rare neurovascular disease. Common variants in the CCM-signaling pathway were consistently protective for CAD risk, despite established pathogenic roles for these genes for neurovascular disease. We showed that two CAD-associated genes—CCM2 and TLNRD1—interact in the CCM complex and regulate many other CAD risk genes and the endothelial shear stress response. Given this success, Project 1 will use our recently developed in vivo Perturb-seq capabilities to rapidly map out disease mechanisms of the CCM pathway in the whole organism and identify CAD gene programs active in diseased arteries. Specifically, we need to understand why mutations in the CCM-signaling pathway are pathogenic for cerebrovascular disease, but protective for CAD. Our experiments will address the central hypothesis that the CCM signaling pathway is a major determinant of CAD risk, with context-dependent effects on ECs that depend on the stage of atherosclerosis, dosage/degree of gene inhibition, and EC subtypes from vascular beds in different organs. Applying new innovations for in vivo Perturb-seq developed by our Scientific Cores, the Aims of Project 1 are to 1) determine context-dependent effects of 250 genes in 3 stages of murine atherosclerosis; 2) characterize the effects of EC-specific TLNRD1 knockout on atherosclerosis, vascular development, arteriogenesis, and angiogenesis; and 3) identify the organotypic effects of CCM2/TLNRD1 knockdown in brain, lung, and aortic ECs. Our study will be highly synergistic with the other Projects in the Program, enabling comparison of causal CAD programs between ECs and smooth muscle cells (with Project 2), and comparisons of the functions of EC genes and programs between two distinct diseases (CAD vs. pulmonary arterial hypertension with Project 3). Through integration across all 3 Projects, we will address the central goal of the Program to identify cell-type specific effects of causal genes in multiple vascular cell types and prioritize the pathways with the greatest translational relevance for therapy and prevention.