Developmental basis for vascular smooth muscle cell dysfunction in Marfan syndrome aortic aneurysm

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Project Summary/Abstract Marfan syndrome (MFS), caused by mutations in the fibrillin-1 (FBN1) gene, is the most common inherited connective tissue disorder, affecting 1 in 5,000 individuals. Aortic root aneurysm leads to reduced life expectancy due to dissection or rupture of the aneurysm unless preventative aortic surgery is performed. Normally, vascular smooth muscle cells (SMCs) maintain homeostasis within the aorta via dynamic contraction/relaxation and extracellular matrix production, however these cells retain significant plasticity to alter their phenotype in response to injury, growth factors, or other stimuli. Dysfunctional SMC phenotype modulation is known to contribute to aneurysm development in MFS. Dysregulated transforming growth factor-beta (TGF-b) signaling also contributes to aortic aneurysm, though the precise role of this pathway remains controversial. Furthermore, mechanisms driving the tendency of the aortic root (the segment most proximal to the aortic valve) to develop focal aneurysm despite systemic effects of FBN1 mutations are poorly understood. Distinct embryonic origins of SMCs populating the aortic root (from the second heart field, SHF) and ascending aortic segments (from neural crest, NC) is hypothesized to contribute to aortic root-specific pathology, however it remains unclear how these embryonic origins affect propensity for aneurysm development. Recently, single-cell RNA sequencing (scRNA- seq) has permitted high-resolution analysis of individual SMC gene expression. My preliminary work applying scRNA-seq to a mouse model of MFS has identified a subset of SMCs with a severely modulated, pathologic phenotype. The proposed study will advance our current understanding of SMC development and dysfunction in MFS aortic aneurysm using two complementary aims. In Aim 1 I will define the distinct phenotypes of thoracic aortic SMCs derived from the second heart field and neural crest lineages by applying single-cell RNA sequencing to an embryonic lineage-tracing mouse model and in vitro studies of TGF-b dysregulation on SHF and NC-derived SMC phenotype. In Aim 2 I will characterize the source and pathologic effects of modulated SMCs in MFS aortic root aneurysm by lineage-tracing early SMCs in a murine MFS model and applying computational transcriptomic analysis tools to scRNAseq data to determine molecular mechanisms driving their phenotype changes. Co-culture experiments of phenotypically modulated SMCs with healthy aortic SMCs will model aortic aneurysm pathology in vitro. These studies will generate important data that will help pinpoint molecular mechanisms driving aortic pathology in MFS and other hereditary aneurysm disorders toward new therapy development. The proposed research training plan features direct mentorship from a committee of accomplished clinician-scientists and access to state-of-the art facilities and techniques. This plan also incorporates professional development and career planning strategies, employing the unique collaborative spirit between Cardiovascular Surgery and Medicine at Stanford University intended to maximize training potential.