Project Summary/Abstract Marfan syndrome (MFS) is the most common inherited connective tissue disorder, caused by mutations in the fibrillin-1 (FBN1) gene, affecting 1 in 5,000 individuals. Aortic root aneurysms lead to aortic dissection or rupture, resulting in reduced life expectancy unless preventative aortic surgery is performed. Normally, aortic wall homeostasis depends on SMC sensing and responding to ECM mechanical force in a process called mechanotransduction. Dysfunctional ECM maintenance results in aortic wall stiffening, but the role of mechanotransduction in aneurysm development remains controversial. Furthermore, mechanisms driving focal aneurysm development restricted to the aortic root (the segment most proximal to the aortic valve) despite systemic effects of FBN1 mutations are poorly understood. SMCs populating the aorta are derived from specific embryologic origins such that the aortic root is derived from the second heart field (SHF) and ascending aortic segments arise from neural crest (NC). We have developed an induced pluripotent stem cell in vitro system to model embryologic derived vascular pathology. Utilizing an iPSC model relinquishes the dependance for surgical tissue specimens and opens the door for personalized precision medicine. My preliminary work showed that iPSC-derived SMC grown on varying stiffness plates demonstrated a distinct embryologic response to increasing ECM stiffness. The proposed study will advance our current understanding in ECM-SMC mechanotransduction during aneurysm formation using two complimentary aims. Aim 1 will assess the transcriptomic effects of ECM stiffness and composition on SMCs from both embryologic origins by applying single cell RNA sequencing to cells grown on varying stiffness and ECM composition. The composition and mechanical properties of ECM produced by each embryologic origin SMC will be compared with atomic force microscopy and mass spectrometry. Aim 2 will investigate embryologic dependent ECM stiffening by utilizing iPSC-derived SMCs transduced to overexpress mannose receptor 2 (MRC2) to characterized intracellular collagen recycling in vitro. A transgenic lineage traced murine model will be used to characterize TGF-b effects on MRC2 induced ECM pathology in vivo. These studies will generate a greater understanding of how altered ECM composition and stiffness influences ECM-SMC mechanotransduction to provide insight into new therapeutic targets to prevent aneurysm formation. The proposed research training plan features direct mentorship from a diverse committee of clinician-scientist and access to state-of-the-art facilities and techniques. The plan incorporates professional development and career planning strategies, utilizing collaborative resources between Cardiothoracic Surgery, Cardiovascular Medicine, and the Cardiovascular Institute to maximize my training potential.