PROJECT SUMMARY Marfan syndrome (MFS), caused by mutations in the fibrillin-1 gene, is the most common inherited connective tissue disorder. Patients typically develop aortic root aneurysms with ensuing aortic dissection and rupture remaining the leading cause of death. Without prophylactic surgery, life-expectancy is reduced to age 40 years. Novel targeted drug therapies have been absent, likely due to limitations in mechanistic understanding. Thus, there is an urgent need to dissect the pathway(s) involved in MFS aortic wall extracellular matrix (ECM) breakdown resulting in aneurysm formation. We hypothesize that distinct embryonic origins of SMCs populating the aortic root contribute to aortic root-specific aneurysm pathophysiology. In the recent funding period, we created and characterized induced-pluripotent stem cells (iPSCs) from MFS patients and differentiated them into SMCs from each embryologic origin. We learned that the microRNA, miR-29b and related downstream genes were dependent on SMC embryologic origin. After applying high-throughput proteomics, we also discovered that iPSC-derived SMCs from MFS patients express distinct, lineage-specific proteomic profiles affecting critical biologic processes including ECM synthesis/degeneration and SMC contraction/motility. Utilizing single-cell RNA sequencing, we identified a distinct, disease-specific SMC cluster that develops transcriptomic alterations regionally in both murine models and human aortic root aneurysms. In complimentary MFS Fbn1C1041G/+ mouse in vivo studies, we discovered several regional upstream TGF-β-dependent triggers that ultimately increase matrix metalloproteinase-mediated ECM remodeling, including: (a) Ras (GTPase) signaling; (b) NADPH activation leading to reactive oxygen species production; and (c) androgen potentiation of TGF-β signaling in male mice. Drug blockade of each discovered pathway reduced aneurysm formation. In this renewal application, we rigorously expand our research progress by proposing several innovative experiments to study the relative contributions of SMC lineages to regional ECM remodeling, investigate the upstream triggers and pathologic role of modulated SMCs during aneurysm formation, and advance the translation of disease-specific iPSC modeling for novel pathway discovery. Specific Aim 1 seeks whether interactions between MFS SHF- and neural crest-SMCs induce synthetic SMC phenotype and ECM remodeling. We investigate the novel role of reduced SHF-SMC mannose receptor 2 (MRC2) on ECM composition, proteolysis and SMC contractile function. We will also elucidate the effect of ECM biomechanical properties on lineage-specific SMC responses, including SMC transcriptomic modulation and single cell mechanical properties. Specific Aim 2 strives to determine the effects of modulated SMC populations on ECM remodeling in vitro using our innovative MFS decellularized matrix scaffolds. Novel lineage-tracing transgenic mouse models will be utilized to iden...