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Introduction: Mutation in the extracellular matrix (ECM) protein fibrillin-1 lead to aortic root aneurysm in Marfan Syndrome (MFS). ECM composition influences vascular smooth muscle cell (SMC) phenotype, contractility, and signaling through mechanotransduction. Induced pluripotent stem cell-derived SMCs (iPSC-SMCs) are emerging as an attractive approach to model patient-specific vascular disease, but the use of iPSC-SMCs to investigate extracellular matrix biology remains limited. Hypothesis: Diseased MFS ECM induces phenotype modulation in healthy Control cells, while Control ECM will rescue contractile phenotype in MFS. Methods: Label-free mass spectroscopy core matrisome proteomic profiling of MFS and Control iPSC-SMCs from ammonia hydroxide decellularized ECM was performed. Decellularized ECMs were repopulated with MFS or Control iPSC-SMCs (N=3) (Figure A) and RNA extracted from whole cell lysate for RNA sequencing after four days of culture. Results: 24 core matrisome proteins were differentially expressed including overexpressed Collagen 1A1 and Tenascin C in MFS and enriched Thrombospondin 1 and Latent TGF-β Binding Protein 2 in Control (Figure B). Control iPSC-SMCs dramatically decreased contractile gene expression ( ACTA2 and CNN1 ) and ECM synthesis ( COL1A1 and FN1 ) when cultured on the diseased MFS ECM compared to the native Control ECM (Figure C). Conversely, the MFS cells expressed equally low contractile genes and the synthetic phenotype was exacerbated when cultured on the Control ECM (Figure D). Conclusions: Control iPSC-SMC mechanotransduction remains intact when interacting with the MFS ECM resulting in phenotype modulation into a MFS-like cell. Irreversible reprogramming of the MFS iPSC-SMC prevents rescue of the contractile phenotype when grown on a healthy ECM, despite the absence of MFS specific matrisome proteins. The decellularized iPSC-SMC ECM system effectively captures cell-ECM interactions and signaling.

Introduction: Vascular smooth muscle cells (SMC) undergo phenotype modulation (silenced contractile genes and activated proteolytic/synthetic genes) in Marfan syndrome (MFS) aortic aneurysm. Aortic root SMCs develop from the second heart field (SHF) proximally and neural crest (NC) distally. We hypothesize that both developmental lineage and hemodynamic factors dictate SMC vulnerability to phenotype change, promoting focal aortic root aneurysm. Methods/Results: Aortic roots from adult MFS and littermate control mice (n=4 each sex) with a knockin fluorescent SHF tracing construct ( Nkx2-5 cre ) were sorted for lineage-resolved single cell RNA sequencing (scRNAseq). Modulated SMCs (modSMC) were enriched in MFS vs. controls (18.1% of all SMCs vs. 2.2%) and originated from both SHF (15.5%) and NC (20.2%) lineages, ruling out complete lineage specificity. NC-derived modSMCs expressed more aggrecan ( Acan ) and sclerostin ( Sost ) while small proteoglycans decorin ( Dcn ) and lumican ( Lum ) were enriched in SHF modSMCs. Insulin-like growth factor binding protein-2 ( Igfbp2 ) was a lineage-independent modSMC marker. RNA in situ hybridization localized Igfbp2 + SMCs within SHF/NC overlap in the aortic root. To study regional SMC propensity for modulation due to hemodynamic stresses, we generated aortic pressure overload via 27-gauge transverse aortic arch constriction (TAC) or sham surgery on healthy Myh11 cre / Rosa tdT (SMC-traced) mice. TAC induced mild root dilation vs . sham (1.93±0.07 mm vs. 1.69±0.09 mm, n=4, p Conclusions: Embryologic origin does not dictate aortic SMC propensity for pathologic phenotype change in MFS but does promote distinct ECM proteoglycan gene activation. Aortic pressure overload incites root SMC modulation, suggesting hemodynamic effects may promote regional susceptibility to SMC-mediated aortic remodeling.

Introduction: Marfan syndrome (MFS) patients classically develop focal aortic root aneurysms. Aortic root smooth muscle cells (SMCs) develop predominately from second heart field (SHF), whereas ascending aorta/arch SMCs originate from cardiac neural crest (CNC). We previously demonstrated induced pluripotent stem cell (iPSC)-derived SHF SMCs overexpress integrin αV (ITGAV). Therefore, we hypothesize that ITGAV promotes SHF SMC proliferation, contributing to thoracic aortic root aneurysm development. Methods: iPSCs from MFS patients (n=2) and healthy control (n=1) were differentiated into SHF and CNC SMCs. ITGAV-pFAK-pAkt Thr308 levels were detected with western blot and ELISA at baseline and following recombinant TGF-β (2ng/ml) and/or an ITGAV antagonist (GLPG0187, 10nM) for 24 hours. Cell proliferation was measured with quantitative BrdU assay. Results: Baseline ITGAV-pFAK-pAkt Thr308 activation were significantly elevated in MFS SHF lines (n=5 experimental replicates) compared to MFS CNC (n=5) or control SHF (n=4, pThr308 levels in MFS SHF. Treatment with GLPG0187 reduced pFAK-pAkt Thr308 in MFS SHF and CNC, confirming ITGAV-mediated signaling promotes pathologic FAK- Akt Thr308 activation. SMCs from MFS SHF (n=6) had increased proliferation compared to other SMCs (p Conclusions: The integrin-FAK-Akt Thr308 signaling pathway is activated in iPSC SMCs from MFS patients specifically from the SHF lineage, which is further exacerbated by TGF-β. Mechanistically, this pathway promotes SMC proliferation in vitro . ITGAV blockade via GLPG0187 may be a promising therapeutic approach for MFS aneurysmal growth.

Introduction: Mutations in TGF-beta (TGF-ß) signaling genes lead to aortic root aneurysm in Loeys Dietz syndrome (LDS). Smooth muscle cells (SMCs) in the proximal aorta develop from two embryologic origins: second heart field (SHF) and neural crest (NC). Induced pluripotent stem cell (iPSC) models simulate these lineages, but direct correlation to clinical disease is lacking. Hypothesis: iPSC-derived SMCs accurately model lineage-specific aortopathy in LDS. Methods: We generated SMC lines from root and ascending aortic surgical tissue and iPSC-derived SMCs through SHF and NC-specific pathways from an LDS patient ( TGFBR1 mutation). Lineage-specific TGF-ß responses were determined by western blot/ELISA. RNA sequencing and RT-PCR identified SMC transcriptomes. Results: Aortic root SMCs showed greater canonical TGF-ß activation (p-SMAD2/3) versus ascending at baseline and with TGF-ß stimulation ( Figure ). Synonymous results were seen in SHF versus NC SMCs from the iPSC pathway. RNAseq identified 1,600 differentially expressed genes between iPSC lineages, including altered TGF-ß receptor and ligand expression profiles. Primary aortic lines validated iPSC data: root SMCs showed enriched TGF-ß receptor 1/2/3 expression (1.7-, 3.9- and 5.9-fold) while ascending SMCs overexpressed TGFB1 and TGFB2 ligands (1.8- and 3.5-fold). Despite discordant TGF-ß activation, SMC contractile gene expression was similar between lineages in aortic and iPSC-SMCs, suggesting alternative downstream effects in LDS aneurysm. Conclusion: iPSC-derived SMCs effectively model lineage-specific aortic root aneurysm pathology, validating this model as a tool for mechanistic testing and therapy discovery.