Abstract Body: Background: Vascular Ehlers-Danlos Syndrome (vEDS) is a rare genetic disorder caused by mutations in COL3A1. The disorder manifests primarily as vascular fragility, resulting in aneurysms and spontaneous arterial ruptures. Although viewed as a defect arising from abnormal extracellular matrix composition, emerging evidence suggests a critical role for intrinsic cellular defects within vascular smooth muscle cells (vSMCs).
Objective: To identify dysregulated cell signaling pathways in human induced pluripotent stem cell-derived vSMCs (hiPSC-vSMCs) from vEDS patients that promote vSMC dysfunction.
Methods: RNA-seq datasets were analyzed from vEDS and control fibroblasts (GSE239914). Synthetic and contractile vSMCs (induced by the MEK inhibitor (MEKi)) differentiated from hiPSC lines harboring vEDS-associated COL3A1 mutations (c.755G>T) and isogenic controls were analyzed. We quantified the protein abundance for p38 MAPK isoforms and SMAD2/3 signaling pathways ± pharmacological interventions by Western blotting. Contractile phenotypes were characterized by assessing smooth muscle and contractile-regulatory markers (CNN1, ACTA2, TAGLN and MLC2). Single cell forces were measured using micropost array detectors (mPADs).
Results: In vitro differentiated vSMCs were highly proliferative, and MEKi treatment significantly reduced proliferation and induced a contractile phenotype in both vEDS and isogenic hiPSC-vSMCs. Contractile protein markers and single cell forces were also increased in synthetic vEDS hiPSC-vSMCs relative to isogenic control vSMCs (p<0.05, n = 4). RNA-seq analysis suggested reduced activity of several signaling pathways in vEDS cells. Using synthetic vEDS hiPSC-vSMCs, western blotting showed no significant alterations in phosphorylation of SMAD2/3; however, vEDS hiPSC-vSMCs exhibited significantly decreased phosphorylation of the p38 MAPK isoforms compared to isogenic controls. These data and pharmacological modulation of p38 MAPK signaling implicate this pathway in the hypercontractile phenotype of synthetic vSMCs. The role of specific isoforms in this vEDS-restricted dysregulation is under investigation.
Conclusion: Our findings show that synthetic vEDS hiPSC-vSMCs have dysregulated p38 MAPK signaling, increased contractile protein levels and increased force generation relative to isogenic controls. These data are consistent with the formation of hypercontractile synthetic vSMCs that may contribute to the vascular fragility of patients with vEDS.