Background: The Activin A and bone morphogenetic protein (BMP) pathways are critical regulators of the immune\nsystem and of bone formation. Inappropriate activation of these pathways, as in conditions of congenital heterotopic\nossification, are thought to activate an osteogenic program in endothelial cells. However, if and how this occurs in\nhuman endothelial cells remains unclear.\nMethods: We used a new directed differentiation protocol to create human induced pluripotent stem cell (hiPSC)-\nderived endothelial cells (iECs) from patients with fibrodysplasia ossificans progressiva (FOP), a congenital disease of\nheterotopic ossification caused by an activating R206H mutation in the Activin A type I receptor (ACVR1). This strategy\nallowed the direct assay of the cell-autonomous effects of ACVR1 R206H in the endogenous locus without the use of\ntransgenic expression. These cells were challenged with BMP or Activin A ligand, and tested for their ability to activate\nosteogenesis, extracellular matrix production, and differential downstream signaling in the BMP/Activin A pathways.\nResults: We found that FOP iECs could form in conditions with low or absent BMP4. These conditions are not normally\npermissive in control cells. FOP iECs cultured in mineralization media showed increased alkaline phosphatase staining,\nsuggesting formation of immature osteoblasts, but failed to show mature osteoblastic features. However, FOP iECs\nexpressed more fibroblastic genes and Collagen 1/2 compared to control iECs, suggesting a mechanism for the tissue\nfibrosis seen in early heterotopic lesions. Finally, FOP iECs showed increased SMAD1/5/8 signaling upon BMP4\nstimulation. Contrary to FOP hiPSCs, FOP iECs did not show a significant increase in SMAD1/5/8 phosphorylation upon\nActivin A stimulation, suggesting that the ACVR1 R206H mutation has a cell type-specific effect. In addition, we found\nthat the expression of ACVR1 and type II receptors were different in hiPSCs and iECs, which could explain the cell typespecific\nSMAD signaling.Conclusions: Our results suggest that the ACVR1 R206H mutation may not directly increase the formation of\nmature chondrogenic or osteogenic cells by FOP iECs. Our results also show that BMP can induce endothelial cell\ndysfunction, increase expression of fibrogenic matrix proteins, and cause differential downstream signaling of the\nACVR1 R206H mutation. This iPSC model provides new insight into how human endothelial cells may contribute\nto the pathogenesis of heterotopic ossification.
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