title: The ACVR1 R206H mutation found in fibrodysplasia ossificans progressiva increases human induced pluripotent stem cell-derived endothelial cell formation and collagen production through BMP-mediated SMAD1/5/8 signaling.
abstract: The Activin A and bone morphogenetic protein (BMP) pathways are critical regulators of the immune system and of bone formation. Inappropriate activation of these pathways, as in conditions of congenital heterotopic ossification, are thought to activate an osteogenic program in endothelial cells. However, if and how this occurs in human endothelial cells remains unclear.
  
  
  
  We used a new directed differentiation protocol to create human induced pluripotent stem cell (hiPSC)-derived endothelial cells (iECs) from patients with fibrodysplasia ossificans progressiva (FOP), a congenital disease of heterotopic ossification caused by an activating R206H mutation in the Activin A type I receptor (ACVR1). This strategy allowed the direct assay of the cell-autonomous effects of ACVR1 R206H in the endogenous locus without the use of transgenic expression. These cells were challenged with BMP or Activin A ligand, and tested for their ability to activate osteogenesis, extracellular matrix production, and differential downstream signaling in the BMP/Activin A pathways.
  
  
  
  We found that FOP iECs could form in conditions with low or absent BMP4. These conditions are not normally permissive in control cells. FOP iECs cultured in mineralization media showed increased alkaline phosphatase staining, suggesting formation of immature osteoblasts, but failed to show mature osteoblastic features. However, FOP iECs expressed more fibroblastic genes and Collagen 1/2 compared to control iECs, suggesting a mechanism for the tissue fibrosis seen in early heterotopic lesions. Finally, FOP iECs showed increased SMAD1/5/8 signaling upon BMP4 stimulation. Contrary to FOP hiPSCs, FOP iECs did not show a significant increase in SMAD1/5/8 phosphorylation upon Activin A stimulation, suggesting that the ACVR1 R206H mutation has a cell type-specific effect. In addition, we found that the expression of ACVR1 and type II receptors were different in hiPSCs and iECs, which could explain the cell type-specific SMAD signaling.
  
  
  
  Our results suggest that the ACVR1 R206H mutation may not directly increase the formation of mature chondrogenic or osteogenic cells by FOP iECs. Our results also show that BMP can induce endothelial cell dysfunction, increase expression of fibrogenic matrix proteins, and cause differential downstream signaling of the ACVR1 R206H mutation. This iPSC model provides new insight into how human endothelial cells may contribute to the pathogenesis of heterotopic ossification.
authors.affiliation: Institute for Human Genetics and the Division of Endocrinology and Metabolism, University of California, 513 Parnassus Avenue, HSE901G, San Francisco, CA, 94143-0794, USA.
authors.forename: Emilie
authors.initials: E
authors.lastname: Barruet
authors.affiliation: Institute for Human Genetics and the Division of Endocrinology and Metabolism, University of California, 513 Parnassus Avenue, HSE901G, San Francisco, CA, 94143-0794, USA.
authors.forename: Blanca M
authors.initials: BM
authors.lastname: Morales
authors.affiliation: Institute for Human Genetics and the Division of Endocrinology and Metabolism, University of California, 513 Parnassus Avenue, HSE901G, San Francisco, CA, 94143-0794, USA.
authors.forename: Wint
authors.initials: W
authors.lastname: Lwin
authors.affiliation: Gladstone Institute of Cardiovascular Disease, 1650 Owens Street, San Francisco, CA, 94158, USA.
authors.forename: Mark P
authors.initials: MP
authors.lastname: White
authors.affiliation: Gladstone Institute of Cardiovascular Disease, 1650 Owens Street, San Francisco, CA, 94158, USA.
authors.forename: Christina V
authors.initials: CV
authors.lastname: Theodoris
authors.affiliation: Institute for Human Genetics and the Division of Endocrinology and Metabolism, University of California, 513 Parnassus Avenue, HSE901G, San Francisco, CA, 94143-0794, USA.
authors.forename: Hannah
authors.initials: H
authors.lastname: Kim
authors.affiliation: Institute for Human Genetics and the Division of Endocrinology and Metabolism, University of California, 513 Parnassus Avenue, HSE901G, San Francisco, CA, 94143-0794, USA.
authors.forename: Ashley
authors.initials: A
authors.lastname: Urrutia
authors.affiliation: School of Dentistry, Oral and Craniofacial Sciences Program, University of California, 707 Parnassus Avenue, San Francisco, CA, 94143, USA.
authors.forename: Sarah Anne
authors.initials: SA
authors.lastname: Wong
authors.affiliation: Gladstone Institute of Cardiovascular Disease, 1650 Owens Street, San Francisco, CA, 94158, USA.
authors.forename: Deepak
authors.initials: D
authors.lastname: Srivastava
authors.affiliation: Institute for Human Genetics and the Division of Endocrinology and Metabolism, University of California, 513 Parnassus Avenue, HSE901G, San Francisco, CA, 94143-0794, USA. Edward.Hsiao@ucsf.edu.|Department of Endocrinology, Diabetes, and Metabolism, Institute for Human Genetics, University of California, 513 Parnassus Avenue, HSE901G, UCSF Box 0794, San Francisco, CA, 94143-0794, USA. Edward.Hsiao@ucsf.edu.
authors.forename: Edward C
authors.initials: EC
authors.lastname: Hsiao
mesh_terms: D030201:Activin Receptors, Type I / Q000235:genetics*; D028341:Activins / Q000378:metabolism; D055415:Bone Morphogenetic Protein 4 / Q000378:metabolism*; D002454:Cell Differentiation / Q000502:physiology; D002460:Cell Line; D020219:Chondrogenesis / Q000235:genetics / Q000502:physiology; D003094:Collagen / Q000378:metabolism*; D042783:Endothelial Cells / Q000378:metabolism* / Q000502:physiology; D005109:Extracellular Matrix / Q000378:metabolism; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000378:metabolism* / Q000502:physiology; D008024:Ligands; D009154:Mutation; D009221:Myositis Ossificans / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D009999:Ossification, Heterotopic / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D010006:Osteoblasts / Q000378:metabolism / Q000502:physiology; D010012:Osteogenesis / Q000502:physiology; D010766:Phosphorylation / Q000235:genetics / Q000502:physiology; D015398:Signal Transduction / Q000502:physiology; D051785:Smad Proteins / Q000378:metabolism*
chemical_list: D055415:Bone Morphogenetic Protein 4; D008024:Ligands; D051785:Smad Proteins; C422405:activin A; D028341:Activins; D003094:Collagen; C425423:ACVR1 protein, human; D030201:Activin Receptors, Type I
keywords: ACVR1; Activin A signaling; BMP; Fibrodysplasia ossificans progressiva; Tissue fibrosis; hiPS-derived endothelial cells