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

Background: 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.

Methods: 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.

Results: 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.

Conclusions: 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.

Keywords: ACVR1; Activin A signaling; BMP; Fibrodysplasia ossificans progressiva; Tissue fibrosis; hiPS-derived endothelial cells.

Fig. 1

Differentiation of human induced pluripotent stem cell (hiPSC)-derived endothelial cells (iECs) from wild type (WT) and fibrodysplasia ossificans progressiva (FOP) hiPSC lines. a Endothelial cell differentiation protocol adapted from White et al. [17]. WT and FOP hiPSCs were induced to form mesodermal progenitors using BMP4, Activin A, and bFGF. Embryoid bodies (EBs) were cultured in medium supplemented with VEGF and bFGF on day 4 and plated onto collagen IV on day 5. iEC precursors were identified by KDR/PECAM positivity. Scale bars, 200 μm (hiPSCs, EBs, and EBs at day 6) and 1 μm (hiPS-derived endothelial cells). b Mean percentage of cells expressing both PECAM and KDR by fluorescence-activated cell sorting (FACS) analysis on day 6 of endothelial differentiation of one WT hESC line, three WT hiPSC lines, and four FOP hiPSC lines. Error bars represent the mean ± one standard deviation of at least three independent replicates for each of the three WT and four FOP cell lines. Mean values were not statistically different. c FACS analysis of WT and FOP hiPSCs on day 0 and day 6 of endothelial differentiation. iECs co-expressing PECAM and KDR were sorted by FACS on day 6 and then cultured in endothelial cell medium. Analysis of PECAM and VE-Cadherin expression by FACS of sorted iECs after one passage is shown on the right. d WT and FOP hiPSCs were differentiated as in Fig. 1A and sorted on day 6. iECs were immunostained for endothelial markers PECAM and VE-Cadherin. Scale bars, 100 μm

Fig. 2

Loss of endothelial lineage commitment of fibrodysplasia ossificans progressiva (FOP) human induced pluripotent stem cell-derived endothelial cells (iECs). a Gene expression analysis of endothelial markers by quantitative PCR of wild type (WT) and FOP iECs. b Mesenchymal/fibroblastic (FSP-1, CD34), mature chondrogenic (COL2A1), and osteogenic (COL1A1, ALPL) gene expression were statistically different by Student’s t test: * p < 0.05, ** p < 0.01. Error bars represent the mean ± one standard deviation of at least three independent replicates for each of the three WT and four FOP iEC lines

Fig. 3

Osteogenic capacity of fibrodysplasia ossificans progressiva (FOP) human induced pluripotent stem cell-derived endothelial cells (iECs). a FOP iECs showed increased alkaline phosphatase (ALPL) staining at day 15. b Quantification of ALPL staining from at least three independent replicates for each of the three wild type (WT) and four FOP iECs lines. Error bars represent the mean ± one standard deviation; ** p < 0.01 by Student’s t test. c Osteogenic and chondrogenic gene expression analysis by quantitative PCR of WT and FOP iECs cultured in 50 % endothelial cell medium/50 % OB medium for 15 days. COL1A1 and COL2A1 gene expression were statistically different by Student’s t test: * p < 0.05. Error bars represent the mean ± one standard deviation of at least three independent replicates for each of the three WT and four FOP iEC lines

Fig. 4

Fibrodysplasia ossificans progressiva (FOP) human induced pluripotent stem cell (hiPSC)-derived endothelial cells (iECs) form in conditions not permissive for controls and respond differently upon stimulation. a Wild type (WT) and FOP hiPSCs were differentiated as illustrated in Fig. 1d using different concentration of BMP4 (0, 1, 4, and 12 ng/ml). Shown is the mean percentage of PECAM⁺/KDR⁺ cells analyzed by fluorescent-activated cell sorting (FACS) at day 6 from three WT and four FOP hiPSC lines in at least three separate experiments for each group. Mean values were statistically different for 0, 1, and 4 ng/ml of BMP4. b WT and FOP hiPSCs were differentiated with or without supplemental Activin A. FACS analysis of PECAM⁺/KDR⁺ cells at day 6 did not show significant differences. c Representative western blot showing activation of SMAD pathways upon BMP4, Activin A, or TGFβ1 stimulation in WT and FOP iECs. d Western blot quantification of p-SMAD2 and p-SMAD1/5/8. Phosphorylation of SMAD2 was significantly increased in WT and FOP iECs treated with TGFβ1 compared to untreated cells. p-SMAD2 and p-SMAD1/5/8 protein expression were normalized to GAPDH protein expression. At least three separate experiments were run for WT and FOP iECs for each group. Error bars represent the mean ± one standard deviation. Mean values were statistically different by Student’s t test: * p < 0.05, ** p < 0.01, *** p<0.005

Fig. 5

Cell type-specific expression of SMADs and type I and II receptors. a Expression of the inhibitory SMAD6 and SMAD7 was higher in human induced pluripotent stem cell (hiPSC)-derived endothelial cells (iECs) than in hiPSCs. Also, SMAD6 had significantly higher expression in fibrodysplasia ossificans progressiva (FOP) iECs. b Activin A gene expression levels were higher in iECs than in hiPSCs; however, there was no difference in Activin A levels between FOP and wild type (WT) by quantitative PCR or ELISA. c Gene expression analysis of WT ACVR1, ACVR1 R206H, and the ratio of R206H/WT ACVR1. WT ACVR1 expression was higher in iECs compared to hiPSCs. WT ACVR1 levels were lower in FOP cells and ACVR1 R206H was detected only in FOP cells, as expected. d ACVR2A, ACVR2B, and BMPRII gene expression analysis of WT and FOP hiPSCs, iECs, and iECs cultured in osteogenic media (OB iEC). Type II receptor expression was differentially expressed based on cell type but not on ACVR1 R206H status. WT and FOP hiPSCs, iECs, and iECs cultured in osteogenic media were run in at least three separate experiments for each group. Error bars represent the mean ± one standard deviation. Mean values were statistically different by Student’s t test: * p < 0.05, ** p < 0.01, *** p < 0.005, **** p < 0.0001

Fig. 6

Summary of using human induced pluripotent stem cell (hiPSCs) to understand fibrodysplasia ossificans progressiva (FOP) R206H ACVR1 effects on human endothelial cell (EC) lineages. FOP hiPSCs are able to differentiate into ECs in a non-permissive condition and show increased osteogenic potential [14], which may reflect the increased number of ECs found in FOP lesions. FOP hiPSC-derived ECs (iECs) show increased expression of mesenchymal, extracellular matrix, chondrogenic, and osteogenic markers, which may play a major role in the tissue fibrosis found in patients with FOP during early stages of heterotopic ossification. FOP iECs show increased SMAD6 gene expression and respond differently to different ligands such as BMP, which could be proposed as a mechanism for their loss of cellular commitment and their proficiency in undergoing endothelial-to-mesenchymal transition