Background/Purpose: Systemic sclerosis associated pulmonary arterial hypertension (SSc-PAH) is of particularly clinical interest since its outcomes remain unfavorable despite modern PAH therapies. Previous reports showed the abnormality of endothelial cells (ECs) in SSc-PAH, but details have yet to be shown. We aimed to investigate the pathogenesis of SSc-PAH with disease-specific induced pluripotent stem cells (iPSCs).

Methods: Peripheral blood mononuclear cells were obtained from a patient with SSc-PAH and a healthy donor. Yamanaka factors were transfected to establish iPSCs. ECs were differentiated with the culture system containing bone morphogenetic protein 4, activin, basic fibroblast growth factor, CHIR99021, Y-27632, VEGF and SB431542. Cell proliferation and vasculogenesis were evaluated by the bromodeoxyuridine (BrdU) assay and the tube formation assay, respectively. RNA-sequencing (RNA-seq) was performed to pick up differentially expressed genes (DEGs). Picked up gene was knocked down using siRNA for human umbilical vein endothelial cells (HUVECs) and endothelial to mesenchymal transition (Endo-MT), another possible hallmark of SSc-PAH, was evaluated by immunofluorescence staining of Endo-MT markers. Conversely, the expression of protein encoded by picked up gene was evaluated in untransfected HUVECs in which Endo-MT was induced in the presence of TGF-β.

Results: The cellular uptake of BrdU was increased (0.49±0.05 vs 0.30±0.01 abs, p< 0.05, n=3) while the tube formation was impaired (31.2±2.0 vs 47.0±1.3 mm, p< 0.01, n=3) in SSc-PAH ECs compared to healthy ECs. RNA-seq revealed some DEGs and enriched Gene Ontology terms, including blood vessel development and vascular endothelial growth factor-activated receptor activity. Based on these results, we focused on caveolin1 (CAV1), one of the downregulated genes in SSc-PAH ECs. CAV1 was included in 9 of the top 10 pathways in our Gene Ontology analysis of down-regulated genes, and it is a component molecule of caveolae involved in signal transduction. siRNA-mediated knockdown of CAV1 in HUVECs resulted in increased uptake of BrdU (0.70±0.05 vs 0.46±0.01 abs, p< 0.001, n=6). The expression of mesenchymal cell markers αSMA and vimentin were increased whilst that of endothelial cell marker von Willebrand factor was decreased in CAV1 downregulated HUVECs compared to control HUVECs. Moreover, CAV1 protein was downregulated in HUVECs with TGF-β induced Endo-MT.

Conclusion: We successfully induced SSc-PAH specific ECs through iPSCs. SSc-PAH specific ECs were characterized by facilitated cell proliferation and impaired vasculogenesis. CAV1 may be one of the molecular causes of ECs dysfunction and Endo-MT.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/establishment-of-systemic-sclerosis-associated-pulmonary-arterial-hypertension-specific-endothelial-cells-through-ipscs-and-their-functional-and-molecular-analyses/