Date: Monday, November 9, 2020
Session Type: Poster Session D
Session Time: 9:00AM-11:00AM
Background/Purpose: Pulmonary arterial hypertension (PAH) occurs either primarily or in association with other diseases such as connective tissue diseases. PAH associated with systemic sclerosis (SSc-PAH) is of particularly clinical significance since its outcomes remain unfavorable despite modern PAH therapies, suggesting its distinctive pathogenesis. Previous studies have indicated the abnormality of endothelial cells (ECs) in the lung of SSc-PAH, but its details have yet to be clarified owing to the poor accessibility to lung samples from those patients. To overcome the problem and to elucidate the pathogenesis of SSc-PAH, we established SSc-PAH specific ECs differentiated from induced pluripotent stem cells (iPSc) and performed their functional and molecular analysis.
Methods: Peripheral blood mononuclear cells were obtained from a patient with SSc-PAH and a healthy donor. Yamanaka factors, including OCT3/4, SOX2, KLF4 and L-MYC, were transfected into peripheral blood mononuclear cells using sendai virus vector to establish iPSc. Following more than five passages of iPSc, ECs were differentiated with the culture system containing BMP-4, Activin, bFGF, CHIR99021, Y-27632, VEGF and SB431542. Differentiated ECs were confirmed by immunocytochemistry with specific markers, including VE-cadherin, KDR and CD34. The vasculogenesis and cell proliferative capacity of ECs were evaluated by the tube formation assay and the BrdU assay, respectively. RNA-sequencing was performed to evaluate differentially expressed genes between SSc-PAH ECs and healthy ECs.
Results: SSc-PAH ECs and healthy ECs equally VE-cadherin, KDR and CD34 with similar morphologies, but were functionally distinct. The cellular uptake of BrdU was higher (0.49±0.05(abs) vs 0.30±0.01(abs), p< 0.05, n=3) while the tube formation was impaired (31.2±2.0(mm) vs 47.0±1.3(mm), p< 0.01, n=3) in SSc-PAH ECs compared to healthy ECs, indicating disproportionately reduced vasculogenesis despite accelerated proliferation of SSc-PAH ECs. RNA-sequencing revealed some differentially expressed genes (DEGs) and signiﬁcantly enriched Gene Ontology terms, including blood vessel development, vascular endothelial growth factor-activated receptor activity, regulation of cell proliferation and cell adhesion. From these terms, we detected some important DEGs, geneX, Y and Z, which have a potential to be involved in the pathogenesis of SSc-PAH.
Conclusion: We detected, at a cellular level, the alterations of ECs in patients with SSc-PAH, such as impaired vasculogenesis, facilitated cell proliferation, differentially expressed genes, enriched Gene Ontology terms. The current findings might be related to the pathogenesis of SSc-PAH.
To cite this abstract in AMA style:Kudo Y, Kato M, Shibata Y, Kono M, Fujieda Y, Amengual O, Oku K, Atsumi T. Establishment of iPSc and Differentiated Endothelial Cells of Systemic Sclerosis Associated Pulmonary Arterial Hypertension ; Functional and Molecular Analysis [abstract]. Arthritis Rheumatol. 2020; 72 (suppl 10). https://acrabstracts.org/abstract/establishment-of-ipsc-and-differentiated-endothelial-cells-of-systemic-sclerosis-associated-pulmonary-arterial-hypertension-functional-and-molecular-analysis/. Accessed September 28, 2021.
« Back to ACR Convergence 2020
ACR Meeting Abstracts - https://acrabstracts.org/abstract/establishment-of-ipsc-and-differentiated-endothelial-cells-of-systemic-sclerosis-associated-pulmonary-arterial-hypertension-functional-and-molecular-analysis/