Functional characterization and clinical implications of patient-specific iPSC-derived endothelial cells to unravel the pathogenesis associated with androgen-mediated endothelial dysfunction in polycystic ovarian syndrome

Abstract Study question Whether and how the endothelial cell (EC) dysfunction is involved in the pathogenesis of PCOS revealed by iPSC-based disease modeling? Summary answer PCOS-specific iPSC-derived ECs were successfully established and the results showed that EC proliferation and function were impaired in PCOS through the androgen receptor(AR)-mediated signaling pathway. What is known already ECs have been shown to play critical biological roles in the regulation of vascular tone and inflammatory process and any injury to ECs or disturbance in their homeostasis is called EC dysfunction, which has been recognized as an early marker of atherosclerosis and cardiovascular diseases. Several studies have also revealed indirect evidence of EC dysfunction among women with PCOS, including elevated circulatory markers of endothelial damage and impaired vascular structure. But whether and how EC dysfunction is involved in the pathogenesis of PCOS is still unclear. Study design, size, duration This is an experimental study at tertiary university hospital. iPSCs were established from skin fibroblasts and peripheral blood mononuclear cells from two patients with PCOS and two control participants. Participants/materials, setting, methods iPSCs were differentiated into ECs through monoculture with chemically defined conditions. Single cell RNA sequencing (scRNA-seq) was performed on the iPSCs-derived ECs to compare the differences between PCOS and control. iPSCs-derived ECs were treated with different dosages (1, 10 and 100 nM) of dihydrotestosterone (DHT). Cell cycle phases were analyzed by flow cytometry with BrdU incorporation assay. The expression of AR, cyclin-dependent kinase 1 (CDK1) and vascular endothelial growth factor (VEGF) were analyzed using rtPCR. Main results and the role of chance Up to 90% of iPSC-derived ECs successfully expressed EC markers, including CD31, CD144 and von Willebrand Factor in both PCOS and control groups, showing a high differentiation efficiency in the present study. The pathway enrichment analysis of transcriptomic data of iPSC-derived ECs showed functional differences involving cell cycle process, VEGF signaling and apoptotic process between PCOS and control. Analysis using sc-RNA seq revealed decreased cellular proliferation in the PCOS iPSC-derived ECs compared to control group, which was due to cell cycle arrest revealed by flow cytometry. The modulating effects of androgen on the proliferation and gene expression of iPSC-derived ECs were further investigated. DHT at doses ranging from 1 to 100 nM stimulated iPSC-derived ECs proliferation in the control group through upregulation of AR, CDK1 and VEGF gene expression. In contrast, DHT did not promote the cellular proliferation of PCOS iPSC-derived ECs at physiological concentrations of 1 and 10 nM and the expression of AR, CDK1, and VEGF remained unchanged. These data suggest that DHT-induced cellular proliferation and the expression of associated genes were more sensitive and dose-dependent in the control iPSC-derived ECs and blunted in the PCOS iPSC-derived ECs. Limitations, reasons for caution One of the major limitations of our study was the limited number of cases during the establishment of iPSCs, which was unable to reflect the heterogeneity and complexity of PCOS. Wider implications of the findings Impaired cellular proliferation and differentially expressed genes involved in cell proliferation, immune responses, cardiovascular functions, and apoptosis were observed among PCOS iPSC-derived ECs. Additionally, androgen-induced ECs proliferation was blunted in PCOS through a diminished AR/VEGF/cyclin D1 signaling pathway, which might hinder VEGF-dependent vascular repair and probably increase cardiovascular risks. Trial registration number not applicable