Abstract Background Thoracic aortic aneurysm and dissection (TAAD) is a life-threatening condition often associated with hereditary connective tissue disorders. Endothelial cells (ECs) are key regulators of vascular homeostasis, controlling barrier integrity, inflammation, and nitric oxide (NO) production. EC dysfunction contributes to TAAD progression. Yet, most studies focus on vascular smooth muscle cells, leaving ECs underexplored. Aims This study aims to investigate EC dysfunction in IPO8-related TAAD by generating and functionally characterizing ECs derived from induced pluripotent stem cells (iPSCs) from an IPO8 patient. Methods Two clones with a bi-allelic IPO8 c.819delT (p.Phe273Leufs*22) variant and one clone of isogenic control were subjected to EC differentiation using a standardized protocol. Functional assays were performed to assess proliferation (CytoQUANT), NO production (DAF-FM DA), and oxidative stress levels (CellROX). Additionally, the activation state of iPSC-derived ECs was evaluated by immunocytochemistry (ICC) for ZO-1, caveolin-1, endothelial nitric oxide synthase (eNOS), and ICAM. Results IPSC-derived ECs exhibited the expected morphology, formed a confluent monolayer, and expressed PECAM1 and VE-Cadherin. Functional assays were performed on cells obtained from two independent differentiation rounds of the two IPO8 mutant clones. No differences were observed between iPSC-ECs from the same clone across different differentiation rounds, nor between the two mutant clones in any of the assays. Proliferation analysis revealed decreased mutant iPSC-EC proliferation (p 0.0001), consistent with observations during routine culture. CellROX analysis indicated increased reactive oxygen species (ROS) levels in mutant cells as compared to isogenic control (p = 0.0347), while ICC analysis did not reveal differences in the expression of caveolin-1, ZO-1, ICAM, and eNOS. Importantly, the eNOS results support the NO assay findings, which showed no significant difference in NO levels between the two groups (p = 0.4646). Conclusions IPO8 mutant and isogenic control iPSCs were successfully differentiated into ECs. The results of our functional test battery suggest that EC dysfunction in IPO8-related TAAD is driven primarily by impaired proliferation and oxidative stress rather than NO dysregulation. However, future experiments should investigate other important EC-relevant functions like endothelial barrier integrity in order to fully understand the impact of IPO8 mutation on EC biology. Finally, these observations should be confirmed in an independent IPO8 mutant cell line.
Fedoryshchenko et al. (Fri,) studied this question.