3D-Mesenchymal stromal cells derived VASH2 alleviates oxidative stress-induced endothelial senescence by mediating α-tubulin detyrosination in systemic sclerosis

Endothelial cell (EC) senescence driven by oxidative stress is a pivotal contributor to vasculopathy in systemic sclerosis (SSc), leading to impaired angiogenesis and loss of vascular integrity. Three-dimensional (3D) cultured human umbilical cord-derived mesenchymal stromal cells (MSCs) represent a promising therapeutic strategy for SSc due to their enhanced anti-fibrotic and immunomodulatory properties. However, their potential to mitigate EC senescence remains unclear. This study aims to investigate whether 3D-MSCs confer superior therapeutic effects over conventional MSCs by ameliorating EC senescence and to elucidate the underlying mechanism. The anti-senescent effects of 3D-MSCs and two-dimensional (2D)-MSCs were compared in vitro using a hydrogen peroxide (H₂O₂)-induced EC senescence model and in vivo using a bleomycin-induced SSc mouse model. RNA sequencing identified vasohibin-2 (VASH2) as a key factor, which was further validated using siRNA knockdown. The specific inhibitor EpoY was used to assess the role of VASH2 in microtubule detyrosination in senescent ECs. 3D-MSCs prevented H₂O₂-induced EC senescence and restored angiogenic capacity in vitro. Administration of 3D-MSCs ameliorated skin fibrosis and vasculopathy in SSc mouse models by mitigating EC senescence, thereby improving vascular density and restoring endothelial barrier integrity. Mechanistically, 3D-MSCs secreted VASH2, which then interacted with the small vasohibin-binding protein (SVBP) to restore microtubule detyrosination in senescent ECs under oxidative stress. Inhibition of the VASH/SVBP complex by EpoY impaired the anti-senescent benefits of 3D-MSCs. Our study demonstrates that 3D-MSCs alleviate oxidative stress-induced EC senescence and associated vasculopathy primarily via VASH2 secretion, which restores α-tubulin detyrosination to promote microtubule stability. These findings reveal a novel mechanism against EC senescence and underscore the therapeutic potential of 3D-MSCs in vasculopathy.