Endothelial cell senescence driven by oxidative stress is a pivotal contributor to vasculopathy in systemic sclerosis, leading to impaired angiogenesis and loss of vascular integrity. Three-dimensional cultured human umbilical cord-derived mesenchymal stromal cells represent a promising therapeutic strategy for systemic sclerosis due to their enhanced anti-fibrosis and immunomodulatory properties. However, their potential to mitigate endothelial cell senescence remains unclear. This study aims to investigate whether three-dimensional cultured mesenchymal stromal cells confer superior therapeutic effects over conventional mesenchymal stromal cells by ameliorating endothelial cell senescence and to elucidate the underlying mechanism.

The anti-senescent effects of three-dimensional cultured mesenchymal stromal cells and two-dimensional cultured mesenchymal stromal cells were compared in vitro using a hydrogen peroxide-induced endothelial cell senescence model and in vivo using a bleomycin-induced systemic sclerosis mouse model. RNA sequencing identified vasohibin-2 as a key factor, which was further validated using siRNA knockdown. The specific inhibitor EpoY was used to assess the role of vasohibin-2 in microtubule detyrosination in senescent endothelial cells.

Three-dimensional cultured mesenchymal stromal cells prevented hydrogen peroxide-induced endothelial cell senescence and restored angiogenic capacity in vitro. Administration of three-dimensional cultured mesenchymal stromal cells ameliorated skin fibrosis and vasculopathy in systemic sclerosis mouse models by mitigating endothelial cell senescence, thereby improving vascular density and restoring endothelial barrier integrity. Mechanistically, three-dimensional cultured mesenchymal stromal cells secreted vasohibin-2, which interacted with the small vasohibin-binding protein to restore microtubule detyrosination in senescent endothelial cells under oxidative stress. Inhibition of the vasohibin/small vasohibin-binding protein complex by EpoY impaired the anti-senescent benefits of three-dimensional cultured mesenchymal stromal cells.

Our study demonstrated that three-dimensional cultured mesenchymal stromal cells alleviate oxidative stress-induced endothelial cell senescence and associated vasculopathy primarily via vasohibin-2 secretion, which restored α-tubulin detyrosination to promote microtubule stability. These findings revealed a novel mechanism against endothelial cell senescence and underscored the therapeutic potential of three-dimensional cultured mesenchymal stromal cells in vasculopathy.