Elevated hydrostatic pressure modulates endothelial junctional mechanotransduction through VE-cadherin remodelling and altered association with YAP1, EPS8: an endothelium-on-chip study

Abstract

Endothelial dysfunction is a hallmark of numerous vascular pathologies and is strongly influenced by mechanobiological forces within the vascular microenvironment. While the effects of shear stress have been extensively investigated, the mechanisms by which elevated hydrostatic pressure regulates endothelial junctional organisation remain sparsely investigated. Here, we employed a microfluidic platform to investigate the combined effects of low shear stress (1.4 dyne/cm2) and elevated hydrostatic pressure (~3972 Pa) on endothelial junctional dynamics. Elevated hydrostatic pressure induced marked remodelling of VE-cadherin junctions, characterised by formation of serrated, finger-like structures accompanied by increased YAP1 nuclear localisation and reduced YAP1-VE-cadherin cytoplasmic colocalisation compared to shear stress alone conditions. Further, elevated hydrostatic pressure also demonstrated an increase in cytoplasmic accumulation of EPS8, an actin adaptor protein, and increased cytoplasmic EPS8-VE-cadherin colocalisation. These observations were accompanied by functional changes marked by increased endothelial permeability, and enhanced THP-1 monocyte adhesion, thus suggesting activation of mechanosensitive pathways linked to dynamic junctional reorganisation. Inhibition of PI3K at elevated hydrostatic pressure exhibited a thin VE-cadherin patterning and increased cytoplasmic EPS8-VE-cadherin colocalisation, thus demonstrating a prominent role for PI3K signalling in regulating the junction organisation. Interestingly, Piezo-1 activation using Yoda1 produced context-dependent effects. Under shear stress alone, Yoda1 promoted YAP1 nuclear translocation, reduced YAP1-VE-cadherin colocalisation, increased endothelial permeability but strikingly did not impact THP-1 adhesion compared to shear stress alone conditions. In contrast, under elevated hydrostatic pressure conditions, Yoda1 significantly reduced both endothelial permeability and THP-1 adhesion while increasing YAP1-VE-cadherin colocalisation and decreasing YAP1 nuclear accumulation. Collectively, these findings identify a previously underappreciated elevated hydrostatic pressure-Piezo-1-PI3K signalling axis that regulates endothelial barrier integrity and pro-adhesive endothelial activation through coordinated regulation of VE-cadherin, YAP1, and EPS8. These results highlight elevated hydrostatic pressure as a unique mechanobiological stimulus, distinct from that of shear stress alone and provide novel insights into mechanisms underlying microvascular dysfunction.