Effects of hydrogen peroxide on endothelial function in three-dimensional hydrogel vascular model and regulation...

Three-dimensional (3D) cell cultures better reflect the function of endothelial cells (ECs) than two-dimensional (2D) cultures. In recent years, studies have found that ECs cultured in a 3D luminal structure can mimic the biological characteristics and phenotypes of vascular ECs, thus making it more suitable for endothelial dysfunction research. In this study, we used a 3D model and 2D tissue culture polystyrene (TCP) to investigate the effects of cell polarity on hydrogen peroxide (H₂O₂)-induced endothelial dysfunction and its related mechanisms. We observed the cell morphology, oxidative stress, and barrier and endothelial function of human umbilical vein ECs (HUVECs) in 3D and 2D cultures. We then used Illumina to detect the differentially expressed genes (DEGs) in the 3D-cultured HUVEC with and without H₂O₂stimulation, using clusterProfiler for Gene Ontology function enrichment analysis and Kyoto Encyclopaedia of Genes and Genomes pathway enrichment analysis of DEGs. Finally, we explored the role and mechanism of polar protein partitioning defective protein 3 (Par3) in the regulation of ECs. ECs were inoculated into the 3D hydrogel channel; after stimulation with H₂O₂, the morphology of HUVECs changed, the boundary was blurred, the expression of intercellular junction proteins decreased, and the barrier function of the EC layer was damaged. 3D culture increased the oxidative stress response of cells stimulated by H₂O₂compared to 2D TCPs. The polarity-related protein Par3 and cell division control protein 42 were screened using bioinformatics analysis, and western blotting was used to verify the results. Par3 knockdown significantly suppressed claudin1 (CLDN1) and vascular endothelial cadherin. These results suggest that the polar protein Par3 can protect H₂O₂-induced vascular ECs from damage by regulating CLDN1 and VE-cadherin.