gms | German Medical Science

Objective: Oxidative stress increases endothelial cell dysfunction, which is a potential risk factor for the formation and rupture of aneurysms. Dysfunctional endothelial cells release MCP- 1 and IL-8 which attract macrophages and neutrophils. Both cell types have been reported with increasing evidence to play an important role in aneurysm formation and rupture. This study aims to investigate the effect of Rapalink-1 on cytokine release in dysfunctional endothelial cells after exposure to oxidative stress in human umbilical vein endothelial cells (HUVECs).

Methods: HUVECs (Promocell, Heidelberg, Germany) were treated with different concentrations (100µM, 200µM) of hydrogen peroxide (H₂O₂) in the presence or absence of Rapalink-1 (0.5 nm). The relative mRNAs expression of a panel of cytokines and receptors was investigated by qRT-PCR and protein levels were analyzed by Western blot. Scratch assay was performed to analyze the migration ability of HUVECS. For the scratch assay, 50,000 cells were seeded in 6 well plates. After 90 % confluency, using a 10 µL tip a scratch was made. Images were captured at 6, 12, and 24 hrs. Image J was used for quantification of HUVECs migration in scratch assay. All experiments were performed with three biological replicates and each biological replicate had technical triplicates. For statistical analysis, the Student t-test was applied for two groups and one-way ANOVA to compare more than two groups.

Results: Rapalink-1 reduced the mRNA expression of oxidative stress induced inflammatory cytokines (IL-1β: Control=1±0.25, H₂O₂=6.5±2.5, H₂O₂ + Rapalink-1=0.01±0.01; IL-8: Control=1±0.25, H₂O₂=6.5±2.5, H₂O₂ + Rapalink-1=0.03±0.01; MCP-1: Control=1±0.29, H₂O₂=3.95±0.09, H₂O₂ + Rapalink-1=0.01±0.01) and receptor TLR-4 (Control=1±0.12, H₂O₂=1.23±0.03, H₂O₂ + Rapalink-1=0.36±0.03). Rapalink-1 recovered the migration ability of HUVECs and inhibited endothelial cell dysfunction (ICAM-1: Control=1±0.09, H₂O₂=2.94±0.27, H₂O₂+Rapalink-1=0.27±0.04; e-Selectin: Control=1±0.09, H₂O₂=5±0.86, H₂O₂+Rapalink-1=0.1±0.02). All values are mean±StDev and P<0.05.

Conclusion: Oxidative stress led to endothelial dysfunction and the expression of inflammatory cytokines. Rapalink-1 reversed the oxidative stress-mediated endothelial inflammation dysfunction. Rapalink-1 might be a suitable target in oxidative stress-mediated vascular diseases.