Effects of omentin on endothelial disruption and hydrogen peroxide-induced inflammation in vascular endothelial cell monolayer

• Main Author: Kamaruddin, Nur Aqilah
• Format: Thesis
• Language: English
• Published: 2022
• Subjects: Endothelial Cells; Vascular Endothelial Cells
• Online Access: http://psasir.upm.edu.my/id/eprint/99749/1/NUR%20AQILAH%20BINTI%20KAMAR%20-%20IR3.pdf; http://psasir.upm.edu.my/id/eprint/99749/

The endothelial barrier plays important roles in maintaining vascular homeostasis. In response to oxidative stress, the endothelial barrier breaks down and results in an increase in endothelial permeability, which is followed by cell injury. Vascular damage is characterized by the breakdown of the endothelial barrier and the associated hyperpermeability. An increase in generation of reactive oxygen species (ROS) and the disruption of endothelial intercellular junctions are critical events in oxidative stressinduced endothelial cell injury. Omentin is an adipocytokine abundantly secreted in visceral adipose tissue and has anti-diabetic and anti-inflammatory properties. Withal, it has not been explored whether omentin can ameliorate endothelial injury and barrier dysfunction induced by oxidative stress. Both cytotoxic and cytoprotective effects of omentin were determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The apoptotic activity of human umbilical vein endothelial cells (HUVECs) was investigated using Annexin-V/PI and Hoechst 33258 staining. Omentin antioxidant activity was evaluated by measuring both ROS levels and glutathione peroxidase (GPx) activity. Conducive to understanding the role of omentin in preserving the endothelial barrier, the effect of omentin on endothelial hyperpermeability induced by hydrogen peroxide (H2O2) using sodium fluorescein (NaF), evans blue albumin (EBA) and transendothelial electrical resistance (TEER) has been assessed. Distribution of filamentous (F)-actin, adherens junctions (AJs) and tight junctions (TJs) in cells were investigated using immunocytochemistry and confocal imaging. Total protein expression of F-actin, vascular endothelial (VE)-cadherin, β-catenin, occludin, zona occludens (ZO)-1, Rho and ROCK2 was determined using Western blot analysis. This study indicated that there is no cytotoxic effect observed in HUVECs treated with omentin alone at concentrations of 150 to 450 ng/mL. The result of the MTT assay showed that omentin significantly blocked cell death induced by H2O2 (p<0.001). Hoechst staining and flow cytometry also revealed that omentin notably halted H2O2-induced apoptosis. In addition, omentin significantly inhibited ROS production (p<0.01) and also significantly (p<0.01) increased GPx activity in HUVECs, resulting in protection against oxidative stress-induced cell damage in HUVECs. Besides, pretreatment of omentin also significantly (p<0.001) reduced endothelial hyperpermeability, as demonstrated by increased TEER value and decreased passage of NaF and EBA through the monolayer of the cells. Immunostaining data demonstrated that omentin reversed rearrangement of the cytoskeletal protein, F-actin, enhanced the distribution of AJs and TJs protein such as VE-cadherin, β-catenin, occludin, and ZO-1. In addition, omentin also significantly suppressed increased F-actin levels and prevented the reduced expression level of junctional proteins (p<0.01). Interestingly, omentin abolished the activation of RhoA/ROCK induced by H2O2 (p<0.01), an effect which was similar with the action of a ROCK inhibitor. These in vitro findings demonstrated that the antioxidant and antiinflammatory actions of omentin involve a reduction in production of ROS, reduced endothelial hyperpermeability, the actin cytoskeleton stabilization and increased junction proteins by regulating the localization and protein expression of occludin, ZO-1, VEcadherin and β-catenin. This study also implies that omentin prevents endothelial disruption and inhibits H2O2-induced inflammation in vascular endothelial cell monolayer.