Effects of omentin on endothelial disruption and hydrogen peroxide-induced inflammation in vascular endothelial cell monolayer
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 th...
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| Main Author: | |
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| Format: | Thesis |
| Language: | English |
| Published: |
2022
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| Subjects: | |
| Online Access: | http://psasir.upm.edu.my/id/eprint/99749/1/NUR%20AQILAH%20BINTI%20KAMAR%20-%20IR3.pdf |
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| Summary: | 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. |
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