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Endothelial cell activation in vascular disease mediated by hydrogen peroxide in vitro
Habas, Khaled S.A. Shang, Lijun
Habas, Khaled S.A.
Shang, Lijun
Publication Date
2016
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© 2016 The Authors. Published by the Physiological Society. Reproduced in accordance with the publisher's self-archiving policy.
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Abstract
The development of cardiovascular disease (CVD) is the main cause of death among chronic kidney
disease (CKD) patients (1). Endothelial injury and dysfunction are critical steps in atherosclerosis, a major CVD (2).
Increased production of reactive oxygen species (ROS) has been associated with the pathogenesis of cardiovascular
diseases such as atherosclerosis, hypertension and heart failure (3). However, hydrogen peroxide (H2O2) modulates
endothelial cell function by intricate mechanisms. Ambient production of O2.− and subsequently H2O2 at low levels,
maintained via basal activity of pre-assembled endothelial NAD (P) H oxidases (4). Endothelial cells play an important
regulatory role in the circulation as a physical barrier and as a source of a variety of regulatory substances.
Dysfunction of the vascular endothelium is thus leading to atherosclerosis which is characterised by overexpression of
adhesion molecule expression, comprising vascular cell adhesion molecule 1(VCAM1). This adhesion molecule has
been found to be up-regulation in human atherosclerotic lesions.
The aim of this study is to evaluate the effect of H2O2 on the endothelial cells adhesion molecules expression.
Primary cultures of Human Umbilical Vascular Endothelial Cells (HUVECs) will be maintained in endothelial growth
medium supplemented with penicillin-streptomycin and supplement mix of fetal calf serum in a 37C humidified
incubator in an atmosphere of 5% v/v CO2. HUVECs will be treated with in the presence and absences of 50 μM of H
2O2 for 2, 6, 12 and 24 h. Intracellular superoxide anion production in HUVECs will be detected by using p-Nitro Blue
Tetrazolium (NBT) assay to demonstrate whether H2O2 induce the generation of superoxide anions intracellularly in
HUVECs. The formation of blue formazan will be measured spectrophotometrically at 570 nm. Total RNA will be
extracted from non-treated and treated cells and RNA quantity and quality will be checked by OD260/280
measurements. VCAM-1 mRNA expression will be assessed using RT-PCR. Our results show that H2O2 could
potentially significantly induce EC activation through increased mRNA expression of ICAM-1 adhesion molecules in
cultured HUVECs. Treatment with N-acetyl cysteine (NAC) (bulk/nano form) could significantly attenuate the effect of
H2O2 administration on adhesion molecule protein expression. This strongly suggests the role of ROS in the
endothelial cell damage sustained. Future work is to find reliable methods to test endothelial function. Non-invasive
studies such as brachial ultrasound testing are also needed to determine its predictive value as a potential predictor
for cardiovascular disease.
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Citation
Habas K and Shang L (2016) Endothelial cell activation in vascular disease mediated by hydrogen peroxide in vitro [Proceedings abstract]. Proceedings of the Physiological Society: Cardiovascular, Respiratory and Autonomic Control Abstracts. Physiology 2016 (Dublin, Ireland). 37, PCB083.
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Conference paper