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167 The Mechanism of P22PHOX C242T SNP Inhibition of TNF Alpha-Induced Nox2 Activation in Human Endothelial Cells and Vessels
  1. Daniel Meijles1,
  2. Lampson Fan2,
  3. Maziah Ghazaly3,
  4. Jian-Mei Li1
  1. 1University of Reading
  2. 2Royal Berkshire Hospital
  3. 3University of Surrey

Abstract

NADPH oxidase, by generating reactive oxygen species, is involved in the pathophysiology of many cardiovascular diseases and represents a therapeutic target for the development of novel drugs. A single-nucleotide polymorphism (SNP) C242T of the p22phox subunit of NADPH oxidase has been reported to be negatively associated with coronary heart disease (CHD) and may predict disease prevalence. However, the underlying mechanisms remain unknown.

Using computer molecular modelling we discovered that C242T SNP causes significant structural changes in the extracellular loop of p22phox and reduces its interaction stability with the catalytic Nox2 subunit. Gene transfection of human pulmonary microvascular endothelial cells showed that C242T p22phox reduced significantly Nox2 expression, but had no significant effect on basal endothelial superoxide (O2 .-) production, or the expression of Nox1 and Nox4. When cells were stimulated with TNFalpha (or high glucose), C242T p22phox inhibited significantly TNFalpha-induced Nox2 maturation, O2 .- production, MAPK and NFkB activation, and monocyte adhesion (all p < 0.05; one-way AVOVA). These C242T effects were further confirmed using p22phox shRNA engineered HeLa cells and coronary microvascular endothelial cells isolated from Nox2 knockout mice. Clinical significance was investigated using saphenous vein segments from non-CHD subjects after phlebectomies. Informed consents were obtained from the patients and the project was approved by the local NHS and the university ethical committees according to the UK regulation. TT (C242T) allele was common (prevalence of ~20%) and compared to CC, veins bearing TT allele had significantly lower levels of Nox2 expression and O2 .- generation in response to high glucose challenge (all p < 0.05).

In summary, our study for the first time provides mechanistic insight into the protective effect of the p22phox C242T SNP against the inflammatory oxidative stress-related cardiovascular diseases. p22phox C242T SNP causes p22phox structural changes that alter its interaction with the catalytic subunit Nox2 and inhibits endothelial oxidative response to TNFalpha or high glucose stimulation.

  • NADPH oxidase
  • oxidative stress
  • endothelial dysfunction

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