MilliporeSigma
  • Home
  • Search Results
  • 12/15-Lipoxygenase-dependent ROS production is required for diet-induced endothelial barrier dysfunction.

12/15-Lipoxygenase-dependent ROS production is required for diet-induced endothelial barrier dysfunction.

Journal of lipid research (2015-01-06)
Rima Chattopadhyay, Alexander Tinnikov, Elena Dyukova, Nikhlesh K Singh, Sivareddy Kotla, James A Mobley, Gadiparthi N Rao
ABSTRACT

To understand the mechanisms of 15(S)-HETE-induced endothelial cell (EC) barrier dysfunction, we examined the role of xanthine oxidase (XO). 15(S)-HETE induced junction adhesion molecule A (JamA) phosphorylation on Y164, Y218, and Y280 involving XO-mediated reactive oxygen species production and Src and Pyk2 activation, resulting in its dissociation from occludin, thereby causing tight junction (TJ) disruption, increased vascular permeability, and enhanced leukocyte and monocyte transmigration in vitro using EC monolayer and ex vivo using arteries as models. The phosphorylation of JamA on Y164, Y218, and Y280 appears to be critical for its role in 15(S)-HETE-induced EC barrier dysfunction, as mutation of any one of these amino acid residues prevented its dissociation from occludin and restored TJ integrity and barrier function. In response to high-fat diet (HFD) feeding, WT, but not 12/15-lipoxygenase (LO)(-/-), mice showed enhanced XO expression and its activity in the artery, which was correlated with increased aortic TJ disruption and barrier permeability with enhanced leukocyte adhesion and these responses were inhibited by allopurinol. These observations provide novel insights on the role of XO in 12/15-LO-induced JamA tyrosine phosphorylation and TJ disruption leading to increased vascular permeability in response to HFD.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
DL-Phenylalanine, ReagentPlus®, 99%
Sigma-Aldrich
Fluorescein 5(6)-isothiocyanate, BioReagent, suitable for fluorescence, mixture of 2 components, ≥90% (HPLC)
Sigma-Aldrich
12(S)-Hydroxy-(5Z,8Z,10E,14Z)-eicosatetraenoic acid, ~100 μg/mL in ethanol, ≥95% (HPLC)
Sigma-Aldrich
Fluorescein 5(6)-isothiocyanate, ≥90% (HPLC)
Sigma-Aldrich
Catalase−polyethylene glycol, lyophilized powder, ~40,000 units/mg protein
Phenylalanine, European Pharmacopoeia (EP) Reference Standard
Supelco
Allopurinol, Pharmaceutical Secondary Standard; Certified Reference Material
Sigma-Aldrich
Anti-Phosphotyrosine Antibody, recombinant clone 4G10®, clone 4G10®, Upstate®, from mouse
Sigma-Aldrich
Anti-Src Antibody, clone GD11, clone GD11, Upstate®, from mouse
Isoflurane, European Pharmacopoeia (EP) Reference Standard
Sigma-Aldrich
Fluorescein isothiocyanate isomer I, ≥97.5% (HPLC)
Sigma-Aldrich
Fluorescein isothiocyanate isomer I, suitable for protein labeling, ≥90% (HPLC), powder
Sigma-Aldrich
Fluorescein isothiocyanate–dextran, average mol wt 60,000-76,000
Sigma-Aldrich
Fluorescein isothiocyanate isomer I, ≥97.5% (HPLC)