Importance of cholesterol-rich microdomains in the regulation of Nox isoforms and redox signaling in human vascular smooth muscle cells

Anagnostopoulou, A., Camargo, L. L. , Rodrigues, D., Montezano, A. C. and Touyz, R. M. (2020) Importance of cholesterol-rich microdomains in the regulation of Nox isoforms and redox signaling in human vascular smooth muscle cells. Scientific Reports, 10, 17818. (doi: 10.1038/s41598-020-73751-4) (PMID:33082354) (PMCID:PMC7575553)

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Abstract

Vascular smooth muscle cell (VSMC) function is regulated by Nox-derived reactive oxygen species (ROS) and redox-dependent signaling in discrete cellular compartments. Whether cholesterol-rich microdomains (lipid rafts/caveolae) are involved in these processes is unclear. Here we examined the sub-cellular compartmentalization of Nox isoforms in lipid rafts/caveolae and assessed the role of these microdomains in VSMC ROS production and pro-contractile and growth signaling. Intact small arteries and primary VSMCs from humans were studied. Vessels from Cav-1−/− mice were used to test proof of concept. Human VSMCs express Nox1, Nox4, Nox5 and Cav-1. Cell fractionation studies showed that Nox1 and Nox5 but not Nox4, localize in cholesterol-rich fractions in VSMCs. Angiotensin II (Ang II) stimulation induced trafficking into and out of lipid rafts/caveolae for Nox1 and Nox5 respectively. Co-immunoprecipitation studies showed interactions between Cav-1/Nox1 but not Cav-1/Nox5. Lipid raft/caveolae disruptors (methyl-β-cyclodextrin (MCD) and Nystatin) and Ang II stimulation variably increased O2− generation and phosphorylation of MLC20, Ezrin-Radixin-Moesin (ERM) and p53 but not ERK1/2, effects recapitulated in Cav-1 silenced (siRNA) VSMCs. Nox inhibition prevented Ang II-induced phosphorylation of signaling molecules, specifically, ERK1/2 phosphorylation was attenuated by mellitin (Nox5 inhibitor) and Nox5 siRNA, while p53 phosphorylation was inhibited by NoxA1ds (Nox1 inhibitor). Ang II increased oxidation of DJ1, dual anti-oxidant and signaling molecule, through lipid raft/caveolae-dependent processes. Vessels from Cav-1−/− mice exhibited increased O2− generation and phosphorylation of ERM. We identify an important role for lipid rafts/caveolae that act as signaling platforms for Nox1 and Nox5 but not Nox4, in human VSMCs. Disruption of these microdomains promotes oxidative stress and Nox isoform-specific redox signalling important in vascular dysfunction associated with cardiovascular diseases.

Item Type:Articles
Status:Published
Refereed:Yes
Glasgow Author(s) Enlighten ID:De Lucca Camargo, Dr Livia and Anagnostopoulou, Dr Aikaterini and Montezano, Dr Augusto and Rodrigues, Mr Daniel and Touyz, Professor Rhian
Authors: Anagnostopoulou, A., Camargo, L. L., Rodrigues, D., Montezano, A. C., and Touyz, R. M.
College/School:College of Medical Veterinary and Life Sciences > Institute of Cardiovascular and Medical Sciences
Journal Name:Scientific Reports
Publisher:Nature Research
ISSN:2045-2322
ISSN (Online):2045-2322
Copyright Holders:Copyright © 2020 The Authors
First Published:First published in Scientific Reports 10: 17818
Publisher Policy:Reproduced under a Creative Commons License

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Project CodeAward NoProject NamePrincipal InvestigatorFunder's NameFunder RefLead Dept
167932Vascular Noxs as therapeutic targets and biomarkers in hypertensionRhian TouyzBritish Heart Foundation (BHF)RG/13/7/30099Institute of Cardiovascular & Medical Sciences
190814BHF centre of excellenceRhian TouyzBritish Heart Foundation (BHF)RE/13/5/30177Institute of Cardiovascular & Medical Sciences
300689Vascular Noxs as therapeutic targets and biomarkers in hypertensionRhian TouyzBritish Heart Foundation (BHF)CH/12/4/29762CAMS - Cardiovascular Science