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

Aikaterini Anagnostopoulou, Livia de Lucca Camargo, Daniel Rodrigues, Augusto C. Montezano and Rhian Touyz 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. https://doi.org/10.1038/s41598-020-73751-4

TitleImportance of cholesterol-rich microdomains in the regulation of Nox isoforms and redox signaling in human vascular smooth muscle cells
TypeJournal article
AuthorsAikaterini Anagnostopoulou, Livia de Lucca Camargo, Daniel Rodrigues, Augusto C. Montezano and Rhian Touyz
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.

Article number17818
JournalScientific Reports
Journal citation10
ISSN2045-2322
Year2020
PublisherNature Publishing Group
Publisher's version
License
CC BY 4.0
File Access Level
Open (open metadata and files)
Digital Object Identifier (DOI)https://doi.org/10.1038/s41598-020-73751-4
Web address (URL)http://dx.doi.org/10.1038/s41598-020-73751-4
Publication dates
Published20 Oct 2020

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