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

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.

NADPH Oxidase 5 and Melatonin: Involvement in Ram Sperm Capacitation

“…Regarding NOX5 localization, mainly in the acrosome region and the apical edge, we have demonstrated that it changes during ram sperm capacitation. To explain this redistribution we could hypothesize that NOX5 could be embedded in lipid rafts, as it has been demonstrated in somatic cells (Anagnostopoulou et al, 2020). During the capacitation process, the cholesterol efflux from the sperm membrane allows the reorganization of the lipid rafts (van Gestel et al, 2005), giving new protein distributions, and modulating their activity (Kawano et al, 2011).…”

“…During the capacitation process, the cholesterol efflux from the sperm membrane allows the reorganization of the lipid rafts (van Gestel et al, 2005), giving new protein distributions, and modulating their activity (Kawano et al, 2011). This lipid raft redistribution was demonstrated to be the mechanism by which NOX5 could modulate its activity, in certain somatic cells (Anagnostopoulou et al, 2020). The cytoskeleton plays a fundamental part in the rearrangement of the membrane during sperm capacitation (Brener et al, 2003).…”

Endothelial Nox5 Expression Modulates Glucose Uptake and Lipid Accumulation in Mice Fed a High-Fat Diet and 3T3-L1 Adipocytes Treated with Glucose and Palmitic Acid

“…Finally, the fact that NOXs have been reported to be found within caveolae in endothelium [ 23 , 24 ], as well as in adipose tissue [ 19 ], and that they can interact with CAV1, raises the possibility of a cross-regulation, something that deserves further investigation. In regard to this, it is interesting the finding that ROS may enhance CAV1 transcription in NIH-3T3-L1 fibroblasts [ 50 ].…”

“…A direct interaction between caveolae and NOX enzymes has also been shown. A recent study has revealed that NOX5 is present in caveolae from vascular smooth muscle cells [ 23 ]. In this regard, it has been described that CAV1 can bind to NOX5 promoting a decrease of its activity [ 24 ].…”

Human Vascular Smooth Muscle Function and Oxidative Stress Induced by NADPH Oxidase with the Clinical Implications

“…A recent study has documented that NOX1 and NOX5, but not NOX4, localize in cholesterol-rich fractions in intact human vascular smooth muscle cells from patients undergoing elective craniofacial surgeries [27]. These results suggest an essential role of lipid rafts/caveolae in the signaling platforms for specific NOX isoforms in intact human vascular smooth muscle cells.…”