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.
Highlights
Vascular smooth muscle cell (VSMC) function is regulated by Nox-derived reactive oxygen species (ROS) and redox-dependent signaling in discrete cellular compartments
Sub-cellular mechanisms underlying these processes have not been fully elucidated but compartmentalization in cholesterol-rich microdomains may be important. This is especially relevant for Angiotensin II (Ang II) since the Ang II type 1 receptor (AT1R) and associated signalling molecules localize in lipid rafts/caveolae
In the present study, using a multidisciplinary approach, we identify these microdomains as an important structural element in Nox-ROS regulation and redox-dependent signalling in human VSMCs and show that disruption of these microdomains promotes oxidative stress and aberrant vascular signalling
Summary
Vascular smooth muscle cell (VSMC) function is regulated by Nox-derived reactive oxygen species (ROS) and redox-dependent signaling in discrete cellular compartments. Vascular smooth muscle cell (VSMC) function is regulated by vasoactive factors that signal through multiple membrane-associated receptors and downstream signalling molecules, including kinases, phosphatases, ion channels and transcription factors Many of these processes are influenced by reactive oxygen species (ROS), superoxide ( O2−) and hydrogen peroxide ( H2O2), and involve post-translational modification of signalling proteins, including oxidation and p hosphorylation[1, 2]. Cav-1 contains a scaffolding domain that interacts with several proteins in microdomains and regulates their activation or inhibition[8] It influences Ang II/ Ang II type 1 receptor (AT1R) signalling and trafficking, critically important in the regulation of VSMC function and vascular c ontraction[9, 10]. Processes controlling these differential responses may relate to sub-cellular compartmentalization of Noxs, the oxidases responsible for ROS generation in the vascular system
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