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Hypoxia triggers subcellular compartmental redox signaling in vascular smooth muscle cells.

ABSTRACT: Recent studies have implicated mitochondrial reactive oxygen species (ROS) in regulating hypoxic pulmonary vasoconstriction (HPV), but controversy exists regarding whether hypoxia increases or decreases ROS generation.This study tested the hypothesis that hypoxia induces redox changes that differ among subcellular compartments in pulmonary (PASMCs) and systemic (SASMCs) smooth muscle cells.We used a novel, redox-sensitive, ratiometric fluorescent protein sensor (RoGFP) to assess the effects of hypoxia on redox signaling in cultured PASMCs and SASMCs. Using genetic targeting sequences, RoGFP was expressed in the cytosol (Cyto-RoGFP), the mitochondrial matrix (Mito-RoGFP), or the mitochondrial intermembrane space (IMS-RoGFP), allowing assessment of oxidant signaling in distinct intracellular compartments. Superfusion of PASMCs or SASMCs with hypoxic media increased oxidation of both Cyto-RoGFP and IMS-RoGFP. However, hypoxia decreased oxidation of Mito-RoGFP in both cell types. The hypoxia-induced oxidation of Cyto-RoGFP was attenuated through the overexpression of cytosolic catalase in PASMCs.These results indicate that hypoxia causes a decrease in nonspecific ROS generation in the matrix compartment, whereas it increases regulated ROS production in the IMS, which diffuses to the cytosol of both PASMCs and SASMCs.

SUBMITTER: Waypa GB

PROVIDER: S-EPMC2856085 | biostudies-literature | 2010 Feb

REPOSITORIES: biostudies-literature

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Hypoxia triggers subcellular compartmental redox signaling in vascular smooth muscle cells.

Waypa Gregory B GB Marks Jeremy D JD Guzy Robert R Mungai Paul T PT Schriewer Jacqueline J Dokic Danijela D Schumacker Paul T PT

Circulation research 20091217 3

<h4>Rationale</h4>Recent studies have implicated mitochondrial reactive oxygen species (ROS) in regulating hypoxic pulmonary vasoconstriction (HPV), but controversy exists regarding whether hypoxia increases or decreases ROS generation.<h4>Objective</h4>This study tested the hypothesis that hypoxia induces redox changes that differ among subcellular compartments in pulmonary (PASMCs) and systemic (SASMCs) smooth muscle cells.<h4>Methods and results</h4>We used a novel, redox-sensitive, ratiometr ...[more]

PMID: 20019331

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Project description: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.

Dataset Information

Hypoxia triggers subcellular compartmental redox signaling in vascular smooth muscle cells.

Publications

Hypoxia triggers subcellular compartmental redox signaling in vascular smooth muscle cells.

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