Abstract 5525: Caveolin-1 Downregulation Reduces NADPH Oxidase Assembly and Reverses eNOS Uncoupling
Activation of the renin-angiotensin system generates endothelial dysfunction through increased oxidant stress. We examined the spatial co-localization of eNOS and NADPH oxidase in endothelial caveolae and the role of caveolin-1 (cav1) on eNOS activation and uncoupling in response to Angiotensin II (AII) in vitro and in vivo. NO and O2- production at the membrane of BAECs and HUVECs was measured by EPR spin-trapping (Fe(II)-[DETC]2 and DMPO, respectively). AII increased NO to 158±12% and O2- to 209±5% of control (all from min. 3 exp; P<0.05). AII-stimulated NO production was sensitive to inhibition of NADPH oxidase assembly (with apocynin, to 64±11% of ctl; P<0.05), as well as of the redox-sensitive PI3 kinase (with LY294002), cSrc (with PP2) and cAbl kinases (by siRNA targeting; all P<0.05). Reciprocally, the NOS inhibitor, L-NAME inhibited AII-stimulated O2- (by 47±11%;P<0.05), indicating eNOS uncoupling, as confirmed by increased eNOS monomer/dimer ratio. To resolve the role of eNOS and NADPH compartmentation for uncoupling, endothelial cell extracts were separated by isopycnic ultracentrifugation. Upon AII stimulation, p47phox, a NADPH subunit, was redistributed with eNOS to cav1-enriched fractions. siRNA downregulation of cav1 by ~50% while preserving eNOS confinement, inhibited AII-stimulated p47phox translocation, O2 - production (by 2.5-fold) in cav-1-enriched fractions and abrogated the L-NAME-sensitive O2 - production, indicating reversal of eNOS uncoupling. To test similar effects of moderate cav1 depletion in vivo, heterozygote cav-1+/− mice (and WT) were treated with AII infusion (osmotic minipump), their blood pressure recorded by telemetry and plasma Hb-NO measured by ESR. While AII infusion produced hypertension and decreased Hb-NO in WT (to 55±15% of ctl; p<0.05), cav-1+/− mice were fully preserved. We conclude that, in endothelial cells, co-localization of NADPH oxidase and eNOS in caveolae-enriched fractions activates eNOS through ROS-sensitive PI3K and cSrc/cAbl kinases but produces eNOS uncoupling in response to AII. Moderate downregulation of cav-1 prevents NADPH assembly and eNOS uncoupling, while preserving NO production, underlining the interest for cav-1 modulation to treat endothelial dysfunction.