Abstract 18096: Novel Role of Caveolin-1 in Endothelial Function by Regulating Extracellular SOD Activity and Cu Transporter ATP7A Function

Abstract

Caveolin (Cav)-enriched lipid raft (C/LR) microdomains play an important role for regulating insulin signaling and endothelial function; however underlying mechanisms is incompletely understood. Vascular extracellular superoxide dismutase (ecSOD), a secretory copper (Cu) enzyme synthesized from vascular smooth muscle cells (VSMCs), preserves nitric oxide (NO)-mediated endothelial function by reducing extracellular superoxide anion (O2•-) levels. We previously demonstrated that Cu transporter ATP7A is required for full activity of ecSOD and that both ecSOD and ATP7A are found in C/LR. However, the functional significance of ATP7A-ecSOD pathway in insulin signaling and C/LR remains unknown. Here we show that in organoid culture of mouse aorta, insulin stimulation increases ecSOD specific activity (47%) and ATP7A expression. In cultured VSMCs, insulin stimulation promotes ATP7A translocation to the C/LR (1.7 fold) where ecSOD is activated by ATP7A through their binding. Furthermore, insulin stimulation promotes ATP7A association with Cav1 as shown by immunofluorescence and co-immunoprecipitation assay. Specific activity of ecSOD, but not Cu/Zn SOD, is significantly decreased (60%) in aorta of Cav1 knockout (KO) mice, indicating that Cav1 or C/LR is required for full ecSOD activity. Of note, ATP7A expression is decreased in aorta from Cav1 KO mice or Cav1-depleted VSMCs with siRNA, which is due to enhanced ATP7A ubiquitination. These results suggest that Cav1 binding to ATP7A at C/LR protects ATP7A from proteosomal degradation, thereby increasing ecSOD activity. Functionally, NO-dependent vasorelaxation induced by acetylcholine, but not NO-independent relaxation induced by sodium nitroprusside, is impaired in mesenteric arteries of Cav1 KO mice vs. wild type mice (P<0.05), which is associated with overproduction of O2•- in the vessels and rescued by gene transfer of ecSOD. In summary, Cav1 plays an essential role for insulin-induced full ecSOD activation by regulating ATP7A translocation and expression. Our studies will provide novel insights into the Cav1-ATP7A-ecSOD axis as a potential therapeutic target for promoting endothelial function against oxidative stress-dependent cardiovascular and metabolic diseases.