CAVEOLAE AS SPATIO-TEMPORAL COMPARTMENTS FOR ROS/RNS GENERATION AND NITROXIDATIVE STRESS SIGNALING

Abstract

During inflammatory conditions excessive production of reactive oxygen (ROS) and nitrogen species (RNS), peroxynitrite, is implicated in the development of vascular pathologies. Our previous studies showed that both NADPH oxidase enzyme complexes and eNOS localize to endothelial caveolae microdomains. Additionally, caveolae internalization has been shown as an activating mechanism for enzyme eNOS. However, roles of caveolae in ROS/RNS generation and downstream signaling roles in activating endothelial cells are not well known. Hypothesis: Caveolae act as, a) micro-environments in providing spatio-temporal reaction compartments for ROS/RNS generation, tyrosine nitration of proteins, b) platforms to propagate localized nitroxidative signaling in inducing endothelial cell activation and dysfunction (ICAM-1, VCAM-1 expression), and c) intracellular redox signaling endosomes to regulate adhesion molecule expression. Objectives: The aim of the study was to investigate whether, a) caveolae compartmentalize ROS, regulate localized tyrosine nitration of proteins, b) nitroxidative-signaling in the endothelium is compartmentalized in caveolae, c) dynamin-2-dependent internalization of caveoale is important for activating redox signaling, and d) caveolae compartments can be targeted to reduce endothelial ROS Methods and results: Cultured primary bovine aortic endothelial cells were stimulated with TNFα to generate ROS/RNS. Blockade of NADPH oxidase (gp91ds-tat) or scavenging of peroxynitrite (Uric acid) inhibited TNFα-induced protein tyrosine nitration, activation of the NFkB, and upregulation of ICAM-1/VCAM-1 expression. To test the role of caveolae in this process, cultured cells were depleted of caveolin-1 (siRNA). Similar to inhibitors, TNFα failed to induce protein-tyrosine nitration, activate NFkB or enhance adhesion molecule expression in cells lacking caveolin-1. These findings were corroborated in vivo using Cav1KO animals. Our results show that several caveolar residing proteins were nitrated on tyrosine in response to TNFα. Here, immunoprecipitation of cell lysates with an anti-nitrotyrosine antibody revealed Src-family kinases (SFK) in the precipitated fraction. Moreover, SFK nitration was lost in cells depleted of caveolin-1. Given that SFK nitration is associated with enzyme activation, cells were pretreated with PP2 to inhibit SFK activity. We found that PP2 attenuated the NFkB and adhesion molecule pathway activated by TNFα. Depletion of dynamin-2 (Dyn2siRNA) or inhibiting GTPase activity (Dynasore) also showed reductions in ROS generation, NFkB redox signaling and ICAM-1/VCAM-1 expression. Development of caveolae targeting peptide tagged with gp91ds-tat showed inhibitions in compartmentalized ROS production. Conclusions: Caveolae act as sites for ROS/RNS production where resident redox-sensitive second messengers are activated and propagate signals that regulate endothelial inflammatory phenotype. Targeting NADPH oxidase enzyme specifically in caveolae can be used a therapeutic strategy to limit vascular oxidative stress.