2162Neurofibromin 2 (NF2) drives obesity-related endothelial dysfunction by targeting Caveolin-1: a study in mice and humans

Abstract

Abstract Introduction Endothelial dysfunction (ED) is a key underpinning of cardiovascular disease in obesity, but the underlying molecular mechanisms remain elusive. Neurofibromin 2 (NF2) is a scaffold-like protein implicated in various cellular processes, namely growth, differentiation and survival. NF2 is inactivated by Akt-dependent phosphorylation at Ser518, whereas its dephosphorylation by the myosin phosphatase target subunit 1 (MYPT-1) leads to an active conformation. The role of NF2 in obesity-related alterations of endothelial phenotype remains elusive. Purpose To investigate whether NF2 participates to ED in obesity. Methods Human aortic endothelial cells (HAECs) were exposed to palmitic acid (PA, 200 uM) or vehicle for 48 hours. Gene silencing of NF2 was performed by small interfering RNA (siRNA). Protein expression was assessed by Western blot. Nitric oxide (NO) levels were measured by using a colorimetric assay. The interaction of NF2 with endothelial proteins was investigated by co-immunoprecipitation. To specifically determine NF2 role in the endothelium, we generated mice with endothelium-specific deletion of NF2 (NF2 ECKO) by crossing NF2flox/flox mice with tamoxifen-inducible endothelial-specific cre mice [Cdh5(PAC)-CreERT2]. Endothelium-dependent relaxations to acetylcholine (Ach, 10–9 to 10–5 mol/L) were assessed in aortas isolated from male NF2 ECKO and wild type littermates, fed a control (10 kcal% fat) and a high fat diet (60 kcal% fat) for 20 weeks. NF2 signalling and endothelial function were also assessed in small visceral fat arteries (VFA) isolated from 18 obese and 18 age-matched healthy subjects undergoing bariatric surgery and cholecystectomy, respectively. Results Exposure of HAECs to PA decreased NF2 phosphorylation at Ser518, thus leading to an active protein conformation. Blunted NF2 phosphorylation was explained by a reduction of Akt phosphorylation at Ser473 and a concomitant increase of MYPT-1 phosphorylation at Thr696. Pull-down experiments revealed that NF2 binds and activates Caveolin 1 (Cav-1), a pivotal repressor of endothelial NO synthase (eNOS). NF2 knockdown in PA-treated HAECs prevented eNOS–Cav-1 interaction, thus preserving eNOS activity and NO levels. In aortas from obese mice, we found that NF2-Cav-1 interaction was responsible for impaired eNOS activity, reduced NO levels and endothelial dysfunction. By contrast, Ach-dependent vasorelaxation were preserved in obese mice with endothelium-specific deletion of NF2. Moreover, we found that NF2 is activated in VFA from obese patients as compared to healthy controls, and its activity negatively correlated with Ach-dependent vasorelaxation of isolated VFA, as assessed by organ chamber experiments. Conclusions The present findings – obtained in human endothelial cells, conditional mouse models and visceral fat arteries from obese patients – suggest that targeting NF2 may represent a potential therapeutic strategy to prevent ED in patients with obesity.