Endophilin A2 improves endothelial dysfunction by counteracting eNOS proteasomal degradation

BACKGROUND AND AIMS: Hypertension is a major cardiovascular risk factor arising from endothelial dysfunction driven by dysregulated endothelial nitric oxide synthase (eNOS) activity. Although eNOS turnover is regulated by post-translational modifications, the precise mechanisms remain unclear. Endophilin A2 (EndoA2) is uniquely enriched in the cardiovascular system and may influence eNOS activity and endothelial function. The primary objective of this study was to investigate the function of EndoA2 in blood pressure homeostasis with particular emphasis on elucidating its mechanism in regulating eNOS protein stability. METHODS: Global and endothelial cell-specific EndoA2 knockout mice were generated to determine the effects of EndoA2 on hypertension, employing radiotelemetry, alongside histological, cellular, molecular, and biochemical approaches. RESULTS: Reduced EndoA2 expression was consistently observed in the aortic tissues of three hypertensive animal models. Global or endothelial cell-specific ablation of EndoA2 in mice induced spontaneous hypertension, vascular remodelling, and impaired endothelium-dependent vasodilation. Conversely, endothelial cell-specific EndoA2 overexpression ameliorated vascular remodelling and hypertension by maintaining endothelial homeostasis. Mechanistically, EndoA2 interacted with eNOS through its proline-rich domain (PRD), competing with the E3 ubiquitin ligase Itch for eNOS binding. This competition suppressed the Itch-mediated K48-linked ubiquitination of eNOS at lysine 834, thereby stabilizing eNOS. Notably, the PRD-mimetic peptide recapitulated the beneficial effects of EndoA2 by attenuating eNOS degradation and hypertension in mice. CONCLUSIONS: EndoA2 is a critical regulator of eNOS stability by antagonizing Itch-dependent ubiquitination. The PRD-mimetic peptide preserves eNOS homeostasis and alleviates hypertension, suggesting the therapeutic potential of targeting the EndoA2-Itch-eNOS axis in endothelial dysfunction-related cardiovascular diseases including hypertension.