P67: Role of oxidative stress-induced caveolin-1 S-nitrosylation, ubiquitination, and degradation in endothelial cell dedifferentiation and idiopathic pulmonary arterial hypertension

Abstract

Cav1 −/− mice develop pulmonary arterial hypertension (PAH) which is dependent on eNOS since inhibition or deletion of eNOS abrogates the PAH phenotype. Here, we tested the hypothesis that inflammatory signaling/oxidative stress induces Cav-1 degradation, and that in presence of reduced Cav-1 expression, eNOS hyperactivation promotes endothelial cell dedifferentiation, disorganized postnatal angiogenesis, and vaso-occlusive disease in the lung. In freshly-isolated IPAH patient-derived lung endothelial cells, we observed reduced Cav-1 expression and eNOS hyperphosphorylation. In cultured control human lung endothelial cells, TNF alpaha-induced NO production and S -nitrosylation (SNO) of Cav-1 cysteine 156 was associated with increased Src activity and destabilization of Cav-1 oligomeric chains that could be blocked by the eNOS inhibitor l -NAME or Src inhibitor PP2. Furthermore, Cav-1 SNO was associated with reduced Cav-1 expression following prolonged (72 h) stimulation with TNF alpha or the NO donor DetaNONOate. Moreover, mass spectrometry revealed K86 in the caveolin scaffold domain as a site of Cav-1 ubiquitination (Ub). Thus, these data suggest that Cav-1 SNO at C156 induces Src-dependent Cav-1 phosphorylation at Y14, destabilization of Cav1 oligomers, and Ub of K86 leading to Cav-1 degradation. Furthermore, Cultured Cav1 −/− ECs were observed to be hyperproliferative and dysfunctional in their ability to generate lumenized vessels in Matrigel plugs in vitro and in vivo. The defect in angiogenesis observed in Cav1 −/− ECs was shown to be due to hyperactive eNOS-mediated disruption of endothelial cell–cell junctions and cell contact-dependent Notch signaling. eNOS-dependent peroxynitrite production and resultant Akt and Src hyperactivation in Cav1 −/− ECs not only prevented nuclear Notch signaling, it resulted in increased nuclear beta-catenin signaling that was associated with an “all tip-cell” phenotype. Rescue of Cav-1 null cells with Cav-1 cDNA and in Cav1 −/− × eNOS −/− double knockout ECs, Notch activation and angiogenesis were restored. Therefore, oxidative stress-induced Cav-1 degradation and eNOS hyperactivation-mediated endothelial cell dedifferentiation may play an important role in vaso-occlusive pulmonary vascular disease. Disclosure Nothing to disclose.