NA+/H+ exchanger 1- and aquaporin-1-dependent hyperosmolarity changes decrease nitric oxide production and induce VCAM-I expression in endothelial cells exposed to high glucose

Since diabetic hyperglycaemia causes hyperosmolarity, we investigated the contribution of hyperosmolarity in the proinflammatory endothelial effects of hyperglycemia, and sought to unravel the mechanisms involved. Human aortic endothelial cells (HAEC) were incubated for short-term (1-3 days) or long-term (1-2 weeks) exposures to 5.5 mmol/L glucose (normoglycemia, basal), high glucose (25 and 45 mmol/L, HG), or a hyperosmolar control (mannitol 25 and 45 mmol/L, HM), in the presence or absence of the aquaporin-1 (AQP1) inihibitor dimethylsulfoxide (DMSO), the Na+/H+ exchanger 1 (NHE-1) inihibitor cariporide (CA), the protein kinase C (PKC) inihibitor calphostin C or the PKCβ isoform inhibitor LY379196 (LY). Both short- and long-term exposures to HG and HM decreased the expression of the active, phosphorylated form of endothelial nitric oxide synthase (Ser1146-eNOS) and, in parallel, increased vascular cell adhesion molecule(VCAM)-1 protein at immunoblotting. After 24 h incubation with HG/HM, we observed a significant similar and concentration-dependent enhancement of AQP1 expression. DMSO and CA inhibited hyperosmolarity-induced VCAM-1 expressions, while increasing nitrite levels and Ser1146-eNOS expression. Gene silencing by small interfering RNA reduced the expression of AQP1, and suppressed HG- and HM-stimulated VCAM-1 expression. Calphostin C and LY blunted hyperosmolarity-induced VCAM-1 expression, while increasing the expression of Ser1146-eNOS and nitrite production. Thus HG decreases eNOS activation and induces total VCAM-1 expression in HAEC through a hyperosmolar mechanism. These effects are mediated by activation of the water channels AQP1 and NHE-I, and a PKCβ-mediated intracellular signaling pathway. Targeting osmosignaling pathways may represent a novel strategy to reduce vascular effects of hyperglycemia. Copyright © by BIOLIFE, s.a.s.