Abstract
BackgroundHyperhomocysteinemia (HHcy) causes cardiovascular diseases via regulating inflammatory responses. We investigated whether and how the epithelial sodium channel (ENaC), a recently identified ion channel in endothelial cells, plays a role in HHcy-induced endothelial dysfunction.MethodsCell-attached patch-clamp recording in acute split-open aortic endothelial cells, western blot, confocal imaging, and wire myograph combined with pharmacological approaches were used to determine whether HHcy-mediated inflammatory signaling leads to endothelial dysfunction via stimulating ENaC.ResultsThe data showed that 4 weeks after L-methionine diet the levels of plasma Hcy were significantly increased and the ENaC was dramatically activated in mouse aortic endothelial cells. Administration of benzamil, a specific ENaC blocker, ameliorated L-methionine diet-induced impairment of endothelium-dependent relaxation (EDR) and reversed Hcy-induced increase in ENaC activity. Pharmacological inhibition of NADPH oxidase, reactive oxygen species (ROS), cyclooxygenase-2 (COX-2)/thromboxane B2 (TXB2), or serum/glucocorticoid regulated kinase 1 (SGK1) effectively attenuated both the Hcy-induced activation of endothelial ENaC and impairment of EDR. Our in vitro data showed that both NADPH oxidase inhibitor and an ROS scavenger reversed Hcy-induced increase in COX-2 expression in human umbilical vein endothelial cells (HUVECs). Moreover, Hcy-induced increase in expression levels of SGK-1, phosphorylated-SGK-1, and phosphorylated neural precursor cell-expressed developmentally downregulated protein 4-2 (p-Nedd4-2) in HUVECs were significantly blunted by a COX-2 inhibitor.ConclusionWe show that Hcy activates endothelial ENaC and subsequently impairs EDR of mouse aorta, via ROS/COX-2-dependent activation of SGK-1/Nedd4-2 signaling. Our study provides a rational that blockade of the endothelial ENaC could be potential method to prevent and/or to treat Hcy-induced cardiovascular disease.
Highlights
As an independent risk factor, hyperhomocysteinemia (HHcy) is closely associated with coronary heart disease, venous and arterial thrombosis, atherosclerosis, and hypertension (Rodrigo et al, 2003; Ganguly and Alam, 2015)
We investigated the role of endothelial epithelial sodium channel (ENaC) and underlying mechanisms in HHcy-induced vascular dysfunction, using L-methionine administration-induced HHcy mouse model combined with a variety of experimental approaches
Since our previous data showed that elevation of ENaC activity impaired ACh-induced endothelium-dependent relaxation (EDR) in the salt-sensitive rats (Wang et al, 2018b), we hypothesize that blockade of ENaC may prevent Hcy-induced vascular dysfunction in L-methionine-treated mice
Summary
As an independent risk factor, hyperhomocysteinemia (HHcy) is closely associated with coronary heart disease, venous and arterial thrombosis, atherosclerosis, and hypertension (Rodrigo et al, 2003; Ganguly and Alam, 2015). Hcy induced porcine coronary endothelial dysfunction through ER stress-mediated inhibition of SKCa and IKCa channels (Wang et al, 2015). Studies showed that Hcy significantly inhibited BKCa channels in isolated human and rat artery smooth muscle cells and that the effects of Hcy on BKCa channels were reversed by the DPI, an inhibitor of NADPH oxidase (Cai et al, 2007). Hcy suppressed BKCa channel probably by activating NADPH oxidase in porcine coronary smooth muscle cells. Whether Hcy regulates endothelial epithelial sodium channel (ENaC), a newly identified ion channel in endothelium, remains unclear. We investigated whether and how the epithelial sodium channel (ENaC), a recently identified ion channel in endothelial cells, plays a role in HHcy-induced endothelial dysfunction
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