Downregulated caveolin‑1 expression serves a potential role in coronary artery spasm by inducing nitric oxide production in�vitro

Abstract

The present study aimed to investigate the effects of downregulated caveolin-1 (Cav-1) expression on nitric oxide (NO) production in lipopolysaccharide (LPS)-damaged primary human umbilical vein endothelial cells (HUVECs) in a model of coronary artery spasm (CAS) microenvironment induced by acetylcholine (ACh) treatment. Small interfering RNA (siRNA)-mediated Cav-1 downregulation in HUVECs was confirmed by western blotting. The cell viability and superoxide dismutase (SOD) inhibition in HUVECs incubated with LPS (0, 10, 25, 50, 75 and 100 µg/ml) were measured by cell counting kit-8 assay and a SOD kit, respectively. Intracellular Ca2+ [(Ca2+)i] in Fluo4-acetoxymethyl ester-loaded cells was detected by fluorescence microscopy. NO levels in the cell culture supernatants were measured by the nitrate reductase method. The results indicated that transfection with Cav-1 siRNA, in particular siCav-1 (2), downregulated the Cav-1 protein expression. LPS at a dose of 75 µg/ml induced a significant decrease in HUVECs/si-NC and HUVECs/siCav-1 viability compared with the other concentrations of LPS. Compared with the effects of untreated cells, SOD inhibition in HUVECs/si-NC and HUVECs/siCav-1 was significantly decreased by LPS (75 µg/ml). In addition, ACh stimulation caused a greater increase in [Ca2+]i in HUVECs/si-NC as compared with LPS-treated HUVECs/si-NC. ACh stimulation also induced significantly higher NO levels in LPS-treated HUVECs/siCav-1 compared with LPS-treated HUVECs/si-NC cells (P<0.05). In conclusion, the downregulated Cav-1 expression served a key role in NO production in the in vitro model of CAS induced by ACh stimulation of LPS-damaged HUVECs.

Pathophysiological Role of Caveolae in Hypertension

“…Increased expression of Cav1 is known to appear in patients with insulin resistance and type 2 diabetes (54), associated with impaired acetylcholine-induced NO production and vasodilation (55). Cav1 knockout mice show chronic and dramatic elevation in systemic NO levels and enhanced acetylcholine-induced arterial relaxation (56, 57). Conversely, the Bendhack group (58) reported that caveolae disassembled by methyl-β-cyclodextrin (mβcd) treatment cause an impaired acetylcholine-induced relaxation in the rat isolated aorta.…”