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.

Keywords: acetylcholine; caveolin-1; coronary artery spasm; lipopolysaccharide; nitric oxide.

Figure 1.

Morphological appearance and identification of HUVECs under the inverted microscope. (A) Cells demonstrated a typical ‘cobblestone’ pattern without staining (magnification, ×100). (B) Nuclei were stained with DAPI (magnification, ×200). (C) Immunofluorescent staining of factor VIII expressed in cells (magnification, ×200). (D) Merged image of DAPI and factor VIII staining (magnification, ×200). HUVECs, human umbilical vein endothelial cells; DAPI, 4′,6-diamidino-2-phenylindole.

Figure 2.

Western blot analysis of Cav-1 protein expression in HUVECs transfected with siCav-1(1), siCav-1(2) and siCav-1(3). GAPDH was used as a loading control. ImageLab software was used to quantify the immunoreactive band density, and GraphPad Prism version 5 software was used to generate the histogram. *P<0.05 and ***P<0.001 vs. HUVEC. HUVECs, human umbilical vein endothelial cells; Cav-1, caveolin-1; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; si, small interfering RNA; NC, negative control.

Figure 3.

Effects of treatment with LPS (10–100 µg/ml) for 24 h on the viability of HUVECs/si-NC and HUVECs/siCav-1. Treatment with 75 and 100 µg/ml LPS induced a significant decrease in the viability of HUVECs/si-NC and HUVECs/siCav-1. There was no significant difference in the viability of HUVECs/si-NC and HUVECs/siCav-1 treated with LPS at 75 or 100 µg/ml. *P≤0.001 vs. absence of LPS treatment. HUVECs, human umbilical vein endothelial cells; LPS, lipopolysaccharide; Cav-1, caveolin-1; si, small interfering RNA; NC, negative control.

Figure 4.

Effect of LPS (75 µg/ml) treatment on SOD activity in HUVECs. LPS treatment significantly decreased the inhibition of SOD in HUVECs/si-NC and HUVECs/siCav-1. *P≤0.05 and **P≤0.01. LPS, lipopolysaccharide; SOD, superoxide dismutase; HUVECs, human umbilical vein endothelial cells; Cav-1, caveolin-1; si, small interfering RNA; NC, negative control.

Figure 5.

Effects of 10 µM Ach added at 10 sec on the [Ca²⁺]i responses in Fluo4-acetoxymethyl ester-loaded HUVECs/si-NC with or without LPS treatment. The responses in the eight cell groups were evaluated. The ACh-stimulated changes in the [Ca²⁺]i fluorescence of HUVECs/si-NC are shown in the (A) absence and (B) presence of LPS, at the following time points: (a) 1 sec; (b) 17 sec; (c) 35 sec; (d) 70 sec; (e) 99 sec; (f) 122 sec; (g) 158 sec; (h) 199 sec; (i) 212 sec; (j) 232 sec; (k) 268 sec; and (l) 334 sec. In HUVECs/si-NC without LPS treatment, a characteristic biphasic [Ca²⁺]i response was observed following the addition of ACh (10 µM) at 10 sec, with an initial rapid increase in [Ca²⁺]i, reaching a maximum level at a different time-points for every cell, followed by a sustained plateau in [Ca²⁺]i that declined slowly toward the baseline. In addition, a [Ca²⁺]i oscillation phenomenon was observed with peak and plateau levels occurring regularly. In the HUVECs/si-NC group with LPS treatment, the addition of ACh (10 µM) at 10 sec resulted in a smaller peak and plateau compared with those observed in the group without LPS treatment, with certain cells not presenting any response. [Ca²⁺]i, intracellular Ca²⁺; LPS, lipopolysaccharide; ACh, acetylcholine; HUVECs, human umbilical vein endothelial cells; Cav-1, caveolin-1; si, small interfering RNA; NC, negative control.

Figure 6.

Production of NO with or without ACh exposure in HUVECs/si-NC and HUVECs/siCav-1 with or without LPS pretreatment. ACh stimulated the production of NO in HUVECs/si-NC and HUVECs/siCav-1. The NO level was lower when cells were treated with LPS compared with the levels observed without LPS treatment. ACh stimulated the LPS-induced HUVECs/siCav-1 to produce significantly higher NO levels compared with those produced by LPS-induced HUVECs/si-NC. *P≤0.05, **P≤0.01, ***P≤0.001. NO, nitric oxide; HUVECs, human umbilical vein endothelial cells; LPS, lipopolysaccharide; ACh, acetylcholine; Cav-1, caveolin-1; si, small interfering RNA; NC, negative control; IOD, integrated optical density.