Genipin inhibits endothelial exocytosis via nitric oxide in cultured human umbilical vein endothelial cells

Abstract

Aim: Exocytosis of endothelial Weibel-Palade bodies, which contain von Willebrand factor (VWF), P-selectin and other modulators, plays an important role in both inflammation and thrombosis. The present study investigates whether genipin, an aglycon of geniposide, inhibits endothelial exocytosis.

Methods: Human umbilical vein endothelial cells (HUVECs) were isolated from umbilical cords and cultured. The concentration of VWF in cell supernatants was measured using an ELISA Kit. P-selectin translocation on the cell surface was analyzed by cell surface ELISA. Cell viability was measured using a Cell Counting Kit-8. Mouse bleeding times were measured by amputating the tail tip. Western blot analysis was used to determine the amount of endothelial nitric oxide synthase (eNOS) and phospho-eNOS present. Nitric oxide (NO) was measured in the cell supernatants as nitrite using an NO Colorimetric Assay.

Results: Genipin inhibited thrombin-induced VWF release and P-selectin translocation in HUVECs in a dose- and time-dependent manner. The drug had no cytotoxic effect on the cells at the same doses that were able to inhibit exocytosis. The functional study that demonstrated that genipin inhibited exocytosis in vivo also showed that genipin prolonged the mouse bleeding time. Furthermore, genipin activated eNOS phosphorylation, promoted enzyme activation and increased NO production. L-NAME, an inhibitor of NOS, reversed the inhibitory effects of genipin on endothelial exocytosis.

Conclusion: Genipin inhibits endothelial exocytosis in HUVECs. The mechanism by which this compound inhibits exocytosis may be related to its ability to stimulate eNOS activation and NO production. Our findings suggest a novel anti-inflammatory mechanism for genipin. This compound may represent a new treatment for inflammation and/or thrombosis in which excess endothelial exocytosis plays a pathophysiological role.

Figure 1

Chemical structure of genipin.

Figure 2

Effect of genipin on thrombin-induced exocytosis of VWF from HUVECs. HUVECs were pretreated with the indicated concentration of genipin for various times or left untreated and then stimulated with 1 U/mL thrombin for 30 min. The VWF concentration in the cell supernatants was measured using a VWF ELISA Kit. (A) Genipin inhibited VWF release in a concentration-dependent manner. (B) Genipin inhibited VWF release in a time-dependent manner. Data are expressed as the mean±SEM of 3 independent experiments. ^cP<0.01 vs control (Con); ^eP<0.05 vs thrombin (Thr); ⁱP<0.01 vs Thr.

Figure 3

Effect of genipin on thrombin-induced translocation of P-selectin from HUVECs. HUVECs were pretreated with the indicated concentrations of genipin for various times or left untreated and then stimulated with 1 U/mL thrombin for 15 min. P-selectin translocation on HUVEC surfaces was measured using a cell surface ELISA. (A) Genipin inhibited P-selectin translocation in a concentration-dependent manner. (B) Genipin inhibited P-selectin translocation in a time-dependent manner. Data are expressed as the mean±SEM of 3 independent experiments. ^cP<0.01 vs control (Con); ^eP<0.05 vs thrombin (Thr); ⁱP<0.01 vs Thr.

Figure 4

Effect of genipin on the bleeding time in mice. Mice were injected intravenously with different concentrations of genipin. At various times after injection, the bleeding time was measured. (A) Genipin prolonged the bleeding time in mice in a dose-dependent manner. (B) Genipin prolonged the bleeding time. Data are expressed as the mean±SD of 8 mice. ^bP<0.05, ^cP<0.01 vs control (Con).

Figure 5

Effect of genipin on eNOS-ser (1177) phosphorylation and total eNOS in HUVECs. HUVECs were treated with the indicated concentrations of genipin for the indicated times or left untreated. Total protein samples were electrophoresed and separated on a 10% SDS-PAGE gel. The levels of phosphorylated eNOS-ser (1177) and total eNOS were detected using Western blot analysis. (A) Representative Western blots of eNOS-ser (1177) phosphorylation and total eNOS for the indicated concentration of genipin. (B) Representative Western blots of eNOS-ser (1177) phosphorylation and total eNOS for the indicated lengths of genipin treatment. (C) Semiquantitative analysis of proteins showed that genipin increased eNOS-ser (1177) phosphorylation in a concentration-dependent manner, but had no effect on the total amount of eNOS in HUVECs. (D) Semiquantitative analysis of proteins showed that genipin increased eNOS-ser (1177) phosphorylation in a time-dependent manner, but had no effect on the total amount of eNOS in HUVECs. Data are expressed as the mean±SEM of 3 independent experiments. ^bP<0.05, ^cP<0.01 vs control (Con).

Figure 6

Effect of genipin on nitric oxide production in HUVECs. HUVECs were treated with various concentrations of genipin at different times, and then the NO level was measured in the cell supernatants. (A) Genipin increased NO production in a concentration-dependent manner. (B) Genipin increased NO production in a time-dependent manner. Data are expressed as the mean±SEM of 3 independent experiments. ^bP<0.05, ^cP<0.01 vs control (Con).

Figure 7

Effect of L-NAME on genipin-induced inhibition of VWF exocytosis from HUVECs. HUVECs were pretreated with the indicated concentrations of L-NAME for 1 h and then the indicated concentrations of genipin were added for 2 h. The cells were stimulated with 1 U/mL thrombin for 30 min. The VWF concentration in the cell supernatants was measured using a VWF ELISA Kit. Data are expressed as the mean±SEM of 3 independent experiments. ^bP<0.05, ^cP<0.01 vs L-NAME (0 mmol/L) and genipin (8 μg/mL). ^fP<0.01 vs L-NAME (0 mmol/L) and genipin (1 μg/mL).