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. |
