Hydrogen sulphide donors on endothelial cell adhesion molecule expression

Anna Hsu, Ling Li, Philip Moore. King’s College London, Cardiovascular Division, SE1 9NH, London Waterloo, United Kingdom.

Hydrogen sulphide (H2S) donors are now emerging as a new and potential therapy in cardiovascular and inflammatory diseases. Exogenously administered H2S no doubt has properties to relax vascular smooth muscle cells, possibly in part via the activation of KATP channels. However, the role of H2S in inflammation is uncertain as both pro- and anti-inflammatory effects have been reported. We have previously reported that the novel slow-releasing H2S donor (GYY4137) elicits protective anti-inflammatory effects using in vivo and in vitro models of sepsis (endotoxemia). The expression of adhesion molecules on the endothelial cell surface are implicitly involved in mediating inflammatory responses. The purpose of this study was to directly determine the effect of H2S donors on the regulation of endothelial cell derived adhesion molecule expression.

To this end, human umbilical vein endothelial cells (HUVEC) were pretreated with H2S donors, sodium hydrogen sulphide (NaHS) or GYY4137, for 1 hour prior to stimulation with lipolysaccharide (LPS 100 ng/ml) or IL-1β (10 ng/ml) for 24 hours. The expression of soluble vascular cell adhesion molecule-1 (sVCAM-1), intracellular cell adhesion molecule-1 (sICAM-1) and sE-selectin was quantitated in the cell culture medium by commercial enzyme-linked immunosorbent assay (ELISA). The cell surface expression of these adhesion molecules were futher confirmed by a direct ELISA on HUVEC fixed on a 96-well plate format.

For the first time, we have shown that H2S donors directly and dose dependently reduce LPS and IL-1β induced soluble VCAM-1 expression from HUVEC. In correlation with this GYY4137 and NaHS both signficantly reduced surface expression of VCAM-1. GYY4137 (31.25 – 500 µM) appeared to reduce sICAM-1 expression although this effect was not significant even at the highest concentration used. Neither of the H2S donors affected sE-selectin expression.

The more potent effects of GYY4137 on adhesion molecule expression may be due to the more sustained release of H2S over time (hours) compared to NaHS which releases H2S over a period of seconds. In an attempt to investigate the mechanism underlyingthis effect of H2S on adhesion molecule expression, HUVEC were pretreated with GYY4137 (125 µM) prior to IL-1β stimulation. Interestingly, GYY4137-treated cells showed faster recovery of the NF-kB inhibitory protein, IKB-α at 60 minutes. Futhermore, in other preliminary experiments GYY4137 (500 µM) inhibited the protein expression of cyclooxygenase-2 (COX-2, an enzyme regulated by NF-kB), in IL-1β treated HUVEC.

To conclude, we are the first to show that H2S donors dose dependently reduce VCAM-1 expression in endothelial cells. The inhibitory effects of H2S on cell adhesion molecule expression in HUVEC would appear to be, at least partially, mediated via inhibiting NF-kB translocation into the nucleus. H2S donors may be useful in regulating adhesion molecule expression on endothelial cells where this may be over expressed.