P54 Slow release hydrogen sulfide (H2S) donors prevent hyperglycaemia-induced glycocalyx loss in retinal mirovascular endothelial cells

Abstract

Background Leukocyte adhesion to the vascular endothelium is mediated by endothelial adhesion molecules binding to their leukocyte ligands. The endothelial adhesion molecules are embedded in the glycocalyx, a dense network of membrane-bound proteoglycans and glycoproteins. In health, the glycocalyx contributes to the anti-inflammatory regulation of the vascular wall, effectively ‘hiding’ the adhesion molecules from the circulating leukocytes. However, in pathological conditions associated with glycocalyx loss such as diabetes this protective mechanism is lost, possibly contributing to the associated microangiopathies e.g. diabetic retinopathy. Indeed, the early stages of retinopathy are characterised by increased leukocyte adhesion, contributing to inflammation and vessel blockage. Therefore therapeutic strategies which aim to maintain glycocalyx integrity may be beneficial in the treatment of diabetic retinopathy. We have shown that plasma H 2 S levels are significantly lower in individuals with type 2 diabetes compared with sex/age/BMI matched controls, and that reduced H 2 S levels correlate with microvascular dysfunction in vivo . However, the direct effects of H 2 S on the glycocalyx are unknown and it is possible that diabetes-associated glycocalyx loss was due to reduced bioavailability of H 2 S. Aim To investigate (a) the effects of the slow release H 2 S donors GYY4137 and AP67 on hyperglycaemia-induced changes to the glycocalyx and leukocyte adhesion and (b) potential mechanisms of action of H 2 S donors specifically endothelial K ATP channel activation. Methods Bovine retinal endothelial cells (BREC) were exposed to 5.6 mM glucose (NG) and 25 mM glucose (HG)±GYY4137 for 24 h. A cell based fluorescence assay was used to study glycocalyx changes. Glibenclamide (Glib), gliclazide (Glic) and cromakalim (Crm) were used to test involvement of endothelial K ATP channels. Leukocyte adhesion to BREC was measured at a shear stress of 1 dyn/cm 2 . Results HG-induced glycocalyx degradation was reversed by GYY4137 [HG 93 ± 5% vs HG + GYY 107 ± 8%, p n = 12; control,100%)] and AP67 [HG 92 ± 5% vs HG + AP67 105 ± 8%; p n = 3; control 100%). Crm, Glib or Glic had no effect on glycocalyx changes. HG enhanced leukocyte adhesion and this was attenuated by H 2 S [HG 275 ± 41% vs HG GYY 105 ± 11%; control,100%]. The H 2 S mediated decrease in adhesion was not inhibited by Glib or Glic suggesting no involvement of endothelial K ATP channel activation. H 2 S added simultaneously with HG to BREC pre-treated with Crm did not affect leukostasis. Conclusions Glycocalyx degradation and increased leukocyte adhesion induced by HG were reversed by GYY4137 and AP67, highlighting the therapeutic potential for slow release H 2 S donors in hyperglycaemic conditions.