Hyperglycaemia induced changes in retinal endothelial cell glycocalyx : role of heparanase 1, proteases and hydrogen sulfide

Diabetic retinopathy (OR) is a leading cause of blindness in working-age adults. Increased leukocyte adhesion and capillary non-perfusion, associated with this microvascular complication are early events in development of OR. Although the mechanisms are not fully elucidated, it is likely that the endothelium plays a critical role in this process since its dysfunction is central to the development of diabetic microvascular complications. Healthy endothelium is protected by a negatively charged mesh that occupies a large domain at its luminal site, called a glycocalyx. Thus diabetes-induced loss of the glycoca!yx could contribute to Increased leukostasis. Heparan sulfate proteoglycan side chains form a substantial component of the glycocalyx that can be degraded and modified by heparanase1 (HPSE). Glycocalyx can also be shed by matrix metalloproteinases (MMPs), particularly MMP-2 and MMP-9 which have been implicated in the pathology of diabetic retinopathy. Hydrogen sulfide (H2S) IS a novel gaseous transmitter that plays an important role as a regulator of blood pressure, blood flow and leukocyte adhesion. Decreased plasma H2S levels are associated with endothelial dysfunction and insulin resistance in animal models of diabetes. Furthermore. H2S is decreased in obese patients with type 2 diabetes. The present in vitro study was designed primarily to explore the possible HPSE, MMP and H2S dependent changes in retinal endothelial cell glycocalyx and subsequent alterations in leukocyte adhesion. Given that hyperglycaemia is widely accepted as the primary cause of diabetes-associated endothelial dysfunction this thesis sought to investigate whether the high glucose milieu could affect the endothelial glycocalyx in retinal endothelial cells. Further studies examined whether high glucose altered HPSE expression, secretion and activity in retinal endothelial cells. and also leukocyte adhesion. Additionally, high glucose induced changes in HPSE expression and its intracellular localization in retinal pericytes have been assessed. The profile of MMP secretion was also studied. The final series of experiments investigated whether the slow releasing H2S donor GYY 4137 could prevent hyperglycaemia induced endothelial glycocalyx dysfunction and modulate leukocyte adhesion. The main techniques used in this dissertation include isolation of primary bovine retinal endothelial cells and pericytes (BREC, BRP), immunoprecipitation. immunoblotting, zymography and cell based fluorescence assay. Results indicate that hyperqlycaernia induced glycocalyx changes were accompanied by a marked increase in active HPSE secreted from retinal endothelial cells. Heparanase worked in concert with MMP-2 and increased secretion was accompanied by a decreased glycocalyx in hyperglycaemia. Increased expression of HPSE by hyperglycaemia in the retina was further confirmed in a streptozotocin induced diabetic rat model. Changes within the glycocalyx were associated with increased leukostasis in high glucose under static and shear stress conditions. Intracellular HPSE was found to be uprequlated in pericytes. Addition of exogenous H2S via GYY 4137 reversed hyperglycaemia induced changes in the glycocalyx and decreased leukocyte adhesion. The effect of H2S on endothelial cell glycocalyx has never been reported before. The expression ot cystathionine-B-synthase, endogenous enzyme that can produce H2S, was not affected by high glucose in BREC. The present findings implicate the glycocalyx as a vital component of the vessel wall that is seriously affected in hyperglycaemia. These results suggest that upregulation of HPSE expression and secretion may play an important role in the development of diabetic retinopathy. This study provides also a new insight into the role of H2S in protecting endothelial cells from hyperglycaemia induced injury and suggests increasing H2S bioavailability as a potential opportunity for therapeutic intervention.EThOS - Electronic Theses Online ServiceGBUnited Kingdo