Abstract
Abstract: :
Purpose: Local retinal haemodynamics are altered in diabetic retinopathy. High blood glucose is integral in initiating many of these alterations, which affect the blood flow pattern in the microcirculation. Using our novel perfused trancapillary co–culture system we examined the effect of hyperglycaemia on pulsatile flow induced eNOS expression. Methods: Cell Culture Bovine Retinal Endothelial Cells (BRECs) were cultured in DMEM supplemented with 10% heat–inactivated fetal bovine serum (FBS) plus 100 units/ml penicillin and 100 µg/ml streptomycin in a humidified atmosphere of 5% CO2, 95% air. BRECs were routinely used between passages 5 and 15. 25mM glucose simulated hyperglycaemia with 25mM mannitol as an osmotic control. Western Blot Endothelial cell lysates were separated via SDS–PAGE and probed using specific antibodies for eNOS (Cayman Chemicals) and phospho–eNOS (cell signalling) and Cox–1 (Cayman) Transcapillary Co–Culture The Cellmax artificial capillary module (polypropylene cell–PPF70 from Spectrum Laboratories Inc.) was used to expose cells to ‘low flow’ (0.3 mls/min; 6 mm Hg, Pulse pressure 0.5 (dyne/cm2) or ‘high flow’ (25 mls/min; 56 mmHg, Pulse pressure 23 dyne/cm2) for 24 hours. The system consists of a bundle of 50 semipermeable, fibronectin coated polyethylene tubes (outer diameter 630 µm, wall thickness 150 µm, luminal area 70 cm2, outer surface area 100 cm2, Extracapillary volume 1.4 mls, 95% MWCO 0.5 µm) encased in a plastic holding. Cell culture medium is pumped at the chosen flow rate from a reservoir bottle through the lumen of the capillaries via silicone tubing.BREC monocultures were used. ELISA Assays Endothelin–1 and PGI–2 activity determined with commercially avaialable kits. Nitrate Assay A fluorometric assay for the measurement of nitrate in culture media was used. Results: In controls high pulsatile flow increased ppeNOS expression 6 fold from baseline, with a two fold increase in NO activation. Total eNOS protein expression remained unchanged. PGI–2 activation increased fourfold. Hyperglycaemia decreased total eNOS protein expression 4 fold. This was accompanied by no activation of NO beyond baseline and only partial activation of ppeNOS expression. Cox–1 activation remained unchanged. ET–1 levels increased in both. Conclusions: High glucose suppresses pulsatile flow induced activation of eNOS. Altering the vasoactive balance may favour a constricted and therefore more ischaemic microcirculation in diabetic retinopathy.
Keywords: diabetic retinopathy • blood supply • nitric oxide