Role of NADPH oxidase in retinal microvascular permeability increase by RAGE activation.
OBJECTIVE
The accumulation of advanced glycation end products (AGEs) within the retina in diabetes is associated with a chronic increase in retinal microvascular permeability. Isolated perfused retinas were used to examine the acute effects of AGEs on retinal microvascular permeability.


METHODS
Retinas were dissected from eyes obtained from male Wistar rats, pinned out flat, and perfused with the low-molecular-weight fluorescent dye sulforhodamine B. Microvascular permeability was determined from the rate of decrease in fluorescence gradient across a vessel under conditions of zero flow. The production of reactive oxygen species (ROS) in JG2.1 retinal endothelial cells was also assessed with a fluorescent probe working solution.


RESULTS
A 30-second application of AGE-modified bovine serum albumin (AGE-BSA) to the abluminal surface of the retinal vasculature produced a rapid dose-dependent increase in retinal capillary permeability that was inhibited by pretreatment with anti-RAGE IgG. The permeability response also required ROS generated by NADPH oxidase because pretreatment with apocynin and the free radical scavengers superoxide dismutase and catalase significantly reduced the response. Pretreatment with calphostin C, SKF-96365, and U-73122 also significantly reduced the permeability response. In addition, the permeability response to bradykinin increased permeability through ROS and was potentiated after pretreatment with AGE-BSA. This potentiation was blocked by apocynin.


CONCLUSIONS
Acute activation of NADPH oxidase by phospholipase C-mediated activation of Ca(2+)-dependent PKC occurs downstream of RAGE activation to acutely increase retinal capillary permeability in the isolated perfused rat retina.
Warboys
Christina M
CM
Cardiovascular Division, King's College London, London, United Kingdom.
Toh
Hong-Boon
HB
Fraser
Paul A
PA
D000098:Acetophenones / Q000494:pharmacology; D000818:Animals; D001920:Bradykinin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D002199:Capillary Permeability / Q000187:drug effects / Q000502:physiology*; D002374:Catalase / Q000494:pharmacology; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D004730:Endothelium, Vascular / Q000187:drug effects / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism; D017127:Glycation End Products, Advanced / Q000494:pharmacology; D008297:Male; D019255:NADPH Oxidases / Q000502:physiology*; D011493:Protein Kinase C / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D017382:Reactive Oxygen Species / Q000378:metabolism; D000067759:Receptor for Advanced Glycation End Products; D011971:Receptors, Immunologic / Q000378:metabolism*; D012171:Retinal Vessels / Q000187:drug effects / Q000201:enzymology*; D012235:Rhodamines / Q000378:metabolism; D012710:Serum Albumin, Bovine / Q000494:pharmacology; D013482:Superoxide Dismutase / Q000494:pharmacology
D000098:Acetophenones; D005456:Fluorescent Dyes; D017127:Glycation End Products, Advanced; D017382:Reactive Oxygen Species; D000067759:Receptor for Advanced Glycation End Products; D011971:Receptors, Immunologic; D012235:Rhodamines; C081628:advanced glycation end products-bovine serum albumin; C022027:lissamine rhodamine B; D012710:Serum Albumin, Bovine; C056165:acetovanillone; D002374:Catalase; D013482:Superoxide Dismutase; D019255:NADPH Oxidases; D011493:Protein Kinase C; D001920:Bradykinin; D002118:Calcium