Diabetes-induced activation of caspase-3 in retina: effect of antioxidant therapy.
Apoptosis of retinal endothelial cells and pericytes is postulated to contribute to the development of retinopathy in diabetes. The goal of this study is to investigate diabetes-induced activation of retinal caspase-3, an apoptosis executer enzyme, in retina, and examine the effects of antioxidants on the activation. Caspase-3 activation was determined in the retina of alloxan diabetic rats (2-14 months duration) and in the isolated retinal capillary cells (endothelial cells and pericytes) by measuring cleavage of caspase-3 specific fluorescent substrate, and cleavage of caspase-3 holoenzyme and poly (ADP ribosyl) polymerase. Effect of antioxidants on the activation of caspase-3 was determined by feeding a group of diabetic rats diet supplemented with a comprehensive mixture of antioxidants, including Trolox, alpha-tocopherol, N-acetyl cysteine, ascorbic acid, beta-carotene and selenium for 2-14 months, and also under in vitro conditions by incubating isolated retinal capillary cells with antioxidants with wide range of actions. Caspase-3 was activated in the rat retina at 14 months of diabetes (P < 0.05 vs. normal), but not at 2 months of diabetes, and administration of antioxidants for the entire duration inhibited this activation. In the isolated retinal capillary cells incubated in 25 mM glucose medium, caspase-3 activity was increased by 50% compared to the cells incubated in 5 mM glucose (P < 0.02), and antioxidants or caspase-3 inhibitor inhibited this increase. Our results suggest that increased oxidative stress in diabetes is involved in the activation of retinal caspase-3 and apoptosis of endothelial cells and pericytes. Antioxidants might be inhibiting the development of diabetic retinopathy by inhibiting microvascular apoptosis.
Kowluru
Renu A
RA
Kresge Eye Institute, Wayne State University, 4717 St. Antoine, Detroit, MI 48201, USA. rkowluru@med.wayne.edu
Koppolu
Prashant
P
D000111:Acetylcysteine / Q000008:administration & dosage; D000818:Animals; D000975:Antioxidants / Q000627:therapeutic use*; D017209:Apoptosis; D001205:Ascorbic Acid / Q000008:administration & dosage; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism*; D003921:Diabetes Mellitus, Experimental / Q000201:enzymology*; D003930:Diabetic Retinopathy / Q000201:enzymology / Q000473:pathology / Q000517:prevention & control*; D004730:Endothelium, Vascular / Q000201:enzymology / Q000473:pathology; D004789:Enzyme Activation; D005947:Glucose / Q000378:metabolism; D066298:In Vitro Techniques; D008297:Male; D018384:Oxidative Stress; D020286:Pericytes / Q000201:enzymology / Q000473:pathology; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D012160:Retina / Q000201:enzymology*; D012643:Selenium / Q000008:administration & dosage; D017392:Thiobarbituric Acid Reactive Substances / Q000378:metabolism; D024502:alpha-Tocopherol / Q000008:administration & dosage; D019207:beta Carotene / Q000008:administration & dosage
D000975:Antioxidants; D017392:Thiobarbituric Acid Reactive Substances; D019207:beta Carotene; D011065:Poly(ADP-ribose) Polymerases; C505410:Casp3 protein, rat; D053148:Caspase 3; D020169:Caspases; D024502:alpha-Tocopherol; D012643:Selenium; D005947:Glucose; D001205:Ascorbic Acid; D000111:Acetylcysteine