Homocysteine Inhibition of Endothelium-Dependent Nitric Oxide-Mediated Dilation of Porcine Retinal Arterioles via Enhanced Superoxide Production

PURPOSE: Elevated plasma concentration of homocysteine, a sulfur-containing amino acid, is an emerging risk factor for cardiovascular diseases. Recent epidemiologic studies have confirmed that elevated homocysteine levels are associated with ocular vascular diseases; however, the direct effect of homocysteine on ocular microvascular reactivity remains unknown. We investigated whether homocysteine affects endothelium-dependent nitric oxide (NO)-mediated dilation of retinal arterioles and whether oxidative stress and distinct protein kinase signaling pathways are involved in the homocysteine-mediated effect. METHODS: Porcine retinal arterioles were isolated, cannulated, and pressurized without flow in vitro. Diameter changes were recorded using videomicroscopy techniques. RESULTS: Intraluminal treatment with homocysteine (1 mM, 180 minutes) significantly attenuated arteriolar dilation in response to the endothelium-dependent NO-mediated agonists bradykinin and A23187 but not in response to the endothelium-independent NO donor sodium nitroprusside. In the presence of the superoxide scavenger 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPOL), the nicotinamide adenine dinucleotide phosphate-oxidase (NAD(P)H oxidase inhibitor apocynin, p38 kinase inhibitor SB203580, and peroxisome proliferator-activated receptor-γ (PPAR-γ) agonist pioglitazone, the detrimental effect of homocysteine on bradykinin-induced dilation was prevented; however, neither the xanthine oxidase inhibitor allopurinol, the JNK inhibitor SP600125, or pioglitazone with PPAR-γ inhibitor GW9662 had that effect. CONCLUSIONS: Homocysteine inhibits endothelium-dependent NO-mediated dilation in the retinal arterioles by producing superoxide from NAD(P)H oxidase, which appears to be linked with p38 kinase. By impairing endothelium-dependent NO-mediated vasoreactivity, homocysteine potentially facilitates development of retinal vascular diseases. In addition, pioglitazone can prevent homocysteine-induced endothelial dysfunction possibly by activating PPAR-γ.