Effects of asymmetric dimethylarginine on bovine retinal capillary endothelial cell proliferation, reactive oxygen species production, permeability, intercellular adhesion molecule-1, and occludin expression

摘要

Purpose: Asymmetric dimethylarginine(ADMA), an endogenous competitive inhibitor of nitric oxide synthase,is associated with impaired endothelial dysfunction, such as chronicheart failure, hypertension, diabetes, and pulmonary hypertension. Theeffects of ADMA on cell proliferation, reactive oxygen species (ROS)production, cell permeability, intercellular adhesion molecule-1(ICAM-1), and tight-junction protein occludin levels in bovine retinalcapillary endothelial cells (BRCECs) were investigated. Methods: A cell proliferation assay wasperformed using the novel tetrazolium compound3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazoliumand an electron coupling reagent. Intracellular ROS levels weredetermined using the fluorescent probe CM-H2DCFDA.Horseradish peroxidase was used for a permeability assay. ICAM-1 andtight-junction protein occludin were assessed by western blotting andquantitative real-time PCR. Results: Cell proliferation wassignificantly inhibited by ADMA. ADMA increased intracellular ROSgeneration in BRCECs. The increased ROS production induced by ADMA wasmarkedly inhibited by the angiotensin II receptor-blocker telmisartan,the angiotensin-converting enzyme inhibitor benazepril, the reducedform of nicotinamide-adenine dinucleotide phosphate (NADPH) oxidaseinhibitor diphenyliodonium (DPI), or the antioxidant and free-radicalscavenger N-acetyl-l-cysteine (NAC). ADMA significantly increasedhorseradish peroxidase (HRP) permeability in BRCECs. Benazepril,telmisartan, DPI, and NAC downregulated cell permeability. ADMAmarkedly upregulated ICAM-1 expression in BRCECs, which weredownregulated by telmisartan, DPI, and NAC. ADMA significantlydownregulated occludin expression in BRCECs. Benazepril and telmisartanupregulated occludin expression in BRCECs exposed to ADMA. Conclusions: Our results provide thefirst reported evidence that ADMA has potent adverse effects on cellproliferation, intracellular ROS generation, cell permeability, levelsof ICAM-1, and the tight-junction protein occludin.Angiotensin-converting enzyme inhibitors, angiotensin II receptorblockers, and antioxidants are effective inhibitors of the adverseeffects of ADMA.