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

Abstract

Purpose: Asymmetric dimethylarginine (ADMA), an endogenous competitive inhibitor of nitric oxide synthase, is associated with impaired endothelial dysfunction, such as chronic heart failure, hypertension, diabetes, and pulmonary hypertension. The effects 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 retinal capillary endothelial cells (BRCECs) were investigated.

Methods: A cell proliferation assay was performed using the novel tetrazolium compound 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium and an electron coupling reagent. Intracellular ROS levels were determined using the fluorescent probe CM-H(2)DCFDA. Horseradish peroxidase was used for a permeability assay. ICAM-1 and tight-junction protein occludin were assessed by western blotting and quantitative real-time PCR.

Results: Cell proliferation was significantly inhibited by ADMA. ADMA increased intracellular ROS generation in BRCECs. The increased ROS production induced by ADMA was markedly inhibited by the angiotensin II receptor-blocker telmisartan, the angiotensin-converting enzyme inhibitor benazepril, the reduced form of nicotinamide-adenine dinucleotide phosphate (NADPH) oxidase inhibitor diphenyliodonium (DPI), or the antioxidant and free-radical scavenger N-acetyl-L-cysteine (NAC). ADMA significantly increased horseradish peroxidase (HRP) permeability in BRCECs. Benazepril, telmisartan, DPI, and NAC downregulated cell permeability. ADMA markedly upregulated ICAM-1 expression in BRCECs, which were downregulated by telmisartan, DPI, and NAC. ADMA significantly downregulated occludin expression in BRCECs. Benazepril and telmisartan upregulated occludin expression in BRCECs exposed to ADMA.

Conclusions: Our results provide the first reported evidence that ADMA has potent adverse effects on cell proliferation, intracellular ROS generation, cell permeability, levels of ICAM-1, and the tight-junction protein occludin. Angiotensin-converting enzyme inhibitors, angiotensin II receptor blockers, and antioxidants are effective inhibitors of the adverse effects of ADMA.

Figure 1

Cell proliferation analysis of bovine retinal capillary endothelial cells. Cell proliferation was significantly inhibited by asymmetric dimethylarginine (ADMA) for 24–72 h. In group A, bovine retinal capillary endothelial cells (BRCECs) were cultured in endothelial cell medium (ECM) with 0.4% fetal bovine serum (FBS). In groups B throught E, BRCECs were cultured in ECM with 0.4% FBS plus 10 μM, 50 μM, 100 μM, or 200 μM ADMA. Results are expressed as absorbance at 490 nm and represent means±SEM, n=8: *p<0.05 versus group A, **p<0.01 versus group A, #p<0.05 versus group B, ##p<0.01 versus group B, +p<0.05 versus group C, ++p<0.01 versus group C, &p<0.05 versus group D, &&p<0.01 versus group D.

Figure 2

Intracellular reactive oxygen species generation in bovine retinal capillary endothelial cells for 24 h, as determined using the fluorescent probe CM-H₂DCFDA. symmetric dimethylarginine (ADMA) increased intracellular reactive oxygen species (ROS) generation in bovine retinal capillary endothelial cells (BRCECs). The increased reactive oxygen species (ROS) production induced by asymmetric dimethylarginine (ADMA) was markedly inhibited by benazepril, telmisartan, diphenyliodonium, or N-acetyl-l-cysteine. In group C, BRCECs were cultured in endothelial cell medium with 0.4% fetal bovine serum. In group A, BRCECs were cultured in endothelial cell medium with 0.4% fetal bovine serum plus ADMA (100 μM). In group “A+B,” BRCECs were cultured in the same media as A and 10 μM benazepril. In group “A+T,” BRCECs were cultured in the same media as A and 10 μM telmisartan. In group “A+D,” BRCECs were cultured in the same media as A and 10 μM diphenyliodonium. In group “A+N,” BRCECs were cultured in the same media as group A and 10 mM N-acetyl-l-cysteine (mean±SD, n=3). **p<0.01 versus group C, ##p<0.01 versus group A.

Figure 3

Horseradish peroxidase (HRP) permeability assay of bovine retinal capillary endothelial cells (BRCECs). Asymmetric dimethylarginine (ADMA) significantly increased HRP permeability in BRCECs. Treatment with benazepril and NAC for 15 min decreased HRP permeability in BRCECs. The permeability increase by ADMA was significantly downregulated by benazepril, telmisartan, diphenyliodonium (DPI), or N-acetyl-l-cysteine (NAC) for 30 min, 45 min, and 1 h. The group C bars are that BRCECs were cultured in endothelial cell medium (ECM) with 0.4% FBS. In group A, BRCECs were cultured in ECM with 0.4% FBS plus ADMA (100 μM). In group “A+B,” BRCECs were cultured in the same media as A and 10 μM benazepril. Group “A+T” was the same as for group A and 10 μM telmisartan. In group “A+D,” BRCECs were cultured in the same media as group A and 10 μM DPI. In group “A+N,” BRCECs were cultured in the same media as group A and 10 mM NAC (mean±SD, n=3). *p<0.05 versus group C, **p<0.01 versus group C, #p<0.05 versus group A, ##p<0.01 versus group A.

Figure 4

Intercellular adhesion molecule-1 (ICAM-1) protein expression in bovine retinal capillary endothelial cells. A: western blotting analysis showing the presence of ICAM-1 protein in bovine retinal capillary endothelial cells (BRCECs). B: Results of statistical analysis of protein levels relative to β-actin. Asymmetric dimethylarginine (ADMA) markedly upregulated ICAM-1 protein expression in BRCECs. Telmisartan, diphenyliodonium (DPI), or N-acetyl-l-cysteine (NAC) downregulated ICAM-1 protein expression in BRCECs exposed to ADMA. Benazepril had no effect on ICAM-1 expression. In group C, BRCECs wrre cultured in endothelial cell medium (ECM) with 0.4% fetal bovine serum (FBS). In group A, BRCECs were cultured in ECM with 0.4% FBS plus ADMA (100 μM). In group “A+B,” BRCECs were cultured in the same media as A and 10 μM benazepril. In group “A+T,” BRCECs were cultured in the same media as group A and 10 μM telmisartan. In group “A+D,” BRCECs were cultured in the same media as group A and 10 μM DPI. In group “A+N,” BRCECs were cultured in the same media as A and 10 mM NAC (mean±SD, n=3). **p<0.01 versus C, ##p<0.01 versus group A.

Figure 5

Occludin protein expression in bovine retinal capillary endothelial cells. A: western blotting analysis showing the presence of occludin protein in bovine retinal capillary endothelial cells (BRCECs). B: The results of statistical analysis of protein level relative to β-actin. Asymmetric dimethylarginine (ADMA) significantly downregulated occludin protein expression in BRCECs. Benazepril and telmisartan upregulated protein expression of occludin in BRCECs exposed to ADMA. However, diphenyliodonium (DPI) and N-acetyl-l-cysteine (NAC) had no effect on occludin expression. In group C, BRCECs were cultured in ECM with 0.4% fetal bovine serum (FBS). In group A, BRCECs were cultured in ECM with 0.4% FBS plus ADMA (100 μM). Group “A+B” represents same as for group A and 10 μM benazepril. Group “A+T” represents same as for group A and 10 μM telmisartan. Group “A+D” represents same as for group A and 10 μM DPI. Group “A+N” represents same as for group A and 10 mM NAC (mean±SD, n=3). **p<0.01 versus group C, ##p<0.01 versus group A.

Figure 6

The statistical analysis results of bovine retinal capillary endothelial cells (BRCECs) intercellular adhesion molecule-1 (ICAM-1) gene expression relative to 18S rRNA for 24 h. Asymmetric dimethylarginine (ADMA) markedly upregulated ICAM-1 mRNA expression in BRCECs for 24 h. Benazepril, telmisartan, diphenyliodonium (DPI), or N-acetyl-l-cysteine (NAC) downregulated ICAM-1 mRNA expression in BRCECs exposed to ADMA. In group C, BRCECs were cultured in endothelial cell medium (ECM) with 0.4% fetal bovine serum (FBS). In group A, BRCECs were cultured in ECM with 0.4% FBS plus ADMA (100 μM). “A+B” represents same as for group A and 10 μM benazepril. In group “A+T,” BRCECs were cultured in the same media as group A and 10 μM telmisartan. In group “A+D,” BRCECs were cultured in the same media as group A and 10 μM DPI. In group “A+N,” BRCECs were cultured in the same media as group A and 10 mM NAC (mean±SD, n=3). **p<0.01 versus group C, ##p<0.01 versus group A.

Figure 7

Results of statistical analysis of occludin gene expression relative to 18S rRNA in bovine retinal capillary endothelial cells (BRCECs) for 24 h. ADMA downregulated occludin mRNA expression in BRCECs for 24 h. Benazepril, telmisartan, or diphenyliodonium (DPI) upregulated occludin mRNA level. However, N-acetyl-l-cysteine (NAC) had no effect on occludin mRNA level. In group C, BRCECs were cultured in endothelial cell medium (ECM) with 0.4% fetal bovine serum (FBS). In group A, BRCECs were cultured in ECM with 0.4% FBS plus ADMA (100 μM). In group “A+B,” BRCECs were cultured in the same media as group A and 10 μM benazepril. In “A+T,” BRCECs were cultured in the same media as group A and 10 μM telmisartan. In group “A+D,” BRCECs were cultured in the same media as group A and 10 μM DPI. In group “A+N,” BRCECs were cultured in the same media as group A and 10 mM NAC (mean±SD, n=3). *p<0.05 versus group C, #p<0.05 versus group A.