An endogenous inhibitor of nitric oxide synthase regulates endothelial adhesiveness for monocytes
Rainer H. Böger, MDa,
Stefanie M. Bode-Böger, MDa,
Philip S. Tsao, PhDa,
Patrick S. Lin, BSa,
Jason R. Chan, BSa and
John P. Cooke, MD, PhDa
a Section of Vascular Medicine, Stanford University School of Medicine, Stanford, California, USA
View larger version (17K):
[in a new window]
|
Figure 1 (A) The synthesis of ADMA by the cultured human endothelial cell line ECV 304. Data are mean ± SEM for n = 5 to 6 separate experiments. The synthesis rates of ADMA and SDMA were calculated from the mean differences in dimethylarginine concentrations between the experimental days as 0.34 ± 0.17 µmol ADMA/107 cells/24 h and 0.29 ± 0.14 µmol SDMA/107 cells/24 h. (B) The effect of incubating endothelial cells with nLDL or oxLDL on ADMA elaboration. Concentrations of lipoproteins are given as mg/dL in legend. Data are mean of n = 4 to 6 separate experiments. *p < 0.05 versus control. ADMA = asymmetric dimethylarginine; nLDL = native low-density lipoprotein; oxLDL = oxidized low-density lipoprotein; SDMA = symmetric dimethylarginine.
|
|
View larger version (17K):
[in a new window]
|
Figure 2 The release of NOx from ECV 304 human endothelial cells during 4 h of incubation in the presence or absence of ADMA, SDMA or ADMA + L-arginine (1 mmol/L). Cells were stimulated with calcium ionophore A23187 (1 µmol/L). Values are expressed as percent of the NOx level in conditioned medium from control cells incubated with vehicle alone. Data are mean ± SEM of n = 5 to 6 identical experiments. ADMA = asymmetric dimethylarginine; NOx = nitrogen oxides; SDMA = symmetric dimethylarginine.
|
|
View larger version (42K):
[in a new window]
|
Figure 3 The release of superoxide radicals from ECV 304 human endothelial cells. Cells were incubated for 24 h with the substances indicated before the addition of PMA (2 µmol/L). Data are mean ± SEM of n = 3 to 4 experiments performed in duplicate. ADMA = asymmetric dimethylarginine; nLDL = native low-density lipoprotein; oxLDL = oxidized low-density lipoprotein; SDMA = symmetric dimethylarginine.
|
|
View larger version (20K):
[in a new window]
|
Figure 4 The effect of preincubating ECV 304 human endothelial cells with ADMA, SDMA or ADMA in the presence of 1 mmol/L L-arginine on the adhesion of THP-1 human monocytoid cells. Endothelial cells were coincubated with monocytoid cells for 30 min on a rocking platform. Adhering cells were fixed in glutaraldehyde and counted using a computer-aided image analysis system. Data are mean ± SEM of three separate experiments performed in triplicate. ADMA = asymmetric dimethylarginine; SDMA = symmetric dimethylarginine.
|
|
View larger version (50K):
[in a new window]
|
Figure 5 Effect of coincubation with L-arginine (1 mmol/L) or with a neutralizing anti-MCP-1 antibody on the effects of ADMA, nLDL (100 mg/dL) or oxLDL (3 mg/dL) on the adhesiveness of ECV 304 human endothelial cells for THP-1 human monocytoid cells. ADMA = asymmetric dimethylarginine; MCP-1 = monocyte chemotactic protein; nLDL = native low-density lipoprotein; oxLDL = oxidized low-density lipoprotein. * = significantly different from vehicle treated control endothelial cells, p < 0.05;  = significantly different from endothelial cells treated with lipoprotein or ADMA.
|
|
View larger version (48K):
[in a new window]
|
Figure 7 Release of monocyte chemotactic protein from ECV 304 human endothelial cells during a 24 h incubation in the presence or absence of the substances indicated. Data are mean ± SEM of n = 4 to 6 experiments measured in duplicate. * = significantly different from vehicle treated control endothelial cells, p < 0.05; = significantly different from endothelial cells treated with ADMA (10–4 m) p < 0.05.
|
|
|
B. Nuclear extracts were isolated from ECV 304 human endothelial cells after incubation with the various substances for 24 h. Binding reactions were carried out by mixing nuclear proteins with a 32P-labeled double-stranded DNA oligonucleotide corresponding to the published NF-