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EXPERIMENTAL STUDY

An endogenous inhibitor of nitric oxide synthase regulates endothelial adhesiveness for monocytes

^a Section of Vascular Medicine, Stanford University School of Medicine, Stanford, California, USA

Manuscript received August 10, 1999; revised manuscript received June 16, 2000, accepted August 16, 2000.

Reprint requests and correspondence to: Dr. John P. Cooke, Division of Cardiovascular Medicine, Stanford University School of Medicine, 300 Pasteur Drive, Stanford, California 94305-5406
john.cooke{at}stanford.edu

OBJECTIVES

We sought to determine whether asymmetric dimethylarginine (ADMA) inhibits nitric oxide (NO) elaboration in cultured human endothelial cells and whether this is associated with the activation of oxidant-sensitive signaling mediating endothelial adhesiveness for monocytes.

BACKGROUND

Endothelial NO elaboration is impaired in hypercholesterolemia and atherosclerosis, which may be due to elevated concentrations of ADMA, an endogenous inhibitor of NO synthase.

METHODS

Human umbilical vein endothelial cells (ECV 304) and human monocytoid cells (THP-1) were studied in a functional binding assay. Nitric oxide and superoxide anion (O₂^–) were measured by chemiluminescence; ADMA by high pressure liquid chromatography; monocyte chemotactic protein-1 (MCP-1) by ELISA and NF-B by electromobility gel shift assay.

RESULTS

Incubation of endothelial cells with ADMA (0.1 µM to 100 µM) inhibited NO formation, which was reversed by coincubation with L-arginine (1 mM). The biologically inactive stereoisomer symmetric dimethylarginine did not inhibit NO release. Asymmetric dimethylarginine (10 µM) or native low-density lipoprotein cholesterol (100 mg/dL) increased endothelial O₂^– to the same degree. Asymmetric dimethylarginine also stimulated MCP-1 formation by endothelial cells. This effect was paralleled by activation of the redox-sensitive transcription factor NF-B. Preincubation of endothelial cells with ADMA increased the adhesiveness of endothelial cells for THP-1 cells in a concentration-dependent manner. Asymmetric dimethylarginine-induced monocyte binding was diminished by L-arginine or by a neutralizing anti-MCP-1 antibody.

CONCLUSIONS

We concluded that the endogenous NO synthase inhibitor ADMA is synthesized in human endothelial cells. Asymmetric dimethylarginine increases endothelial oxidative stress and potentiates monocyte binding. Asymmetric dimethylarginine may be an endogenous proatherogenic molecule.

Abbreviations and Acronyms   ADMA = asymmetric dimethylarginine   BHT = butylated hydroxytoluene   DDAH = dimethylarginine dimethylaminohydrolase   ECV 304 = a line of transformed human umbilical vein endothelial cells   ELISA = enzyme-linked immunosorbent assay   HBSS = Hanks balanced salt solution   HPLC = high-pressure liquid chromatography   LDL = low-density lipoprotein   LNMMA = L, N-monomethylarginine   MCP-1 = monocyte chemotactic protein   nLDL = native low-density lipoprotein   NO = nitric oxide   OPA = o-phthaldialdehyde   oxLDL = oxidized low-density lipoprotein   SDMA = symmetric dimethylarginine   THP-1 = a line of human monocytoid cells

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