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CLINICAL STUDIES

Gene transfer of nitric oxide synthase

Effects on endothelial biology

Josef Niebauer, MD^{* ,1}, J.ózef Dulak, PhD^*,2, Jason R. Chan, BS^*, Philip S. Tsao, PhD^*,3 and John P. Cooke, MD, PhD, FACC^*,4

^* Section of Vascular Medicine, Division of Cardiovascular Medicine, Stanford University, Stanford, California, USA
Herzzentrum der Universität Leipzig, Kardiologie, Leipzig, Germany

Manuscript received September 24, 1998; revised manuscript received May 17, 1999, accepted June 10, 1999.

Reprint requests and correspondence: Dr. John P. Cooke, Director, Vascular Medicine, Falk Cardiovascular Research Center, Stanford University School of Medicine, 300 Pasteur Drive, Stanford, California 94305-5246
John.Cooke{at}stanford.edu

OBJECTIVES

The purpose of the study was to investigate the role of nitric oxide (NO) in monocyte-endothelial interaction by augmenting NO release via transfection of human endothelial cells (ECs) with EC NO synthase (eNOS) DNA.

BACKGROUND

Enhancement of NO synthesis by L-arginine or shear stress reduces endothelial adhesiveness for monocytes and inhibits atherogenesis. To elucidate further the underlying mechanism, we augmented NO synthase expression by transfection of human EC.

METHODS

Liposome-mediated transfection of EC was performed with a plasmid construct containing the gene encoding eNOS. Expression of eNOS was confirmed by reverse transcription–polymerase chain reaction (RT-PCR). Endothelial cells were exposed to human monocytoid cells, and adherent cells were quantitated using a computer-assisted program. Nitric oxide was measured by chemiluminescence.

RESULTS

The NO levels were not different in EC that were either not transfected, transfected with beta-gal or liposomes only. The nitric oxide synthase (NOS) transfection increased NO release by +60% (n = 6), which increased further when EC were stimulated by shear stress (24 h) by +137% (n = 5) as compared with untransfected, unstimulated EC (both p < 0.05). The RT-PCR revealed diminished monocyte chemotactic protein-1 (MCP-1) expression in eNOS transfected EC. There was an inverse relation between NO levels and monocyte binding (r = –0.5669, p < 0.002). Stimulation of EC with tumor necrosis factor-alpha (TNF-alpha; 250 U/ml) led to a decrease in NO synthesis, and an increase in monocyte binding. Cells transfected with NOS were resistant to both effects of TNF-alpha.

CONCLUSIONS

Endothelial cells transfected with eNOS synthesize an increased amount of NO; this is associated with diminished MCP-1 expression and monocyte-endothelial binding. The reduction in monocyte-endothelial binding persists even after cytokine stimulation.

Abbreviations and Acronyms   ANOVA = analysis of variance   EC = endothelial cells   eNOS = endothelial cell nitric oxide synthase   MCP-1 = monocyte chemotactic protein-1   NFB = nuclear factor B   NO = nitric oxide   NOS = nitric oxide synthase   RT-PCR = reverse transcription–polymerase chain reaction   TNF- = tumor necrosis factor-alpha   VCAM = vascular cell adhesion molecule

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