Received for publication July 6, 2007.
Revised January 28, 2008.
Accepted for publication January 29, 2008.

Anti-atherosclerotic effects of small molecular weight compounds enhancing eNOS expression and preventing eNOS uncoupling

Abstract

Many cardiovascular diseases are associated with reduced levels of bioactive NO and an uncoupling of oxygen reduction from NO synthesis in endothelial NO synthase (eNOS uncoupling). In human endothelial EA.hy 926 cells, two small molecular-weight compounds with related structures, AVE9488 and AVE3085, enhanced eNOS promoter activity in a concentration-dependent manner; with the responsible cis-element localized within the proximal 263bp of the promoter region. RNA interference-mediated knockdown of the transcription factor Sp1 significantly reduced the basal activity of eNOS promoter, but did not prevent the transcription activation by the compounds. Enhanced transcription of eNOS by AVE9488 in primary human umbilical vein endothelial cells was associated with increased levels of eNOS mRNA and protein expression, as well as increased bradykinin-stimulated NO production. In both wild type C57BL/6J mice and apolipoprotein E-knockout (apoE-KO) mice, treatment with AVE9488 resulted in enhanced vascular eNOS expression. In apoE-KO mice, but not in eNOS-knockout mice, treatment with AVE9488 reduced cuff-induced neointima formation. A 12-week treatment with AVE9488 or AVE3085 reduced atherosclerotic plaque formation in apoE-KO mice, but not in apoE/eNOS-double knockout mice. Aortas from apoE-KO mice showed a significant generation of reactive oxygen species. This was partly prevented by NOS inhibitor L-NAME indicating eNOS uncoupling. Treatment of mice with AVE9488 enhanced vascular content of the essential eNOS cofactor (6R)-5,6,7,8-tetrahydro-L-biopterin and reversed eNOS uncoupling. The combination of an upregulated eNOS expression and a reversal of eNOS uncoupling is likely to be responsible for the observed vaso-protective properties of this new type of compounds.

Key words: atherosclerosis, cardiovascular disease, eNOS uncoupling, endothelium, gene expression, nitric oxide synthase