Abstract
Many cardiovascular diseases are associated with reduced levels of bioactive nitric oxide (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, 4-fluoro-N-indan-2-yl-benzamide (CAS no. 291756-32-6; empirical formula C16H14FNO; AVE9488) and 2,2-difluoro-benzo[1,3]dioxole-5-carboxylic acid indan-2-ylamide (CAS no. 450348-85-3; empirical formula C17H13F2NO3; AVE3085), enhanced eNOS promoter activity in a concentration-dependent manner; with the responsible cis-element localized within the proximal 263 base pairs of the promoter region. RNA interference-mediated knockdown of the transcription factor Sp1 significantly reduced the basal activity of eNOS promoter, but it 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 nitric-oxide inhibitor Nω-nitro-l-arginine methyl ester, 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 up-regulated eNOS expression and a reversal of eNOS uncoupling is probably responsible for the observed vasoprotective properties of this new type of compounds.
Footnotes
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H.L. and U.F. received grants from the Deutsche Forschungsgemeinschaft, Bonn, Germany (Collaborative Research Center SFB 553, project A1). P.W., T.H., H.S., T.S., and H.R. are employees of sanofi-aventis and involved in early identification and characterization of new chemical entities.
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Parts of this work are taken from the following: Endlich A (2008) Investigation to Explore Targets and Mechanisms of eNOS Transcription Enhancers. Ph.D. thesis, Johann Wolfgang Goethe University of Frankfurt, Germany.
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
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doi:10.1124/jpet.107.128009.
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ABBREVIATIONS: NO, nitric oxide; eNOS, endothelial NO synthase; apoE-KO, apolipoprotein E-knockout; AVE9488, 4-fluoro-N-indan-2-yl-benzamide, CAS no. 291756-32-6, empirical formula C16H14FNO; AVE3085, 2,2-difluoro-benzo[1,3]dioxole-5-carboxylic acid indan-2-ylamide, CAS no. 450348-85-3, empirical formula C17H13F2NO3; HUVEC, human umbilical vein endothelial cell(s); kb, kilobase(s); PBS, phosphate-buffered saline; bp, base pair(s); EMSA, electrophoretic mobility shift assay; siRNA, small interfering RNA; TBST, Tris-buffered saline/Tween 20; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; SOD, superoxide dismutase; GCH1, guanosine triphosphate (GTP)-cyclohydrolase-I; sGC, soluble guanylate cyclase; eNOS-KO, eNOS-knockout; DKO, double knockout; apoE/eNOS-DKO, apoE/eNOS-double knockout; ROS, reactive oxygen species; l-012, 8-amino-5-chloro-7-phenylpyrido[3,4-d]pyridazine-1,4(2H,3H)dione; l-NAME, NG-nitro-l-arginine methyl ester; BH4,(6R)-5,6,7,8-tetrahydro-l-biopterin; DRB, 5,6-dichlorobenzimidazole riboside; EST, expressed sequence tag; RT-PCR, reverse transcription-polymerase chain reaction.
- Received July 6, 2007.
- Accepted January 29, 2008.
- The American Society for Pharmacology and Experimental Therapeutics
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