Antiatherosclerotic effects of small-molecular-weight compounds enhancing endothelial nitric-oxide synthase (eNOS) expression and preventing eNOS uncoupling.
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 N(omega)-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.
Wohlfart
Paulus
P
Department of Pharmacology, Johannes Gutenberg University, Obere Zahlbacher Strasse 67, D-55131 Mainz, Germany. huigeli@uni-mainz.de.
Xu
Hui
H
Endlich
Alexandra
A
Habermeier
Alice
A
Closs
Ellen I
EI
Hübschle
Thomas
T
Mang
Christian
C
Strobel
Hartmut
H
Suzuki
Teri
T
Kleinert
Hartmut
H
Förstermann
Ulrich
U
Ruetten
Hartmut
H
Li
Huige
H
D000818:Animals; D001011:Aorta / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D001057:Apolipoproteins E / Q000235:genetics; D050197:Atherosclerosis / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology; D001549:Benzamides / Q000627:therapeutic use*; D052117:Benzodioxoles / Q000627:therapeutic use*; D002460:Cell Line; D042783:Endothelial Cells / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D007189:Indans / Q000627:therapeutic use*; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D008970:Molecular Weight; D009569:Nitric Oxide / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II / Q000235:genetics / Q000378:metabolism*; D052250:Nitric Oxide Synthase Type III / Q000235:genetics / Q000378:metabolism*; D020011:Protective Agents / Q000627:therapeutic use*; D012333:RNA, Messenger / Q000378:metabolism
C528910:2,2-difluorobenzo(1,3)dioxole-5-carboxylic acid indan-2-ylamide; C528454:4-fluoro-N-indan-2-yl-benzamide; D001057:Apolipoproteins E; D001549:Benzamides; D052117:Benzodioxoles; D007189:Indans; D020011:Protective Agents; D012333:RNA, Messenger; D009569:Nitric Oxide; C496314:NOS3 protein, human; D052247:Nitric Oxide Synthase Type II; D052250:Nitric Oxide Synthase Type III; C496315:Nos3 protein, mouse