e46 14th IVBM Abstracts

through the binding of the homeodomain transcription factorNkx2.5. Subsequent investigation revealed that the repressordEF1 competes with the activator Nkx2.5 for an overlappingsite. No Nkx2.5 message or protein was expressed in normalvessels, however both were detected in atherosclerotic lesions.Staining revealed Nkx2.5 was localised within the atheroscle-rotic fibrous cap and in cells within the media.Conclusion: In atherosclerotic lesions Col1a2 is regulated by anenhancer element, activated by the homeobox transcriptionfactor Nkx2.5.

doi:10.1016/j.vph.2006.08.158

A6.03

Nitric oxide reduces transcriptional promoter activity ofSLC29A1 for human equilibrative nucleoside transporter 1in umbilical vein endothelium from gestational diabetes

Marcelo Farias1, Rody San Martin1, Marcal Pastor-Anglada2,Paola Casanello1, Luis Sobrevia1

1CMPL, Obstet. Ginecol. Pontificia Universidad Catolica de Chile,Santiago, Chile2Universitat de Barcelona, Barcelona, Spain

The endogenous vasodilator adenosine is primarily taken upby equilibrative nucleoside transporters 1 (hENT1) in humanumbilical vein endothelium (HUVEC)(San Martin and Sobre-via, 2006). hENT1 expression and activity are reduced inHUVEC from pregnancies with gestational diabetes, an effectassociated with reduced hENT1 mRNA level and increasednitric oxide (NO) synthesis (Vásquez et al. 2004). We nowstudied whether NO modulates hENT1 promoter activity inprimary cultures of HUVEC. hENT1 mRNA and protein levelswere measured in cultured (passage 2) cells isolated from normalor gestational diabetic pregnancies (Ethic Committee approvaland informed patient consent obtained). Cells were exposed toNG-nitro-L-arginine methyl ester (L-NAME, 100 μM), S-nitroso-N-acetyl-L,D-penicillamine (SNAP, 10 μM) or infectedwith an adenovirus-eNOS siRNA (Ad-eNOS). hENT1 promoteractivity was evaluated in cells transfected (electroporation,320 V, 30 ms) with pGL3 reporter constructs carrying −3100,−2056, −1016 or −69 bp from ATG of promoter sequence.hENT1mRNA and protein levels were reduced in HUVEC fromgestational diabetes, an effect blocked by L-NAME or in cellsinfected with Ad-eNOS, and unaltered by SNAP. Ad-eNOStreated cells exhibit reduced mRNA and protein levels.However, SNAP mimicked the effect of gestational diabetes incells from normal pregnancies. Cells from gestational diabetestransfected with hENT1 promoter region spanning −1016 to−697 bp show a higher (1.8-fold, n=4, P=0.0008) firefly/renillaluciferase activity comparedwith cells from normal pregnancies.However the region −2056 to −1016 bp shows a repressoractivity, which was blocked by L-NAME, in gestational diabetes.Thus, we suggest a transcriptional role for NO in the reduction of

hENT1 expression observed in HUVEC from gestationaldiabetes. Vázquez et al. (2004). J Physiol 560, 111–122. SanMartín R and Sobrevia L (2006). Placenta 27, 1–10.Supportedby FONDECYT 1030781 and 1030607 (Chile). M. F. holdsCONICYT and Faculty of Medicine-PhD (Chile) fellowships.

doi:10.1016/j.vph.2006.08.159

A6.04

Injury-inducible yin yang-1 inhibits vascular smoothmuscle cell growth and intimal thickening by repressingP21WAF1/CIP1 transcription and perturbing cyclinD1-CDK4-P21WAF1/CIP1 assembly

Fernando S. Santiago*,1, Hideto Ishii1, Mark D. Hulett2,Alexander Bobik3, John F. Martin4, Colin N. Chesterman1,Ian C. Zachary4, Levon M. Khachigian1

1Centre for Vascular Research, Sydney, Australia2John Curtin School of Medical Research, Canberra, Australia3Baker Heart Research Institute, Melbourne, Australia4Centre for Cardiovascular Biology and Medicine, London,United Kingdom

Vascular injury initiates a cascade of phenotype-alteringmolecular events. These involve key transcription regulators,although their regulatory functions in the injured blood vesselwall are poorly understood. Here we show that the injury-inducible GLI-Krüppel zinc finger transcription factor, yin yang-1 (YY1) inhibits vascular smooth muscle cell (SMC) prolifer-ation and neointima formation in rabbit and rat blood vessels. Itsuppressed expression of the cell cycle regulator p21WAF1/Cip1, proliferating cell nuclear antigen (PCNA) and Rbphosphorylation. YY1 inhibition of SMC proliferation wasrescued by concurrent overexpression of p21WAF1/Cip1.Conversely, YY1 failed to inhibit SMC growth in SMCstransfected with antisense p21WAF1/Cip1. YY1 blockedp21WAF1/Cip1-Cdk4-cyclin D1 complex assembly andpRbSer249/Thr252 phosphorylation in SMCs, but not inendothelial cells (ECs), in which YY1 did not repressp21WAF1/Cip1 expression or proliferation. YY1 knockdownincreased SMC growth and neointima formation with aconcomitant increase in p21WAF1/Cip1, PCNA andpRbSer249/Thr252. YY1 (either wild-type or GST fusionprotein) binds Sp1 and prevents its occupancy of atypical Sp1recognition element (-1375CCTCCC-1370) in the authenticp21WAF1/Cip1 promoter without YY1 itself binding thepromoter. YY1 perturbed the Sp1-DNA interaction via the C-terminal region of Sp1 (residues 620–778). YY1 inhibition ofcell growth was not confined to SMCs. It also blockedmammaryadenocarcinoma growth in rats, abrogating p21WAF1/Cip1,PCNA and pRbSer249/Thr252 levels in MAT cells and solidtumors without affecting microvascular density. These findingsdefine a new role for YY1 as an indirect repressor oftranscription and p21WAF1/Cip1-Cdk4-cyclin D1 complex