Decoy oligodeoxynucleotide characterization of transcription factors controlling endothelin-B receptor expression in vascular smooth muscle cells.

Endothelin-1 is not only a powerful vasoconstrictor but also a potent mitogen for vascular smooth muscle cells (SMC), acting through both the endothelin-A and endothelin-B receptor (ET(B)-R). Although vascular SMC are known to express the ET(B)-R, its transcriptional regulation has not been studied thus far. Here we demonstrate that the potent inhibitor of nuclear factor kappaB activation, pyrrolidine dithiocarbamate (PDTC; 30-100 microM), induces de novo ET(B)-R expression in rat aortic and mesenteric cultured SMC. Electrophoretic mobility shift analyses revealed that besides inhibition of nuclear factor kappaB, PDTC enhances activator protein-1 (AP-1), CCAAT/enhancer-binding protein (C/EBP), and GATA-2 activity in these cells. Preincubation of PDTC-stimulated cells with appropriate decoy oligodeoxynucleotides confirmed the involvement of these three transcription factors, namely that of AP-1, in ET(B)-R expression. The stimulatory effect of PDTC on ET(B)-R expression was also confirmed functionally by monitoring an enhanced ET-1-induced apoptosis in PDTC-treated cells that was sensitive to the ET(B)-R antagonist, BQ788. Taken together, these findings demonstrate that C/EBP, GATA-2, and in particular AP-1 can control ET(B)-R expression in vascular SMC. They further support the notion that ET(B)-R expression in these cells may play an important role in cardiovascular complications, such as restenosis following angioplasty that in the early phase is characterized by prominent SMC apoptosis.

Oxidized low density lipoprotein inhibits inducible nitric oxide synthase, GTP cyclohydrolase I and transforming growth factor beta gene expression in rat macrophages.

Several studies have already demonstrated that oxidized- LDL decreases nitric oxide (NO) generation by cytokine-stimulated macrophages. However, the mechanisms of such an inhibition have not been yet elucidated. NO generation by inducible nitric oxide synthase (iNOS) is dependent on the presence of cofactors for NO generation, tetrathydrobiopterin (BH4) among them. The NO generation by these cells is also regulated by some endogenous inhibitors, like TGF-beta. Therefore, the aim of our recent study was to investigate the influence of ox-LDL on the expression of iNOS and GTP cyclohydrolase I (GTP-CH I), the key enzyme involved in the BH4 synthesis as well as the ox-LDL effect on TGF-beta expression in rat macrophages stimulated with IFNgamma (250 U/ml) and LPS (500 ng/ml). Macrophages, activated in this way, express iNOS, GTP-CH I, and TGF-beta mRNA. This expression was inhibited when the macrophages were preincubated for 24 hours with ox-LDL (100 microg/ml). Quantitative PCR revealed about 10-fold inhibition of iNOS gene expression by ox-LDL. As a consequence of down-regulation of iNOS and GTP-CH I genes, almost 3-fold diminished generation of NO2- by rat macrophages was observed. An inhibition of the TGFbeta mRNA expression was also found. Our studies indicate that decreased NO generation by ox-LDL treated macrophages may be the result of the diminished expression of both iNOS and GTP-CH I genes. This effect may be mediated by the activity of certain endogenous inhibitors of gene expression, however, our studies exclude the TGF-beta as a candidate for this activity.

Cytokine-inducible CD40 gene expression in vascular smooth muscle cells is mediated by nuclear factor kappaB and signal transducer and activation of transcription-1.

The interaction of T-lymphocytes expressing the CD40 ligand (CD154) and cells of the vessel wall expressing the corresponding receptor protein (CD40) may play an important role in chronic inflammation including arteriosclerosis. One way of interfering with CD40-CD154 signalling is to prevent CD40 expression, the regulation of which, however, has yet to be elucidated. Therefore, we studied CD40 expression in rat aortic cultured smooth muscle cells. Both CD40 mRNA and protein expression in these cells was markedly enhanced as early as 6 h after exposure to different pro-inflammatory cytokines. Experiments with actinomycin D and subsequent run-on analyses revealed that CD40 expression in response to these cytokines was regulated at the level of transcription. Moreover, electrophoretic mobility shift analyses along with the employment of transcription factor decoy oligodeoxynucleotides demonstrated that tumor necrosis factor alpha via nuclear kappaB and interferon-gamma via signal transducer and activator of transcription-1 up-regulate CD40 gene expression in rat aortic cultured smooth muscle cells.

Influence of oxy-LDL and interleukin-8 on the platelet/PMNs adhesion and on chemotaxis-mediated induction of iNOS in neutrophils.

Selectin-P expression on platelets stimulated with thrombin was measured in terms of formation of "rosettes" according to Jungi et al. [9]. Selectin-P-mediated platelet/PMNs adhesion was inhibited by iloprost (IC50 = 5.0 nM), sodium nitroprusside (NaNP, IC50 = 0.93 microM) and interleukin-8 (IL-8, IC50 = 88 ng/ml), but activated dose-dependently by oxy-LDL (3-15 micrograms/ml). Glycogen-induced peritonitis in rats up-regulated the iNOS activity measured by the 2,3-[3H]-citrulline formation by the abdominal cavity PMNs up to 6 h after insult. Pretreatment with IL-8 (3 micrograms/300 g iv) decreased the amount of PMNs in ascites as well as its iNOS activity. Chemotaxis mediated iNOS gene expression of PMNs were measured by Northern blot hybridization. IL-8 (100 ng/ml) did not influence the PMNs iNOS gene expression induced by chemotaxis. We conclude that the decrease in iNOS activity by IL-8 involves postranslational modification of the enzyme activity.