We investigated the mechanisms by which H2O2 increases intercellular adhesion molecule 1 (ICAM-1; CD54) expression in endothelial cells. The H2O2-induced increase in ICAM-1 mRNA was inhibited by actinomycin D, by the antioxidant N-acetylcysteine, and by 3-amino-benzamide (which blocks oxidant-induced AP-1 activity), but not by pyrrolidine dithiocarbamate (which blocks oxidant-induced NF-kappa B activity). Nuclear run-on and transient transfections of ICAM-1 promoter constructs indicated that H2O2 stimulated ICAM-1 gene transcription by activation of a distinct region of the ICAM-1 promoter. The H2O2-responsive element was localized to sequences between -981 and -769 (relative to start codon). Located within this region are two 16-base pair repeats, each containing binding sites for the transcription factors AP-1 and Ets. A similar composite AP-1/Ets element isolated from the macrophage scavenger receptor gene conferred H2O2 responsiveness to a minimal promoter. Mutation of the 16-base pair repeats within the ICAM-1 promoter prevented H2O2-induced DNA binding activity, and their deletion abrogated the H2O2-induced transcriptional activity. In contrast, TNF alpha induced ICAM-1 transcription via activation of promoter sequences between -393 and -176, a region with C/EBP and NF-kappa B binding sites. The results indicate that H2O2 activates ICAM-1 transcription through AP-1/Ets elements within the ICAM-1 promoter, which are distinct from NF-kappa B-mediated ICAM-1 expression induced by TNF alpha.
Reactive oxygen radicals (ROS) generated by phagocytes promote human polymorphonuclear leukocyte (PMN) adhesion to human umbilical vein endothelial cells (EC). We determined the effects of hydrogen peroxide (H2O2), a phagocyte-derived ROS, on EC adhesiveness by determining steady-state intracellular adhesion molecule 1 (ICAM-1) mRNA and ICAM-1 protein expression. The adhesion of PMN to H2O2-treated EC was concentration dependent with maximal adhesion achieved at 0.1 mM H2O2. PMN adhesion occurred rapidly, reaching its maximum value within a 1-h treatment time. The PMN adhesion was dependent on the interaction between CD11/CD18 integrins on PMN and ICAM-1 on EC, since either anti-CD18 or anti-ICAM-1 monoclonal antibodies (MAbs) inhibited (by > 90%) the adhesive interaction between PMN and EC. In parallel with the increases in EC adhesivity, we detected a two- to threefold increase in EC surface expression of ICAM-1 between 0.5 and 1 h after H2O2 challenge. Northern analysis revealed an increase in the steady-state ICAM-1 mRNA signal within 0.5 h after H2O2 exposure, and the response was sustained up to 2 h. Inhibition of intracellular catalase in H2O2-treated EC by 3-amino-1,2,3-triazole augmented the PMN adhesion by 20%, whereas enhancement of EC H2O2-scavenging activity by addition of catalase abrogated the H2O2-induced PMN adhesion, indicating that oxidant-antioxidant balance at the EC interface is a critical factor modulating PMN-EC adhesive interactions. The results suggest that H2O2-induced PMN adhesion is dependent on the rapid induction of the ICAM-1 mRNA signal and the surface expression of ICAM-1 on EC.(ABSTRACT TRUNCATED AT 250 WORDS)
Endothelin-1 (ET-1), a 21-amino acid peptide released from the endothelium, elicits a variety of biological effects that include vascular smooth muscle cell (VSMC) contraction, release of secondary mediators, and cell proliferation. The present study was undertaken to examine the proliferative potential of ET-1 toward pulmonary artery VSMC in culture. In the presence of low serum and epidermal growth factor (EGF), ET-1 stimulated marked DNA synthesis and proliferation of VSMC. The contributing factor from serum appeared to be platelet-derived growth factor (PDGF) because the antibody to PDGF eliminated the stimulatory activity. The antibody to EGF also prevented the stimulation, suggesting that both PDGF and EGF are required for the full expression of the VSMC growth-promoting activity of ET-1. A paradoxical aspect of ET-1 effect on VSMC was the ability of ET-1 to inhibit the EGF-stimulated DNA synthesis when the two factors were added together to a high baseline DNA synthetic activity. The inhibition was prevented if ET-1 was added 12-18 h after the addition of EGF or if ET-1 and EGF were added to a protein kinase C-depleted VSMC. The inhibition by ET-1 may be mediated by protein kinase C activation followed by inhibition of EGF binding to its receptor. The results indicate that ET-1 under appropriate conditions can modulate the growth of pulmonary artery VSMC in both positive and negative directions.
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