The recruitment of monocytes into the arterial wall is one of the earliest events in the pathogenesis of atherosclerosis. Since monocyte chemoattractant protein 1 (MCP-1) plays a key role in the subendothelial recruitment of monocytes, we tested whether nitric oxide (NO) modulates the expression of MCP-1 in cultured human endothelial cells. Inhibition of basal NO production by NG-nitro-L-arginine (L-NAG) upregulates endothelial MCP-1 mRNA expression (250 +/- 20%) and protein secretion. Exogenous addition of NO dose-dependently decreased MCP-1 mRNA expression and secretion. Changes in MCP-1 mRNA expression and protein secretion were paralleled by corresponding changes in chemotactic activity of cell-conditioned media for monocytes. An MCP-1 antibody reduced monocyte chemotactic activity by 85% and completely abolished the increased monocyte chemotactic activity induced by the inhibition of NO production. Elevation of endothelial cGMP levels had no significant effect on MCP-1 mRNA expression. Inhibition of basal endothelial NO production by L-NAG increased binding activity of a nuclear factor kappa B (NF-kappa B)-like transcriptional regulatory factor, whereas exogenous addition of NO decreased NF-kappa B-like binding activity during stimulation with tumor necrosis factor-alpha. Thus, NO modulates MCP-1 expression and monocyte chemotactic activity secreted by human umbilical vein endothelial cells (HUVECs) in culture. The activation of NF-kappa B-like transcriptional regulatory proteins by inhibition of NO suggests a molecular link between an oxidant-sensitive transcriptional regulatory mechanism and NO synthesis in HUVECs.

We investigated whether sodium diethyldithiocarbamate @ETC), an inhibitor of the nuclear transcription factor kappa B (NFkappaB), modulates induction of NO synthase (NOS) in murine bone marrow-derived macrophages. A short exposure (between 1 and 16 h) of L929-cell mediumpreconditioned macrophages to E. coli lipopolysaccharide (LPS) significantly increased the level of NOS mRNA, and elicited NO formation as detected by electron spin resonance spectroscopy and by the release of nitrite. DETC (0.1-l mM) present during stimulation with LPS prevented the increase in NOS mRNA and the expression of NOS activity. These findings suggest that NFkappaB is involved in the signal transduction pathway linking stimulation of macrophages by LPS with transcription of the gene encoding inducible NOS.NO synthase; Transcription; NFkappaB; Diethyldithiocarbamate; Bone marrow-derived macrophage 1, INTRODUCTION Intra-and extracellular pathogens, their breakdown products, several cytokines and a plethora of other noxious agents and conditions induce the expression of NO synthase in mammalian cells (for review see [l-3]). NO generated from L-arginine by the inducible NO synthase (iNOS) is regarded as a defense effector molecule with cytotoxic/cytostatic and microbicidal/microbiostatic activity [1,2]. To date little or nothing is known about the transduction cascade that unifies all these pathogenic signals translating them into activation of the iNOS gene. Eukaryotic gene expression is controlled by promotor and enhancer DNA sequences, which are activated by specific protein factors. One of the nuclear transcription factors initially identified in B-cells and monocytes, the multiprotein complex NFkappaB [4], is rapidly activated upon perturbation of cells by conditions similar to those known to induce NOS. NFkappaB is known to mediate the immediate-early gene response by enhancing the transcription of a multitude of genes encoding defense and signalling proteins, such as cytokines and cytokine receptors [4]. Activation of NFkappaB can be specifically prevented by antioxidants, thiols and iron chelators, and especially by dithiocarbamates [4]. To clarify whether NFkappaB participates in the induction of NOS, we assessed the effect of diethyldithiocarbamate (DETC) on iNOS mRNA levels and expression of NOS activity in lipopolysaccharide (LPS)-stimulated murine bone marrow-derived macrophages. MATERIALS AND METHODSBone marrow cells from the femurs of Balb/c mice were isolated and cultured in L929-cell conditioned medium as described recently [5]. Cells grown on culture dishes (3.5 cm diameter; 10' cells/dish) were exposed to LPS (0.1 &ml; E. coli serotype 055:B5), DETC (0.01 to 1 mM; both from Sigma, Deisenhofen, Germany) and hemin (Normosang, Leiras, Turku, Finland) according to the protocols described in section 3.Messenger RNA encoding iNOS was assessed by Northern blot technique using a HincIIlSSP I fragment of the mouse iNOS [6] as a cDNA probe. The cDNA probe was a generous gift of Drs. Q. Xie and C.F. Nathan, Cornell University Medi...

Experiments were designed to examine whether or not insulin-like growth factor I (IGF-I), which is produced by vascular cells in response to injury, affects the production of nitric oxide evoked by the inducible nitric oxide synthase in cultures of smooth muscle cells from the rat aorta. Nitric oxide production was assessed indirectly by the measurement of nitrite accumulation and nitric oxide synthase activity by determining the formation of L-citrulline from L-arginine. Nitric oxide synthase was induced in vascular smooth muscle cells that had been exposed to interleukin-1 beta (IL-1 beta) or tumor necrosis factor-alpha (TNF-alpha). IGF-I inhibited, in a concentration-dependent manner, the production of nitrite and L-citrulline evoked by IL-1 beta or TNF-alpha. The inhibition caused by IGF-I required the presence of the growth factor during the induction of nitric oxide synthase. Two IGF-I-related proteins, IGF-II and insulin, also inhibited, but to a smaller extent, the release of nitrite and the formation of L-citrulline stimulated by IL-1 beta. Under bioassay conditions, the perfusates from columns containing IL-1 beta-treated smooth muscle cells relaxed rings of rat aorta without endothelium that had been contracted with phenylephrine; these relaxations were reversed by nitro-L-arginine. Addition of IL-1 beta-treated vascular smooth muscle cells to indomethacin-treated platelets inhibited their aggregation to thrombin; methylene blue prevented this inhibition. Control smooth muscle cells or cells exposed to IGF-I alone did not have such effects.(ABSTRACT TRUNCATED AT 250 WORDS)