Search

The induction of vascular cell adhesion molecule-1 (VCAM-1) expression by tumor necrosis factor (TNF)-alpha requires the activation of nuclear factor-kappaB (NF-kappaB) via a process involving the phosphorylation and degradation of its cytoplasmic inhibitor, IkappaBalpha. We have shown that nitric oxide (NO) decreases VCAM-1 expression via inhibition of NF-kappaB activation. To determine how NO inhibits NF-kappaB, we studied the fate of IkappaBalpha following TNF-alpha stimulation in the presence of NO donors S-nitrosoglutathione and sodium nitroprusside. Activation of NF-kappaB by TNF-alpha occurred within 15 min and coincided with rapid degradation of IkappaBalpha. Co-treatment with NO donors did not prevent IkappaBalpha phosphorylation or degradation. However, after 2 h of TNF-alpha stimulation, NO donors inhibited NF-kappaB activation and augmented IkappaBalpha resynthesis and nuclear translocation by 2.5- and 3-fold, respectively. This correlated with a 75% reduction in TNF-alpha-induced VCAM-1 expression. In a time-dependent manner, NO donors alone caused the nuclear translocation of IkappaBalpha. To confirm that NO donors have similar effects as endogenously derived NO, murine macrophage-like cells, RAW264.7, were co-cultured with endothelial cells. Induction of RAW264.7-derived NO inhibited lipopolysaccharide-induced endothelial VCAM-1 expression, which was reversed by the NO synthase inhibitor Nomega-monomethyl-L-arginine. These findings indicate that NO inhibits NF-kappaB activation and VCAM-1 expression by increasing the expression and nuclear translocation of IkappaBalpha.

To test the hypothesis that nitric oxide (NO) limits endothelial activation, we treated cytokine-stimulated human saphenous vein endothelial cells with several NO donors and assessed their effects on the inducible expression of vascular cell adhesion molecule-1 (VCAM-1). In a concentration-dependent manner, NO inhibited interleukin (IL)-1 alpha-stimulated VCAM-1 expression by 35-55% as determined by cell surface enzyme immunoassays and flow cytometry. This inhibition was paralleled by reduced monocyte adhesion to endothelial monolayers in nonstatic assays, was unaffected by cGMP analogues, and was quantitatively similar after stimulation by either IL-1 alpha, IL-1 beta, IL-4, tumor necrosis factor (TNF alpha), or bacterial lipopolysaccharide. NO also decreased the endothelial expression of other leukocyte adhesion molecules (E-selectin and to a lesser extent, intercellular adhesion molecule-1) and secretable cytokines (IL-6 and IL-8). Inhibition of endogenous NO production by L-N-monomethyl-arginine also induced the expression of VCAM-1, but did not augment cytokine-induced VCAM-1 expression. Nuclear run-on assays, transfection studies using various VCAM-1 promoter reporter gene constructs, and electrophoretic mobility shift assays indicated that NO represses VCAM-1 gene transcription, in part, by inhibiting NF-kappa B. We propose that NO's ability to limit endothelial activation and inhibit monocyte adhesion may contribute to some of its antiatherogenic and antiinflammatory properties within the vessel wall.

To investigate the effect and regulation of extracellular signal-regulated kinase (ERK1/2) signaling pathway on the expression of transforming growth factor-beta1 (TGF-beta1) in human embryonic lung fibroblasts induced by SiO2.Human alveolar macrophages were collected from a silicotic patient by bronchoalveolar lavage and in the presence or absence of SiO2 (50 ug/ml) exposition for 18h, and then the conditioned supernatants were used to incubate HELF. The expressions of TGF-beta1, of the HELF acted with the conditioned AM supernatant fluid were detected with the immunocytochemistry method after treatment with PD98059 of inhibitor of ERK.The expression of TGF-beta1 in HELF of the SiO2 treatment group (OD value is 0.322 7 +/- 0.023 8) exceed blank group (OD value is 0.163 7 +/- 0.019 6) and AM control group (OD value is 0.240 6 +/- 0.022 5) by the immunocytochemistry method. But the expression of TGF-beta1 had reduction in some extent in the PD98059 intervention group (OD value is 0.271 1 +/- 0.022 9). The values were statistically different (P0.05).ERK inhibitor PD98059 have inhibition effect on the expression of transforming growth factor-beta1 and expression of cytokine of human embryonic lung fibroblasts stimulated by SiO2. The study indicate that the proliferation and collagen production of HELF activated by SiO2 are mediated by ERK/MAPK signal pathway in some extent. PD98059 may antagonizes silica-induced lung fibrosis by inhibiting the expression of transforming growth factor-beta1.

To study the proliferation effect of the AM supernatant incubated activation of p38 mitogen activated protein kinases (p38MAPK) signal transduction pathway in human embryonic lung fibroblasts, and to participate in the development of fibrosis in silicosis.The silicotic alveolar macrophages were collected by bronchoalveolar lavage and incubated in vitro in the DMEM medium containing SiO₂ (50 µg/ml) and DMEM medium without SiO₂ for 18 h. Then the AM supernatant incubated for 18 h was collected. HELFs were isolated by organize paste block method, and incubated with AM supernatants. HELFs were divided into four groups: blank control groups, AM groups, SiO₂ + AM groups, SB203580 + SiO₂ + AM groups. The proliferation in the HELF was detected with MTT method and Flow cytometry.The proliferation in the HELF acted with the conditioned AM supernatant fluid were more than blank control groups, AM groups and SB203580 + SiO₂ + AM groups [average optical density: (0.48 ± 0.03) vs (0.29 ± 0.01), (0.38 ± 0.02), (0.33 ± 0.03)], the values with MTT method were statistically different (P0.05); Proliferous index with flow cytometry in SiO₂ + AM groups (18.12 ± 0.82) was bigger than blank control groups (9.24 ± 0.48), AM groups (14.76 ± 0.43) and SB203580 + SiO₂ + AM groups (11.71 ± 0.70) and the values were statistically different(P0.05).The AM supernatant stimulated by silicon dioxide can accelerate the proliferation in the HELF by activation of p38MAPK signal transduction pathway.

To observe the effect of ERK1/2 signal pathway activated by SiO₂ in the proliferation of human embryonic lung fibroblast mediated by silicotic alveolar macrophages.The alveolar macrophages (AM) harvested from silicotic sufferers by bronchoalveolar lavage (BAL) were interacted with SiO₂ suspension once more. HELF, pretreated with the inhibitor PD98059 (50 µmol/L) for 1 hour, were stimulated by conditional supernatant fluid of silicotic sufferers. The experimentation have been classificated four group: blank group, AM control group, SiO₂ treatment group, PD98059 intervention group. The proliferation activity and expressions of Phospho-ERK1/2 of lung fibroblast activated by AM supernatant fluids of silicotic are detected with the MTT assay, flow cytometry and Western blot method after being pretreatmented with PD98059.The A values of cell proliferation in SiO₂ treatment group and AM control group are 2.6 and 2.0 times that of blank group, in which the difference was statistically significant (P0.05). Comparing with SiO₂ treatment group, the A values of every concentrations of PD98059 intervention group decreased with a dose-response relationship, after 10, 25 and 50 µmol/L PD98059 intervention. The 25 and 50 µmol/L PD98059 intervention group were 72.1% and 48.5% of SiO₂ treatment group, which the difference is statistic (P0.05). The expression of phospho-ERK1/2 in SiO₂ treatment group was up, which appeared in 15 min and apparent activated in 30 min (A value is 0.4653 ± 0.0265), and then still in the higher state afterwards declined after 60 min. In addition to 15 min, the expression of phospho-ERK1/2 protein in SiO₂ treatment group at each time point are 1.25, 1.23, 1.25 times over the same period AM control group respectively, the differences were statistically significant (P0.05).The silicotic supernatant of alveolar macrophages have promote proliferation of HELF and activation of ERK1/2, which may involve in the development of silicosis pathogenesis by ERK1/2 signal pathway.

Macrophage-colony stimulating factor (M-CSF) contributes to atherogenesis by regulating macrophage-derived foam cells in atherosclerotic lesions. Here we report that nitric oxide (NO) inhibits the expression of M-CSF in human vascular endothelial cells independent of guanylyl cyclase activation. The induction of M-CSF mRNA expression by either oxidized low density lipoprotein (ox-LDL) or tumor necrosis factor-alpha (TNF alpha) was attenuated by NO donors, S-nitrosoglutathione (GSNO), sodium nitroprusside (SNP), and 3-morpholinosydnonimine, but not by cGMP analogues, glutathione, or nitrite. Inhibition of endogenous NO production by N-monomethyl-L-arginine (L-NMA) also increased M-CSF expression in control and TNF alpha-stimulated cells. Nuclear run-on assays and transfection studies using M-CSF promoter constructs linked to chloramphenicol acetyltransferase reporter gene indicated that NO repressed M-CSF gene transcription through nuclear factor-kappa B (NF-kappa B). Electrophoretic mobility shift assays demonstrated that activation of NF-kappa B by L-NMA, ox-LDL, and TNF alpha was attenuated by GSNO and SNP, but not by glutathione or cGMP analogues. Since the induction of M-CSF expression depends upon NF-kappa B activation, the ability of NO to inhibit NF-kappa B activation and M-CSF expression may contribute to some of NO's antiatherogenic properties.