Effects of antioxidants and nitric oxide on TNF-alpha-induced adhesion molecule expression and NF-kappaB activation in human dermal microvascular endothelial cells

Cell adhesion molecules expressed on endothelial cells in inflamed skin appear to be controlled by the actions of cytokines and reactive oxygen species. However, molecular mechanisms of the expression of adhesion molecules during skin inflammation are currently not well understood. To evaluate the role of antioxidants and nitric oxide in modulating inflammatory processes in the skin, we examined the effects of pyrrolidine dithiocarbamate (PDTC, 0.1 mM) and spermine NONOate (Sper-NO, 1 mM) on adhesion molecule expression and nuclear factor kappa B (NF-kappaB) activation induced by TNF-alpha (10 ng/ml) in cultured human dermal microvascular endothelial cells (HDMEC). Treatment of cells with TNF-alpha for 4 h significantly induced the surface expression of E-selectin and intercellular adhesion molecule-1 (ICAM-1). Treatment with TNF-alpha for 8 h significantly induced the surface expression of E-selectin, ICAM-1 and vascular cell adhesion molecule-1 (VCAM-1). The up-regulation of these adhesion molecules was suppressed significantly by pretreatment with PDTC or Sper-NO for 1 h. The mRNA expression of E-selectin, ICAM-1 and VCAM-1, and activation of NF-kappaB induced by TNF-alpha for 2 h were significantly decreased by the above two pretreatments. N-acetylcysteine (10 mM) and S-nitroso-N-acetylpenicillamine (1 mM) had no significant inhibitory effects on the cell surface and mRNA expression of these adhesion molecules stimulated by TNF-alpha. These findings indicate that both cell surface and mRNA expression of adhesion molecules in HDMEC induced by TNF-alpha are inhibited significantly by pretreatment with PDTC or Sper-NO, possibly in part through blocking the activation of NF-kappaB. These results suggest a potential therapeutic approach using antioxidant agents or nitric oxide pathway modulators in the treatment of inflammatory skin diseases.     

Monochloramine inhibits the expression of E-selectin and intercellular adhesion molecule-1 induced by TNF-alpha through the suppression of NF-kappaB activation in human endothelial cells

Reactive oxygen species have various effects on the expression of cell adhesion molecules induced by proinflammatory cytokines, such as tumor necrosis factor a (T-NF-alpha). We studied the effects of monochloramine (NH2Cl), a physiological oxidant derived from activated neutrophils, on the TNF-alpha-induced expression of E-selectin and intercellular adhesion molecule-1 (ICAM-1) in human umbilical vein endothelial cells (HUVEC). HUVEC were pretreated with or without NH2Cl (20-90 microM for 20 min), then stimulated with TNF-alpha (10 ng/ml), and the expression of E-selectin and ICAM-1 was measured. Without NH2Cl, TNF-alpha induced marked expression of e-selectin and ICAM-1. Pretreatment with NH2Cl resulted in a significant, but transient inhibition of the expression of adhesion molecules. Higher dose of NH2Cl showed more pronounced inhibition, and the inhibitory effect lasted for 8h when 70 microM of NH2Cl was added. TNF-alpha stimulation also induced marked activation of nuclear factor KB (NF-kappaB). Notably, NH2Cl also inhibited this NF-kappaB activation in a dose- and time-dependent manner, which was similar to the inhibition of E-selectin and ICAM-1 expression. In addition, IkappaB-alpha phosphorylation and degradation were also inhibited by NH2Cl pretreatment. These observations indicated that NH2Cl inhibited TNF-alpha-induced expression of E-selectin and ICAM-1 through the inhibition of NF-kappaB activation. We speculate that neutrophil-derived chloramines may have a regulatory role in the recruitment of leukocytes.     

Inhibition of phosphoinositide 3 kinase-Akt (protein kinase B)-nuclear factor-kappa B pathway by lovastatin limits endothelial-monocyte cell interaction

Integrity of the blood-brain barrier is essential for the normal functioning of CNS. Its disruption contributes to the pathobiology of various inflammatory neurodegenerative disorders. We have shown that the HMG-CoA reductase inhibitor (lovastatin) attenuated experimental autoimmune encephalomyelitis (EAE, an inflammatory disease of CNS) in rodents by inhibiting the infiltration of mononuclear cells into the CNS. Here, using an in vitro system, we report that lovastatin inhibits endothelial-monocyte cell interaction by down-regulating the expression of vascular cell adhesion molecule-1 and E-selectin by inhibiting the phosphoinositide 3 kinase (PI3-kinase)/protein kinase B (Akt)/nuclear factor-kappa B (NF-kappaB) pathway in endothelial cells. It inhibits tumor necrosis factor alpha (TNFalpha)-induced PI3-kinase, Akt and NF-kappaB activation in these cells. Co-transfection of constitutively active forms of PI3-kinase and Akt reversed the lovastatin-mediated inhibition of TNFalpha-induced adhesion, as well as activation of NF-kappaB, indicating the involvement of the PI3-kinase/Akt pathway in the interaction of adhesion molecules and the process of adhesion. This study reports that lovastatin down-regulates the pathway affecting the expression and interaction of adhesion molecules on endothelial cells, which in turn restricts the migration and infiltration of mononuclear cells thereby attenuating the pathogenesis of inflammatory diseases.     

4-Hydroxy-2-nonenal is a powerful endogenous inhibitor of endothelial response

There is increasing evidence that lipid peroxidation is involved in many of the pathophysiologies associated with cardiovascular diseases, such as atherosclerosis and the long-term complications of diabetes. Among the products which originate from the peroxidation of cellular membrane lipids, 4-hydroxy-2-nonenal (HNE) is believed to be largely responsible for the cytopathological effects observed during oxidative stress in vivo. Here we found that HNE dramatically inhibited the expression of adhesion molecules induced by inflammatory stimuli in human aortic endothelial cells. The inhibition was found to be accompanied by a significant reduction of NF-kappaB activation followed by nuclear localization. This and the observation that the HNE treatment of the cells resulted in a rapid reduction of intracellular glutathione levels suggest that redox regulation of NF-kappaB may be involved in the modulation of the endothelial response by reactive aldehydes.     

Suppression by 17beta-estradiol of monocyte adhesion to vascular endothelial cells is mediated by estrogen receptors

Several observational studies have shown that estrogen replacement therapy decreases cardiovascular mortality and morbidity in postmenopausal women. However, The Women's Health Initiative (WHI) study has found that women receiving estrogen plus progestin had a significantly higher risk of breast cancer, coronary heart disease, stroke, and pulmonary embolus. In the present study, we examined whether estrogen prevents mechanisms that relate to plaque formation by inhibiting monocyte adhesion to endothelial cells. ECV304 cells, an endothelial cell line that normally expresses minimal estrogen receptor (ER)alpha, were transfected with an ERalpha expression plasmid. Treatment with tumor necrosis factor (TNF)-alpha increased expression of vascular cell adhesion molecule (VCAM)-1 mRNA, activation of nuclear factor-kappaB (NF-kappaB), and U937 cell adhesion in ECV304 cells. These effects of TNF-alpha were not significantly inhibited by pretreatment of native ECV304 cells with 17beta-estradiol (E(2)). In ECV304 cells overexpressing ERalpha, E(2) significantly inhibited the effects of TNF-alpha on NF-kappaB activation, VCAM-1 expression, and U937 cell adhesion. These findings suggest E(2) suppresses inflammatory cell adhesion to vascular endothelial cells that possess functional estrogen receptors. The mechanism of suppression may involve inhibition of NF-kappaB-mediated up-regulation of VCAM-1 expression induced by atherogenic stimuli. E(2) may prevent plaque formation, as first stage of atheroscrelosis through inhibiting adhesion monocytes to endothelial cell. Actions of estrogen replacement therapy can be assessed in terms of densities of functional ERalpha.     

The 15-lipoxygenase-modified high density lipoproteins 3 fail to inhibit the TNF-alpha-induced inflammatory response in human endothelial cells

Endothelial dysfunction represents one of the earliest events in vascular atherogenesis. Proinflammatory stimuli activate endothelial cells, resulting in an increased expression of adhesion molecules and chemoattractants that mediate leukocyte and monocyte adhesion, migration, and homing. High density lipoproteins (HDL) inhibit endothelial cell expression of adhesion molecules in response to proinflammatory stimuli. In the present work, we demonstrate that the modification of HDL(3) (the major and the most antiatherogenic HDL subfraction) by 15-lipoxygenase (15-LO), an enzyme overexpressed in the atherosclerotic lesions, impairs the anti-inflammatory activity of this lipoprotein. The 15-LO-modified HDL(3) failed to inhibit TNF-alpha-mediated mRNA and protein induction of adhesion molecules and MCP-1 in several models of human endothelial cells, and promoted inflammatory response by up-regulating the expression of such mediators of inflammation and by increasing monocyte adhesion to endothelial cells. Moreover, 15-LO-modified HDL(3) were unable to contrast the formation of reactive oxygen species in cells incubated with TNF-alpha, and increased the reactive oxygen species content in unstimulated cells. Activation of NF-kappaB and AP-1 was mainly involved in the expression of adhesion molecules and MCP-1 induced by 15-LO-HDL(3). Altogether, these results demonstrate that enzymatic modification induced by 15-LO impaired the protective role of HDL(3), generating a dysfunctional lipoprotein endowed with proinflammatory characteristics.     

Effects of antioxidant and nitric oxide on chemokine production in TNF-alpha-stimulated human dermal microvascular endothelial cells

Chemokines have been implicated convincingly in the driving of leukocyte emigration in different inflammatory reactions. Multiple signaling mechanisms are reported to be involved in intracellular activation of chemokine expression in vascular endothelial cells by various stimuli. Nevertheless, redox-regulated mechanisms of chemokine expression in human dermal microvascular endothelial cells (HDMEC) remain unclear. This study examined the effects of pyrrolidine dithiocarbamate (PDTC, 0.1 mM) and spermine NONOate (Sper-NO, 1 mM) on the secretion and gene expression of chemokines, interleukin (IL)-8, monocyte chemotactic protein (MCP)-1, regulated upon activation normal T cell expressed and secreted (RANTES), and eotaxin. This study also addresses PDTC and Sper-NO effects on activation of nuclear factor kappa B (NF-kappaB) induced by TNF-alpha (10 ng/ml). Treatment with TNF-alpha for 8 h significantly increased secretion of IL-8, MCP-1, and RANTES, but not of eotaxin, in cultured HDMEC. Up-regulation of these chemokines was suppressed significantly by pretreatment with PDTC or Sper-NO for 1 h, but not by 1 mM 8-bromo-cyclic GMP. The mRNA accumulation of IL-8, MCP-1, RANTES, and eotaxin, and activation of NF-kappaB were induced by TNF-alpha for 2 h; all were suppressed significantly by the above two pretreatments. These findings indicate that both secretion and mRNA accumulation of IL-8, MCP-1, and RANTES in HDMEC induced by TNF-alpha are inhibited significantly by pretreatment with PDTC or Sper-NO, possibly via blocking redox-regulated NF-kappaB activation. These results suggest that restoration of the redox balance using antioxidant agents or nitric oxide pathway modulators may offer new opportunities for therapeutic interventions in inflammatory skin diseases.     

Phosphatidylethanolamines modified by γ-ketoaldehyde (γKA) induce endoplasmic reticulum stress and endothelial activation

Peroxidation of plasma lipoproteins has been implicated in the endothelial cell activation and monocyte adhesion that initiate atherosclerosis, but the exact mechanisms underlying this activation remain unclear. Lipid peroxidation generates lipid aldehydes, including the γ-ketoaldehydes (γKA), also termed isoketals or isolevuglandins, that readily modify the amine headgroup of phosphatidylethanolamine (PE). We hypothesized that aldehyde modification of PE could mediate some of the proinflammatory effects of lipid peroxidation. We found that PE modified by γKA (γKA-PE) induced THP-1 monocyte adhesion to human umbilical cord endothelial cells. γKA-PE also induced expression of adhesion molecules and increased MCP-1 and IL-8 mRNA in human umbilical cord endothelial cells. To determine the structural requirements for γKA-PE activity, we tested several related compounds. PE modified by 4-oxo-pentanal induced THP-1 adhesion, but N-glutaroyl-PE and C(18:0)N-acyl-PE did not, suggesting that an N-pyrrole moiety was essential for cellular activity. As the N-pyrrole headgroup might distort the membrane, we tested the effect of the pyrrole-PEs on membrane parameters. γKA-PE and 4-oxo-pentanal significantly reduced the temperature for the liquid crystalline to hexagonal phase transition in artificial bilayers, suggesting that these pyrrole-PE markedly altered membrane curvature. Additionally, fluorescently labeled γKA-PE rapidly internalized to the endoplasmic reticulum (ER); γKA-PE induced C/EBP homologous protein CHOP and BiP expression and p38 MAPK activity, and inhibitors of ER stress reduced γKA-PE-induced C/EBP homologous protein CHOP and BiP expression as well as EC activation, consistent with γKA-PE inducing ER stress responses that have been previously linked to inflammatory chemokine expression. Thus, γKA-PE is a potential mediator of the inflammation induced by lipid peroxidation.     

Visfatin enhances ICAM-1 and VCAM-1 expression through ROS-dependent NF-kappaB activation in endothelial cells

Visfatin has recently been identified as a novel visceral adipokine which may be involved in obesity-related vascular disorders. However, it is not known whether visfatin directly contributes to endothelial dysfunction. Here, we investigated the effect of visfatin on vascular inflammation, a key step in a variety of vascular diseases. Visfatin induced leukocyte adhesion to endothelial cells and the aortic endothelium by induction of the cell adhesion molecules, ICAM-1 and VCAM-1. Promoter analysis revealed that visfatin-mediated induction of CAMs is mainly regulated by nuclear factor-kappaB (NF-kappaB). Visfatin stimulated IkappaBalpha phosphorylation, nuclear translocation of the p65 subunit of NF-kappaB, and NF-kappaB DNA binding activity in HMECs. Furthermore, visfatin increased ROS generation, and visfatin-induced CAMs expression and NF-kappaB activation were abrogated in the presence of the direct scavenger of ROS. Taken together, our results demonstrate that visfatin is a vascular inflammatory molecule that increases expression of the inflammatory CAMs, ICAM-1 and VCAM-1, through ROS-dependent NF-kappaB activation in endothelial cells.     

CB2-receptor stimulation attenuates TNF-alpha-induced human endothelial cell activation, transendothelial migration of monocytes, and monocyte-endothelial adhesion

Targeting cannabinoid-2 (CB(2)) receptors with selective agonists may represent a novel therapeutic avenue in various inflammatory diseases, but the mechanisms by which CB(2) activation exerts its anti-inflammatory effects and the cellular targets are elusive. Here, we investigated the effects of CB(2)-receptor activation on TNF-alpha-induced signal transduction in human coronary artery endothelial cells in vitro and on endotoxin-induced vascular inflammatory response in vivo. TNF-alpha induced NF-kappaB and RhoA activation and upregulation of adhesion molecules ICAM-1 and VCAM-1, increased expression of monocyte chemoattractant protein, enhanced transendothelial migration of monocytes, and augmented monocyte-endothelial adhesion. Remarkably, all of the above-mentioned effects of TNF-alpha were attenuated by CB(2) agonists. CB(2) agonists also decreased the TNF-alpha- and/or endotoxin-induced ICAM-1 and VCAM-1 expression in isolated aortas and the adhesion of monocytes to aortic vascular endothelium. CB(1) and CB(2) receptors were detectable in human coronary artery endothelial cells by Western blotting, RT-PCR, real-time PCR, and immunofluorescence staining. Because the above-mentioned TNF-alpha-induced phenotypic changes are critical in the initiation and progression of atherosclerosis and restenosis, our findings suggest that targeting CB(2) receptors on endothelial cells may offer a novel approach in the treatment of these pathologies.     

1,4-dihydroxyxanthone modulates the adhesive property of endothelial cells by inhibiting intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and E-selectin

Cell adhesion molecules, particularly intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule (VCAM-1) and E-selectin, play important roles in the recruitment of leukocytes to the site of inflammation. Blocking the expression of these molecules or preventing their interaction with the receptors has been shown to be important in controlling various inflammatory diseases. These cell adhesion molecules are induced on endothelial cells by various proinflammatory cytokines like IL-1beta and TNF-alpha and also by bacterial LPS. We demonstrate here that 1,4-Dihydroxyxanthone (1,4 DHX) inhibits the expression of cell adhesion molecules, such as ICAM-1, VCAM-1 and E-selectin, on endothelial cells in a concentration and time dependent manner. The inhibition by 1,4 DHX is reversible. On further analysis, our results also show that 1,4 DHX inhibits the adhesion of peripheral neutrophils to the endothelial cell monolayers. 1,4 DHX, therefore, could be used as a novel target for controlling various pathological conditions associated with upregulation of endothelial leukocyte adhesion molecules.     

Critical role of cAMP response element binding protein expression in hypoxia-elicited induction of epithelial tumor necrosis factor-alpha.

Tissue hypoxia is intimately associated with a number of chronic inflammatory conditions of the intestine. In this study, we investigated the impact of hypoxia on the expression of a panel of inflammatory mediators by intestinal epithelia. Initial experiments revealed that epithelial (T84 cell) exposure to ambient hypoxia evoked a time-dependent induction of the proinflammatory markers tumor necrosis factor-alpha (TNF-alpha), interleukin-8 (IL-8), and major histocompatibility complex (MHC) class II (37 +/- 6.1-, 7 +/- 0.8-, and 9 +/- 0.9-fold increase over normoxia, respectively, each p < 0.01). Since the gene regulatory elements for each of these molecules contains an NF-kappaB binding domain, we investigated the influence of hypoxia on NF-kappaB activation. Cellular hypoxia induced a time-dependent increase in nuclear p65, suggesting a dominant role for NF-kappaB in hypoxia-elicited induction of proinflammatory gene products. Further work, however, revealed that hypoxia does not influence epithelial intercellular adhesion molecule 1 (ICAM-1) or MHC class I, the promoters of which also contain NF-kappaB binding domains, suggesting differential responses to hypoxia. Importantly, the genes for TNF-alpha, IL-8, and MHC class II, but not ICAM-1 or MHC class I, contain cyclic AMP response element (CRE) consensus motifs. Thus, we examined the role of cAMP in the hypoxia-elicited phenotype. Hypoxia diminished CRE binding protein (CREB) expression. In parallel, T84 cell cAMP was diminished by hypoxia (83 +/- 13.2% decrease, p < 0.001), and pharmacologic inhibition of protein kinase A induced TNF-alpha and protein release (9 +/- 3.9-fold increase). Addback of cAMP resulted in reversal of hypoxia-elicited TNF-alpha release (86 +/- 3.2% inhibition with 3 mM 8-bromo-cAMP). Furthermore, overexpression of CREB but not mutated CREB by retroviral-mediated gene transfer reversed hypoxia-elicited induction of TNF-alpha defining a causal relationship between hypoxia-elicited CREB reduction and TNF-alpha induction. Such data indicate a prominent role for CREB in the hypoxia-elicited epithelial phenotype and implicate intracellular cAMP as an important second messenger in differential induction of proinflammatory mediators.