Search

We hypothesize that nutrition can modulate the toxicity of environmental pollutants and thus modulate health and disease outcome associated with chemical insult. There is now increasing evidence that exposure to persistent organic pollutants, such as PCBs, can contribute to the development of inflammatory diseases such as atherosclerosis. Activation, chronic inflammation, and dysfunction of the vascular endothelium are critical events in the initiation and acceleration of atherosclerotic lesion formation. Our studies indicate that an increase in cellular oxidative stress and an imbalance in antioxidant status are critical events in PCB-mediated induction of inflammatory genes and endothelial cell dysfunction. Furthermore, we have found that specific dietary fats can further compromise endothelial dysfunction induced by selected PCBs and that antioxidant nutrients (such as vitamin E and dietary flavonoids) can protect against endothelial cell damage mediated by these persistent organic pollutants. Our recent data suggest that membrane lipid rafts such as caveolae may play a major role in the regulation of PCB-induced inflammatory signaling in endothelial cells. In addition, PCB- and lipid-induced inflammation can be down-regulated by ligands of anti-atherogenic peroxisome proliferator-activated receptors (PPARs). We hypothesize that PCBs contribute to an endothelial inflammatory response in part by down-regulating PPAR signaling. Our data so far support our hypothesis that antioxidant nutrients and related bioactive compounds common in fruits and vegetables protect against environmental toxic insult to the vascular endothelium by down-regulation of signaling pathways involved in inflammatory responses and atherosclerosis. Even though the concept that nutrition may modify or ameliorate the toxicity of environmental chemicals is provocative and warrants further study, the implications for human health could be significant. More research is needed to understand observed interactions of PCB toxicity with nutritional interventions.

So-called coplanar polychlorinated biphenyls (PCBs), as well as other environmental contaminants that are aryl hydrocarbon receptor (AhR) agonists, may compromise the normal functions of vascular endothelial cells by activating oxidative stress-sensitive signaling pathways and subsequent proinflammatory events critical in the pathology of atherosclerosis and cardiovascular disease. To test this hypothesis, porcine endothelial cells were exposed to PCB 153 and to three coplanar PCBs (PCB 77, PCB 126, or PCB 169). In contrast to PCB 153, which is not a ligand for the Ah receptor (AhR), all coplanar PCBs disrupted endothelial barrier function. All coplanar PCBs increased expression of the CYP1A1 gene, oxidative stress (DCF fluorescence), and the DNA-binding activity of nuclear factor kappaB (NF-kappaB). PCB-induced oxidative stress was concentration-dependent, with PCB 126 exhibiting a maximal response at the lowest concentration (0.5 microM) tested. The increase in NF-kappaB-dependent transcriptional activity was confirmed in endothelial cells by a luciferase reporter gene assay. In contrast to PCB 153, coplanar PCBs that are AhR ligands increased endothelial production of interleukin-6. At 3.4 microM, expression of the adhesion molecule VCAM-1 was most sensitive to PCB 77 and 169. We also provide in vivo evidence, suggesting that binding to the AhR is critical for the proinflammatory properties of PCBs. Twenty hours after a single administration of PCB 77, VCAM-1 expression was increased only in wild-type mice, while mice lacking the AhR gene showed no increased staining for VCAM-1. These data provide evidence that coplanar PCBs, agonists for the AhR, and inducers of cytochrome P450 1A1, produce oxidative stress and an inflammatory response in vascular endothelial cells. An intact AhR may be necessary for the observed PCB-induced responses. These findings suggest that activation of the AhR can be an underlying mechanism of atherosclerosis mediated by certain environmental contaminants.

Little is known about the requirements and function of zinc in maintaining endothelial cell integrity, especially during stressful conditions, such as the inflammatory response in cardiovascular disease. There is evidence that zinc requirements of the vascular endothelium are increased during inflammatory conditions such as atherosclerosis, where apoptotic cell death is also prevalent. Apoptosis is a morphologically distinct mechanism of programmed cell death which involves the activation of a cell-intrinsic suicide program, and there is evidence that factors such as inflammatory cytokines (e.g., tumor necrosis factor [TNF]) and pure or oxidized lipids are necessary to induce the cell death pathway. Because of its constant exposure to blood components, including prooxidants, diet-derived fats, and their derivatives, the endothelium is very susceptible to oxidative stress and to apoptotic injury mediated by blood lipid components, prooxidants, and cytokines. Thus, it is likely that the cellular lipid environment, primarily polyunsaturated fatty acids, can potentiate the overall endothelial cell injury by increasing cellular oxidative stress and cytokine release in proximity to the endothelium, which then could further induce apoptosis and disrupt endothelial barrier function. Our data suggest that zinc deficiency exacerbates the detrimental effects of specific fatty acids (e.g., linoleic acid) and inflammatory cytokines, such as TNF, on vascular endothelial functions. We propose that a major mechanism of zinc protection against disruption of endothelial cell integrity during inflammatory conditions, is by the ability of zinc to inhibit the pathways of signal transduction leading to apoptosis and especially mechanisms that lead to upregulation of caspase genes.

Polyunsaturated fatty acids such as linoleic acid are well known dietary lipids that may be atherogenic by activating vascular endothelial cells. In the liver, fatty acids can be metabolized by cytochrome P450 (CYP) enzymes, but little is known about the role of these enzymes in the vascular endothelium. CYP 2C9 is involved in linoleic acid epoxygenation, and the major product of this reaction is leukotoxin (LTX). We investigated the role of CYP-mediated mechanisms of linoleic acid metabolism in endothelial cell activation by examining the effects of linoleic acid or its oxidized metabolites such as LTX and leukotoxin diol (LTD).The effect of linoleic acid on CYP 2C9 gene expression was studied by RT-PCR. Oxidative stress was monitored by measuring DCF fluorescence and intracellular glutathione levels, and electrophoretic mobility shift assay was carried out to study the activation of oxidative stress sensitive transcription factors. Analysis of oxidized lipids was carried out by liquid chromatography/mass spectrometry.Linoleic acid treatment for six hours increased the expression of CYP 2C9 in endothelial cells. Linoleic acid-mediated increase in oxidative stress and activation of AP-1 were blocked by sulfaphenazole, a specific inhibitor of CYP 2C9. The linoleic acid metabolites LTX and LTD increased oxidative stress and activation of transcription factors only at high concentrations.Our data show that CYP 2C9 plays a key role in linoleic acid-induced oxidative stress and subsequent proinflammatory events in vascular endothelial cells by possibly causing superoxide generation through uncoupling processes.

Dietary zinc has potent antioxidant and anti-inflammatory properties and is a critical component of peroxisome proliferator-activated receptor (PPAR) gene expression and regulation. To assess the protective mechanisms of PPARgamma in endothelial cell dysfunction and the role of zinc in the modulation of PPARgamma signaling, cultured porcine pulmonary artery endothelial cells were exposed to the membrane-permeable zinc chelator N,N,N'N'-tetrakis (2-pyridylmethyl)-ethylene diamine (TPEN), thiazolidinedione (TZD; PPARgamma agonist) or bisphenol A diglycidyl ether (BADGE; PPARgamma antagonist). Subsequently, endothelial cells were activated by treatment with linoleic acid (90 micro mol/L) for 6 h. Zinc chelation by TPEN increased the DNA binding activity of nuclear factor (NF)-kappaB and activator protein (AP)-1, decreased PPARgamma expression and activation as well as up-regulated interleukin (IL)-6 expression and production. These effects were fully reversed by zinc supplementation. In addition, exposure to TZD down-regulated linoleic acid-induced DNA binding activity of NF-kappaB and AP-1, whereas BADGE further induced activation of these oxidative stress-sensitive transcription factors. Most importantly, the TZD-mediated down-regulation of NF-kappaB and AP-1 and reduced inflammatory response were impaired during zinc chelation. These data suggest that zinc plays a critical role in PPARgamma signaling in linoleic acid-induced endothelial cell activation and indicate that PPARgamma signaling is impaired during zinc deficiency.

Polychlorinated biphenyls (PCBs), especially the more coplanar PCBs, have been shown to induce oxidative stress, various transcription factors, and subsequent inflammatory processes critical to atherosclerosis in vascular endothelial cells. Dietary flavonoids such as catechins and quercetin possess antioxidant and anti-inflammatory properties. To test the hypothesis that flavonoids can modify PCB-mediated endothelial cytotoxicity, endothelial cells were treated with epigallocatechin-3-gallate (EGCG; 5 to 50 muM) or quercetin (10 to 100 muM) with or without PCB 77 (3,3',4,4'-tetrachlorobiphenyl, 3.4 muM) for 6 h. EGCG and quercetin strongly, and in a concentration-dependent manner, inhibited oxidative stress induced by PCB 77 as measured by DCF fluorescence. The role of cytochrome P450 1A1 (CYP1A1) in the PCB-induced toxicity was investigated. EGCG at 50 muM and quercetin at 100 muM concentrations markedly inhibited CYP1A1 mRNA levels and enzyme activity. Furthermore, EGCG and quercetin downregulated the PCB 77-mediated increase in aryl hydrocarbon receptor (AhR)-DNA binding activity. These data suggest that protective effects of EGCG and quercetin are initiated upstream from CYP1A1 and that these flavonoids may be of value for inhibiting the toxic effects of PCBs on vascular endothelial cells.

Vascular endothelial cell activation and dysfunction are critical early events in atherosclerosis. Selected dietary lipids (eg, fatty acids) may be atherogenic by activating endothelial cells and by potentiating an inflammatory response. Due to their prooxidant property, unsaturated fatty acids may play a critical role in endothelial cell activation and injury. To test this hypothesis, porcine endothelial cells were exposed to 18-carbon fatty acids differing in the degree of unsaturation, ie, 90 micromol/L stearic (18:0), oleic (18:1n-9), linoleic (18:2n-6), or linolenic acid (18:3n-3) for 6 to 24 hours and/or tumor necrosis factor alpha ([TNF-alpha] 500 U/L) for up to 3 hours. Compared with control cultures, treatment with 18:0 and 18:2 decreased glutathione levels, suggesting an increase in cellular oxidative stress. Both 18:2 and 18:0 activated the transcription factor nuclear factor kappaB (NF-kappaB) the most and 18:1 the least. This NF-kappaB-dependent transcription was confirmed in endothelial cells by luciferase reporter gene assay. The fatty acid-mediated activation of NF-kappaB was blocked by preenrichment of the cultures with 25 micromol/L vitamin E. All fatty acids except 18:1 and 18:3 increased transendothelial albumin transfer, and 18:2 caused the most marked disruption of endothelial integrity. Preenrichment of endothelial cells with 18:2 followed by exposure to TNF-alpha resulted in a 100% increase in interleukin-6 (IL-6) production compared with TNF-alpha exposure alone. In contrast, cellular preenrichment with 18:0, 18:1, or 18:3 had no effect on TNF-alpha-mediated production of IL-6. Cellular release of radiolabeled arachidonic acid (20:4) was markedly increased only by cell exposure to 18:2 and 18:3, and the release of 20:4 appeared to be mainly from the phosphatidylethanolamine fraction. These data suggest that oleic acid does not activate endothelial cells. Furthermore, linoleic acid and other omega-6 fatty acids appear to be the most proinflammatory and possibly atherogenic fatty acids.

Background Zinc requirements of the vascular endothelium may be increased in inflammatory conditions, ie, atherosclerosis, in which apoptotic cell death is prevalent. Objective We hypothesized that zinc deficiency may potentiate disruption of endothelial cell integrity mediated by fatty acids and inflammatory cytokines by enhancing pathways that lead to apoptosis and up-regulation of caspase genes. Design Endothelial cells were maintained in low-serum medium or grown in culture media containing selected chelators, ie, diethylenetriaminepentaacetate or N,N,N', N'-tetrakis(2-pyridylmethyl)-ethylenediamine (TPEN), with or without zinc supplementation. Subsequently, cells were treated with linoleic acid, tumor necrosis factor alpha (TNF-alpha), or both. We studied the effect of zinc deficiency and supplementation on the induction of apoptosis by measuring caspase-3 activity, cell binding of annexin V, and DNA fragmentation. Results Our results indicated that linoleic acid and TNF-alpha independently, but more markedly in concert, up-regulated caspase-3 activity and induced annexin V binding and DNA fragmentation. Zinc deficiency, especially when induced by TPEN, dramatically increased apoptotic cell death induced by cytokines and lipids compared with control cultures. Supplementation of low-serum- or chelator-treated endothelial cells with physiologic amounts of zinc caused a marked attenuation of apoptosis induced by linoleic acid and TNF-alpha. Morphologic changes of cells observed during zinc deficiency were prevented by zinc supplementation. Media supplementation with other divalent cations (eg, calcium and magnesium) did not mimic the protective role of zinc against apoptosis. Conclusions Our data indicate that zinc is vital to vascular endothelial cell integrity, possibly by regulating signaling events to inhibit apoptotic cell death.

The objective of this study was to test the hypothesis that zinc deficiency in endothelial cells may potentiate the inflammatory response mediated by certain lipids and cytokines, possibly via mechanisms associated with increased cellular oxidative stress. Our experimental approach was to compare conditions of cellular zinc deficiency and zinc supplementation with oxidative stress-mediated molecular and biochemical changes in vascular endothelial cells.To investigate our hypothesis, porcine pulmonary artery-derived endothelial cells were depleted of zinc by culture in media containing 1% fetal bovine serum for eight days. Subsequently, endothelial cells were exposed to media enriched with or without zinc (10 microM) for two days, followed by exposure to either tumor necrosis factor-alpha (TNF, 500 U/mL) or linoleic acid (90 microM), before measurement of oxidative stress (DCF fluorescence), activation of nuclear factor kappaB (NF-kappaB) or activator protein-1 (AP-1) and production of the inflammatory cytokine interleukin 6 (IL-6).Oxidative stress was increased markedly in zinc-deficient endothelial cells following treatment with fatty acid or TNF. This increase in oxidative stress was partially blocked by prior zinc supplementation. The oxidative stress-sensitive transcription factor NF-kappaB was up-regulated by zinc deficiency and fatty acid treatment. The up-regulation mediated by fatty acids was markedly reduced by zinc supplementation. Similar results were obtained with AP-1. Furthermore, endothelial cell production of IL-6 was increased in zinc-deficient endothelial cells following treatment with fatty acids or TNF. This increase in production of inflammatory cytokines was partially blocked by zinc supplementation.Our previous data clearly show that zinc is a protective and critical nutrient for maintenance of endothelial integrity. The present data suggest that zinc may in part be antiatherogenic by inhibiting oxidative stress-responsive events in endothelial cell dysfunction. This may have implications in understanding mechanisms of atherosclerosis.