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Journal of the American College of Nutrition, Vol. 18, No. 2, 152-158 (1999)
Published by the American College of Nutrition

Antioxidant-Like Properties of Zinc in Activated Endothelial Cells

Bernhard Hennig, PhD, FACN, Purushothaman Meerarani, PhD, Michal Toborek, MD, PhD, FACN and Craig J. McClain, MD, FACN

Departments of Nutrition and Food Science (B.H., P.M.), Neurosurgery (M.T.), and Medicine (C.J.M.), University of Kentucky, Lexington, Kentucky

Address reprint requests to: Bernhard Hennig, PhD, FACN, Cell Nutrition Group, Department of Nutrition and Food Science, 204 Funkhouser Building, University of Kentucky, KY 40506-0054


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Objective: 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.

Methods: 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 µM) for two days, followed by exposure to either tumor necrosis factor-{alpha} (TNF, 500 U/mL) or linoleic acid (90 µM), before measurement of oxidative stress (DCF fluorescence), activation of nuclear factor {kappa}B (NF-{kappa}B) or activator protein-1 (AP-1) and production of the inflammatory cytokine interleukin 6 (IL-6).

Results: 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-{kappa}B 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.

Discussion: 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.

Key words: Zinc, antioxidant, NF-{kappa}B, AP-1, cytokines, TNF, IL-6, atherosclerosis


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Zinc is a critical component of biomembranes and is essential for proper membrane structure and function and the activity of numerous enzymes [1,2]. Evidence indicates that a low zinc concentration may have important implications in the pathogenesis of atherosclerosis [3]. Mechanisms of the protective function or functions of zinc in the pathogenesis of atherosclerosis, including vascular cell injury or dysfunction and the inflammatory response, are not clear. There is evidence suggesting that zinc can act as an endogenous protective factor against atherosclerosis by inhibiting the oxidation of LDL by cells or transition metals [4,5]. Because of its antioxidant and membrane-stabilizing properties, zinc appears to be crucial for protection against cell-destabilizing agents such as inflammatory cytokines and polyunsaturated lipids. We have shown that zinc can protect endothelial cells against tumor-necrosis-factor-(TNF)-induced cell injury [6], and one of the underlying mech-anisms could be the ability of zinc to down-regulate oxidative stress-sensitive transcription factors [7]. The implications of these findings are significant, since lipid-induced endothelial cell injury may increase the production of circulating concentrations of cytokines involved in the atherosclerotic disease process. Furthermore, during inflammation there is an internal redistribution of zinc, with zinc being lost from some tissues, such as plasma, and accumulating in other tissues, such as liver. The endothelium may be one tissue from which zinc is lost during the inflammatory or acute phase response.

Atherosclerosis is one of the leading causes of death in the Western world. The primary step in the formation of atherosclerotic lesions involves the adherence of circulating monocytes to the endothelium with subsequent migration into the arterial intima where they differentiate into macrophages. Inflammatory cytokines, such as TNF or interleukin-6 (IL-6), and adhesion molecules are critical in orchestrating these events. For example, TNF production by activated leukocytes was increased in patients with atherosclerosis, and the presence of this cytokine was detected in atherosclerotic lesions [8,9]. Plasma levels of IL-6 were elevated in patients with ischemic heart disease [10], and the significant presence of IL-6 in atherosclerotic arteries strongly suggests the involvement of this cytokine in inflammatory events during the initiation and progression of atherosclerosis [11,12]. Expression of the gene encoding for IL-6 is regulated by oxidative stress-sensitive transcription factor, NF-{kappa}B.

NF-{kappa}B is a transcription factor which is implicated in many endothelial cell activation responses to injury and stress [13]. NF-{kappa}B plays a central role in regulating the cytokine network and hence its activation may be a major factor contributing to the pathogenesis of diseases such as atherosclerosis or adult respiratory distress syndrome. Stimuli known to activate the NF-{kappa}B complex include TNF, IL-1, lipopolysaccharide, and the like, with the common denominator apparently being reactive oxygen species [14]. TNF-induced oxidative stress also activates oxidative stress-responsive genes of the Immediate Early Gene (IEG) family, among them c-jun and c-fos. The Fos and Jun proteins, products of the c-jun and c-fos genes, compose another potent transcription factor, AP-1. The role of c-fos, c-jun and AP-1 in endothelial cell metabolism is not fully understood, but they may be involved in expression of adhesion molecules, for example the intracellular adhesion molecule 1 (ICAM-1) [15]. Hence, modulation of oxidative stress-sensitive transcription factors with antioxidants or by changing the cellular redox cycle may have a significant impact on the overall inflammatory cytokine response and endothelial cell dysfunction.

High fat/calorie diets are a risk factor for atherosclerosis, and free fatty acids derived from lipoprotein lipase-mediated hydrolysis of triglyceride-rich lipoproteins may contribute significantly to endothelial cell activation or dysfunction [16]. In fact, activity of lipoprotein lipase is increased in atherosclerotic lesions [17–19]. Activated lipoprotein lipase induces TNF gene expression in macrophages and TNF production by this type of cell [20]. Furthermore, lipoprotein lipase may be a chemoattractant for activated macrophages [21]. Recent evidence suggests that linoleic acid may play a critical role in the pathogenesis of atherosclerosis [16]. An increase in linoleic acid levels has been reported in the phospholipid fractions of human coronary arteries in cases of sudden cardiac death due to ischemic heart disease [22]. Additionally, concentrations of linoleic acid in adipose tissue were positively correlated with the degree of coronary artery disease [23]. Lipoprotein, lipase-derived remnants of lipoproteins isolated from hypertriglyceridemic subjects as well as selective unsaturated fatty acids, such as linoleic acid, were demonstrated to disrupt endothelial integrity [24,25]. Furthermore, recent evidence indicated that linoleic acid can activate NF-{kappa}B as well as stimulate NF-{kappa}B-dependent transcription [26,27].

The objective of the present 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 related to increased cellular oxidative stress. Our data provide further support to the hypothesis that zinc may act as an antiatherogenic nutrient through its function as an antioxidant and/or membrane stabilizer.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Cell Culture and Experimental Media
Porcine pulmonary artery-derived endothelial cells were isolated from porcine pulmonary arteries and cultured as described previously [28]. Cells were subcultured in M-199 containing 10% fetal bovine serum (FBS; HyClone Laboratories, Inc., Logan, UT) using standard techniques. Purity of the cultures was determined by morphological criteria and by quantitatively measuring angiotensin-converting enzyme (ACE) activity or by their uptake of fluorescent-labeled acetylated LDL (1,1'-dioctadecyl-3,3,3'33-tetramethyl-indocarbocyanine perchlorate; Molecular Probes Inc., Eugene, OR).

The experimental media comprised M199 enriched with 1% FBS and either linoleic acid (90 µM) or TNF-{alpha} (500 U/mL; Knoll Laboratories, Whippany, NJ), and/or supplemental zinc (10 µM, as zinc acetate; Sigma, St. Louis, MO). Linoleic acid (>99% pure) was obtained from Nu-Chek Prep. (Elysian, MN). Preparations of experimental media with 18:2 and/or TNF were performed as described earlier [27,28]. For most experimental settings, cells were grown in 1% FBS concentration for eight days. At the end of this period, the designated groups were supplemented with zinc for 48 hours. Appropriate groups were treated with fatty acid for six to 24 hours or TNF for 1.5 hours prior to termination.

Zinc concentrations in endothelial cells or surrounding culture media were measured using a Perkin-Elmer 5,000 atomic absorption spectrophotometer (Perkin-Elmer, Norwalk, CT) as described previously [7,29]. Examples of cellular zinc levels under experimental conditions, whereby cells were cultured for several days in zinc-deficient (low serum) media and subsequently supplemented with zinc, were 0.08 ± 0.004 and 0.15 ± 0.02 µg zinc/mL/mg protein, respectively. Thus, zinc supplemented cultures demonstrates a marked increase in cellular zinc content compared with unsupplemented cultures. Medium zinc concentrations were significantly lower when M199 was supplemented with 1% FBS (0.026 ± 0.005 ppm) than when M199 was supplemented with 5% FBS (0.133 ± 0.004 ppm) or 10% FBS.

Measurement of Intracellular Oxidative State
Oxidative stress in viable cells was measured by 2,7-dichlorofluorescein (DCF) fluorescence. An oxidation-sensitive compound 2',7'-dichlorofluorescin diacetate (DCF-DA, Molecular Probes Inc., Eugene, OR) is converted into a nonfluorescent polar derivative by cellular esterases following incorporation into cells. Dichlorofluorescin is then oxidized to the fluorescent DCF by peroxidase and peroxides, including hydrogen peroxide. DCF fluorescence in 18:2 and/or TNF treated cell cultures was quantified using imaging methods as described by Goodman and Mattson [30]. Briefly, cells were loaded with DCF (50 µM in H2O) during the remaining 50 minutes of the experiment and followed by three washes in HANKS buffer. The relative fluorescence intensity of cells was assessed by a confocal laser scanning microscope system consisting of a Nikon Diaphot microscope using 488 nm excitation and 510 nm emission filters. Average pixel intensity was measured within each field and expressed in relative units of DCF fluorescence.

Transcription Factor (NF-{kappa}B and AP-1) Activation Studies: Electrophoretic Mobility Shift Assay
These transcription factors, which bind to enhancer elements on DNA, were determined in endothelial cells by an electrophoretic mobility-shift assay as described by Sen and Baltimore [31]. Nuclear extracts containing the NF-{kappa}B or AP-1 active proteins were prepared from cells according to the method of Dignam et al. [32]. Nuclear extracts were incubated for 20 to 30 minutes with a 32P-end-labeled oligonucleotide probe (Gibco/BRL, Gaithersburg, MD) containing the {kappa}B enhancer DNA element with a tandem duplicate of a NF-{kappa}B binding site (-GGGGACTTTCC-). The oligonucleotide containing the AP-1 binding site was a tandem repeat of the consensus sequence (-TGACTCA-) (Gibco/BRL). Incubation at room temperature was carried out in the presence of nonspecific competitor DNA. Following binding, the complexed DNA and the uncomplexed DNA in the mixture were resolved by electrophoresis in a 5% low-ionic-strength non-denaturing polyacrylamide gel and visualized by autoradiography. Control reactions using a 200-fold molar excess of unlabeled oligonucleotide probes were induced to demonstrate the specificity of the shifted DNA-protein complexes for NF-{kappa}B and AP-1.

Interleukin-6 Production
After exposure to linoleic acid or TNF, media were removed from the wells and frozen immediately at -80°C until IL-6 analysis was performed. The remaining cells were trypsinized and washed with phosphate-buffered saline (PBS) twice and resuspended in 0.2% SDS with 0.2 M NaOH for protein analysis [33] or washed with PBS and stained with trypan blue to determine cell viability. Each experimental group was done in triplicate, and total protein as well as cell viability were measured. IL-6 production and release into the medium was determined using the murine hybridoma cell line B9 (kindly supplied by Dr. L.A. Aarden, Emeryville, CA) as described by Helle et al. [34]. Media were collected from endothelial cultures following the treatment period and stored at -80°C until the IL-6 analysis. Viability of the B9 cell line is IL-6 dependent; thus, the incorporation of 3H thymidine by viable cells was a reflection of the quantity of IL-6 produced by endothelial cells. The final IL-6 concentration was determined from the standard curve, where eight pg of IL-6 per mL media corresponds to 12.6 x 107 units/mg recombinant IL-6.

Statistical Analysis
Data were analyzed statistically using a one-way analysis of variance (ANOVA). For each endpoint, the treatment means were compared in pairs using Fisher’s least significant difference procedure [35]. Statistical probability of p <= 0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
ACKNOWLEDGMENTS
 REFERENCES
 
Fig. 1 shows oxidative stress measurement in endothelial cells during zinc deficiency and supplementation and after exposure to linoleic acid (FA) or TNF. Both linoleic acid and TNF contributed significantly to cellular oxidative stress, which was partially blocked by zinc supplementation. Interestingly, zinc supplementation to control cultures alone (1% serum) also resulted in a small reduction of measurable oxidative stress.



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  		Fig. 1. Oxidative stress measurement in endothelial cells during zinc deficiency and supplementation and after exposure to linoleic acid (FA) or TNF. Cells were cultured for 8 days in media supplemented with only 1% FBS and subsequently enriched either with zinc (10 µM) for 48 hours and/or treated with FA (90 µM) for 6 hours or TNF (500 U/mL) for 1.5 hours. Values are mean±SEM (n=6). *Significantly different from respective cultures without added zinc.

 
The effect of zinc supplementation on linoleic acid-mediated activation of NF-{kappa}B in nuclear extracts from endothelial cells is shown in Fig. 2. Zinc supplementation markedly attenuated linoleic acid-mediated activation of NF-{kappa}B (Lane 4 compared to Lane 3). Our data also suggest that zinc deficiency alone, in the absence of additional oxidative stress, does increase slightly the capacity of NF-{kappa}B binding. Similar results were observed when measuring AP-1 (Fig. 3, Lane 5 compared to Lane 4). AP-1, however, was not up-regulated by zinc deficiency alone (Lane 3 compared to Lane 4).



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  		Fig. 2. Effect of linoleic acid (FA) and added zinc on activation of NF-{kappa}B. Cells were cultured for 8 days in media supplemented with 1% FBS and appropriate groups enriched with zinc (10 µM) for 48 hours and treatment with FA (90 µM) for 6 hours. Lane 1 = positive control (HeLa cell extract); lane 2 = competitor (unlabeled oligonucleotide); lane 3 = cells cultured in M199 enriched with 1% FBS (control); lane 4 = control + zinc (10 µM); lane 5=control + FA (90 µM); lane 6=control + zinc + FA.

 


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  		Fig. 3. Effect of linoleic acid (FA) and added zinc on activation of AP-1. Cells were cultured for 8 days in media supplemented with 1% FBS and appropriate groups enriched with zinc (10 µM) for 48 hours and treatment with FA (90 µM) for 6 hours. Lane 1=positive control (HeLa cell extract); lane 2=competitor (unlabeled oligonucleotide); lane 3=cells cultured in M199 enriched with 1% FBS (control); lane 4=control + zinc (10 µM); lane 5=control + FA (90 µM); lane 6=control + zinc + FA.

 
Fig. 4 shows IL-6 production in endothelial cells during zinc deficiency and supplementation and after exposure to linoleic acid or TNF. These data show that TNF exposure to endothelial cells resulted in a much higher production of IL-6 than did fatty acid exposure. However, in both cases, zinc supplementation caused a marked decrease in IL-6 expression by endothelial cells in response to TNF or fatty acid-mediated cell activation. In addition, zinc supplementation to control cultures alone (1% serum) also resulted in a small reduction in IL-6 production.



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  		Fig. 4. IL-6 production in endothelial cells during zinc deficiency and supplementation and after exposure to linoleic acid (FA) or TNF. Cells were culture for 8 days in media supplemented with only 1% FBS and subsequently enriched either with zinc (10 µM) for 48 hours and/or treated with FA or TNF for 12 hours. Values are mean±SEM (n=6). *Significantly different from respective cultures without added zinc.

 

   DISCUSSION
 
There is evidence that certain antioxidant nutrients and adequate antioxidant enzyme activities may protect against atherosclerosis by preventing metabolic and physiologic derangements of the vascular endothelium [36]. Of particular interest is zinc, because it may function as an antioxidant and membrane stabilizer [1,2,37]. Mechanisms of the protective function or functions of zinc in the pathogenesis of atherosclerosis, including vascular cell injury or dysfunction and the inflammatory response, are not clear. Epidemiological studies suggest that in some population groups lower serum levels of zinc are inversely associated with coronary artery disease [38]. Furthermore, zinc concentrations were significantly lower in atherosclerotic plaques of abdominal aortas of deceased patients with ischemic heart disease and acute myocardial infarction [39]. As mentioned earlier, there is evidence suggesting that zinc can act as an endogenous protective factor against atherosclerosis by inhibiting the oxidation of LDL by cells or transition metals [4,5]. In fact, compared with zinc-adequate rats, zinc-deficient rats fed a highly unsaturated fat diet (linseed oil) had increased plasma levels of total lipids and cholesterol and an increased susceptibility of LDL to copper-induced lipid peroxidation [40]. In addition, feeding a zinc-deficient diet to mice induced oxidative stress in hepatic and brain tissues as well as in peritoneal macrophages [41]. Our data clearly support the hypothesis that zinc can have antioxidant properties. Both linoleic acid and TNF contributed significantly to cellular oxidative stress, which was partially blocked by zinc supplementation. It has been suggested that zinc may protect sulfhydryl groups against oxidation and inhibit the production of reactive oxygen by transition metals, in addition to its function as a membrane stabilizer [37,42]. Dietary zinc deficiency was also reported to lower lymphatic absorption of vitamin E [43] and decrease concentrations of this vitamin in plasma [44] and selected organs [45], suggesting that dietary zinc deficiency may increase the nutritional requirement for vitamin E necessary to maintain adequate plasma and tissue concentrations.

Little is known about the requirements and functions of zinc in maintaining the integrity of the vasculature and particularly the vascular endothelium. We have shown that zinc is vital to endothelial integrity and that zinc deficiency causes a severe impairment of endothelial barrier function [46]. Media supplemented with physiological concentrations of zinc completely restored the cell integrity. We now also provide evidence that a critical sign of zinc "deficiency" may be a compromised control of activation of transcription factors, cytokine activity and endothelial cell inflammatory response. By inhibiting fatty acid or cytokine-mediated activation of NF-{kappa}B and AP-1, our data support the concept that zinc can have distinct protective properties during the inflammatory response in atherosclerosis [3,47].

Similarly to our previous work [46], the present study was designed to utilize physiological concentrations of supplemental zinc that are sufficient to restore metabolic functions of endothelial cells cultured in zinc-deficient media. We have experimented with several methods to produce zinc deficiency in endothelial cells. Included among these methods are cells cultured in low-serum medium (where serum was the only source of zinc), as well as in media previously exposed to different types of chelating agents (e.g., 1,10-orthophenathroline or chelex). All these techniques resulted in depletion of intracellular zinc levels and similar metabolic changes, such as decreased activities of zinc-dependent membrane-bound enzymes and endothelial barrier dysfunction [6,46].

Pathological conditions related to increased activity of TNF, such as inflammation or infection, may significantly influence zinc metabolism. It is known that during inflammation or infection there is an internal redistribution of zinc, with zinc being lost from some tissues such as plasma and zinc accumulating in other tissues, such as liver. The endothelium may be one tissue from which zinc is lost during an inflammatory response, and it is probable that similar depletion of zinc in endothelial cells may occur in atherosclerosis. We have shown previously that there is a depletion of cellular zinc in association with TNF-mediated endothelial cell injury which may lead to disruption of normal membrane integrity [6].

The nutritional status of the endothelium is likely to influence its response to TNF [3,48], and a marginal status of protective nutrients (e.g., zinc) may increase the susceptibility of the endothelial cell towards TNF-induced injury. Cellular enrichment with zinc has been shown to attenuate or prevent TNF-induced endothelial cell injury [6]. The protective mechanism or mechanisms of zinc against cytokine-induced injury still requires further clarification, but our new evidence suggests that TNF-mediated activation of transcription factors, such as NF-{kappa}B and AP-1, can be attenuated by zinc. Our data show that certain dietary lipids (e.g., unsaturated fatty acids, such as linoleic acid) also can activate these transcription factors, suggesting that the cellular lipid environment can greatly influence the inflammatory response of the endothelium. While NF-{kappa}B is activated in the response to oxidative stress, it appears that regulation of AP-1 activation might be more complex. For example, antioxidants might also lead to elevated levels of AP-1 through increased expression of c-fos and c-jun mRNA [49]. However, AP-1 binding sites were identified in the promotor regions of a variety of genes involved in inflammatory reactions, including genes encoding for ICAM-1 [50], VCAM-1 [51], and monocyte chemoattractant protein-1 (MCP-1; [52]). Evidence indicates that cooperation between activated AP-1 and NF-{kappa}B is required to stimulate the expression of these genes [51,52]. It is also not clear if zinc can attenuate transcription factors directly or possibly indirectly through activation of zinc finger proteins such as A20. Interestingly, A20 has been shown to block TNF-induced signal-transduction pathways and specifically to inhibit activation of NF-{kappa}B and AP-1 in carcinoma cells [53]. Also, in bovine aortic endothelial cells, expression of A20 down-regulated the expression of genes associated with TNF, LPS or hydrogen peroxide-induced endothelial-cell activation [54]. Interestingly, zinc supplementation can down-regulate NF-{kappa}B and AP-1, independently of the source of activation (e.g., linoleic acid or TNF). The fact that zinc can also in part block genes encoding for inflammatory cytokines, such as IL-6 or IL-8 [7], in endothelial cells strongly supports the hypothesis that adequate zinc nutrition may protect against inflammatory diseases such as atherosclerosis by inhibiting the activation of oxidative stress-responsive transcription factors, as well as expression of inflammatory cytokines.


   ACKNOWLEDGMENTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 ACKNOWLEDGMENTS
REFERENCES
 
Supported in part by grants from NRICGP/USDA (97-35200-4231), NIEHS/EPA (1 P42 ES 07380), and the Kentucky Agricultural Experimental Station.

Received July 1, 1998. Accepted November 1, 1998.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 ACKNOWLEDGMENTS
 REFERENCES
 

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