Antioxidant effects of dihydrocaffeic acid in human EA.hy926 endothelial cells

https://doi.org/10.1016/j.jnutbio.2004.07.002Get rights and content

Abstract

Dihydrocaffeic acid (DHCA) is a metabolite of caffeic acid with potent antioxidant properties. Since DHCA has been detected in human plasma following coffee ingestion, we tested the hypothesis that DHCA protects the endothelium from oxidative stress in a model in human-derived EA.hy926 endothelial cells. During culture for 16–24 hours, the cells accumulated DHCA against a concentration gradient to low millimolar concentrations. In α-tocopherol-loaded cells, DHCA spared α-tocopherol during overnight culture in a dose-dependent manner. In response to oxidant stress induced by a water-soluble free radical initiator, both α-tocopherol and DHCA diminished oxidation of cis-parinaric acid that had been incorporated into the cells, although their antioxidant activities were not additive. DHCA also decreased intracellular oxidation of dihydrofluorescein due to redox cycling by menadione. This suggests that the protective effects of DHCA were caused by scavenging of intracellular reactive oxygen species. DHCA also increased nitric oxide synthase activity in a dose-dependent manner in cultured cells, which was associated with a comparable increase in endothelial nitric oxide synthase protein. Although the DHCA concentrations required for these effects are higher than those likely to be present in plasma or the interstitium, these results indicate that DHCA can function as an intracellular antioxidant.

Introduction

Dietary plant polyphenols and flavonoids have been suggested to prevent colorectal cancer [1] and atherosclerosis [2], at least in part by providing defense against the oxidant stress thought to initiate and perpetuate these diseases. Of the polyphenols, chlorogenic acid (CGA) and its derivatives have long been of interest because of their relatively high content in coffee [3], fruits, vegetables, and wine that characterize the so-called “Mediterranean” diet [4]. Their benefits have been associated with their ability to function as antioxidants. For example, caffeic acid (CA), which corresponds to the hydroxycinnamic acid or “aglycone” portion of of CGA (Fig. 1), is an excellent scavenger of reactive species containing both nitrogen (e.g., peroxynitrite) and oxygen (e.g., superoxide) [5], [6]. At least part of the antioxidant effectiveness of CA may also relate to its ability to chelate transition metals, including iron and copper [7].

In biological systems, CA has shown promise as an antioxidant in vitro and at the cellular level. In a cell-free system, CA at 5 μmol/L completely protected human low-density lipoprotein (LDL) from peroxidative damage due both to a free radical initiator and to copper ions [7]. In addition, this effect was associated with sparing of α-tocopherol (α-TOC) in LDL [8]. The mechanism of the sparing effect of CA on α-TOC was attributed to direct recycling of α-TOC by CA, since the α-tocopheroxyl radical did not appear until after CA was consumed [8]. At the cellular level, CA has been shown to protect cultured human endothelial cells from apoptosis induced by oxidized LDL [9]. The IC50 (8.3 μmol/L) of the effect of CA on apoptosis was similar to that found to protect LDL [9]. Although phenolic derivatives such as ferulic acid (Fig. 1), which contain only one phenolic hydroxyl group, also protected LDL, CA was twice as potent in this regard [9]. In these studies, the protective effect of CA was due both to prevention of LDL oxidation and to a direct effect at the cellular level. We also recently reported that CGA, CA, and its reduced form dihydrocaffeic acid (DHCA) (Fig. 1) decreased lipid peroxidation in plasma and liposomes incubated with human erythrocytes, and that this was accompanied by a sparing effect on erythrocyte α-TOC [10]. In our study, CGA protected plasma and erythrocytes at concentrations as low as 5 μmol/L. Of relevance to this result is that a diet of 0.8% by weight CA in rats resulted in nonfasting CA concentrations in plasma of about 5 μmol/L  [11].

CA and related compounds may act as antioxidants in endothelial cells, which would have direct relevance to vascular disease and atherosclerosis. For example, CA and flavonols induce endothelium-dependent vasorelaxation in rat thoracic aorta [12]. At the cellular level, CA at the half-maximal concentration of 8 μmol/L prevented apoptosis in endothelial cells induced by oxidized LDL [9]. This was considered due both to prevention of LDL oxidation and to direct protection of the cells. To further investigate the cellular protection afforded by dietary polyphenolics, we studied whether DHCA can prevent the toxic effects of oxidant stress in EA.hy926 endothelial cells. The latter is a permanent cell line derived from human umbilical vein endothelial cells that expresses factor VIII antigen [13], oxidatively modifies human LDL [14], and shows calcium-dependent stimulation of endothelial nitric oxide synthase (eNOS) [15]. DHCA, which is a metabolic product of CA, was chosen for study since it has been detected in human plasma following coffee ingestion [16], and since it has a similar antioxidant potential in human erythrocytes relative to CA [10]. The present results show 1) that DHCA is taken up by EA.hy926 cells; 2) that it spares α-TOC and decreases membrane lipid peroxidation due to a free radical initiator; 3) that it decreases intracellular oxidant stress due to menadione redox cycling; and 4) that it enhances eNOS activity by increasing expression of the protein.

Section snippets

Materials

Menadione, (d,l)-α-tocopherol (α-TOC) and 3,4-dihydroxyhydrocinnamic acid (DHCA) were obtained from Sigma-Aldrich Chemical Co., (St. Louis, MO). α-TOC was prepared by dissolving it in ethanol to a concentration of 10 mmol/L. cis-Parinaric acid (c-PnA) was obtained from Molecular Probes, Inc. (Eugene, OR) and was prepared by dissolving in ethanol to 180 mmol/L. Protein was measured using the BCA kit from Sigma-Aldrich. The 2,2′-azobis(2-amidinopropane) dihydrochloride (AAPH) was obtained from

Results

The ability of EA.hy926 cells to take up and to retain DHCA was measured after overnight incubation in culture. As shown in Fig. 2, intracellular DHCA progressively increased to concentrations more than 10-fold those initially present in the incubations. This suggests that the cells are able to concentrate DHCA against a gradient. At concentrations >400 μmol/L, there was cell death related to toxicity of DHCA; consequently, for subsequent studies of antioxidant effects, lower concentrations of

Discussion

In this work we found that human-derived endothelial cells take up DHCA during overnight incubation in culture, and that they retain it under steady-state conditions against a concentration gradient (Fig. 2). This differs from human erythrocytes in short-term (1-hour) incubations, in which neither CA nor DHCA accumulated to concentrations higher than those present in the incubation medium [10]. The present results suggest that DHCA may be transported into endothelial cells via an active

Acknowledgments

This work was supported by National Institutes of Health grant AT01062, and by the Vanderbilt Institute for Coffee Studies. Dr. Huang is a Nestlé International Scholar at the Vanderbilt Institute for Coffee Studies.

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