Alcohol Enhances Oxysterol-Induced Apoptosis in Human Endothelial Cells by a Calcium-Dependent Mechanism
Abstract—Controversy exists about the net effect of alcohol on atherogenesis. A protective effect is assumed, especially from the tannins and phenolic compounds in red wine, owing to their inhibition of low density lipoprotein (LDL) oxidation. However, increased atherogenesis occurs in subjects with moderate to heavy drinking habits. The purpose of this study was to investigate the influence of alcohol in combination with oxysterols on the endothelium. Cultured human arterial endothelial cells (HAECs) served as an in vitro model to test the cellular effects of various oxysterols. Oxysterols (7β-hydroxycholesterol, 7-ketocholesterol, and cholesterol-5,6-epoxides), which are assumed to be the most toxic constituents of oxidized LDL, induced apoptosis in HAECs through calcium mobilization followed by activation of caspase-3. Ethanol, methanol, isopropanol, tert-butanol, and red wine all potentiated oxysterol-induced cell death up to 5-fold, paralleled by further induction of caspase-3. The alcohol effect occurred in a dose-dependent manner and reached a plateau at 0.05% concentration. Alcohol itself did not affect endothelial cell viability, nor did other solvents such as dimethyl sulfoxide mimic the alcohol effect. So far as the physiologically occurring oxysterols are concerned, this effect was apparent only for oxysterols oxidized at the steran ring. The possibility of alcohol facilitating the uptake of oxysterols into the cell was not supported by the data from an uptake study with radiolabeled compounds. Finally, alcohol in combination with oxysterols did cause a dramatic increase in cytosolic calcium influx. Blockage of calcium influx by the calcium channel blocker aurintricarboxylic acid or the calcium chelator ethylene glycol-bis(β-aminoethyl ether)-N,N,N’,N’-tetraacetic acid abrogated the alcohol-mediated enhancement of oxysterol toxicity. We describe for the first time a mechanistic concept explaining possible adverse effects of alcohol in conjunction with physiologically occurring oxysterols on atherogenesis.
- Received February 10, 2000.
- Accepted September 18, 2000.
Atherosclerotic lesions contain oxidized LDL, which has been shown to promote the development of atherosclerosis1 2 3 4 Most of the cytotoxicity of freshly isolated human LDL is attributable to a minor fraction that has been oxidatively modified and highly enriched in cholesterol oxides (oxysterols).5 Oxysterols contribute to the atherosclerotic process by promoting smooth muscle cell proliferation as well as by causing injury to endothelial cells.6 7 8
Cell death can be divided in 2 distinct morphological entities, necrosis and apoptosis. Whereas necrosis is characterized by cellular swelling with subsequent rupture of the plasma membrane, apoptosis, or programmed cell death, involves activation of so-called caspases, ie, cysteine proteases cleaving specifically after aspartate residues.9 10 11 12 A cascade of activated caspases ultimately leads to nuclear condensation, DNA fragmentation, and destruction of cell structures such as the lamina.11 There is growing evidence that mitochondria are pivotal in this process of controlling life and death through their role in (1) disrupting electron transport with subsequent loss of ATP production; (2) releasing caspase-activating proteins such as cytochrome c; and (3) generating reactive oxygen species.13 Preceding these events, the mitochondrial inner transmembrane potential collapses, induced by oxidants or elevations in cytosolic calcium ([Ca2+]i).14 Oxidized LDL induces apoptosis of human endothelial cells by activation of CPP32- (or caspase-3)-like proteases.15 16 Apoptosis and necrosis induced by oxidized LDL are also calcium dependent and can be inhibited by blocking mitochondrial permeability.17 18
Low amounts of alcohol consumed on a regular basis have been shown to protect against cardiovascular disease and death.19 Phenolic compounds and tannins in red wine but not ethanol have been shown to exert antioxidant activity, leading to less oxidized LDL.20 The recent Bruneck study demonstrated a lower risk for light drinkers (<50 g alcohol per day) to develop carotid atherosclerosis (odds ratio 0.63), whereas higher alcohol intake (51 to 100 g/d) already led to a significant risk increase (odds ratio 2.04).21 Fifty grams of alcohol is equivalent to two 500-mL glasses of beer. Although the protective effect of moderate alcohol intake could be explained by attenuation of LDL oxidation, no previous study has investigated possible mechanisms for a direct adverse effect of alcohol on atherogenesis.
Endothelial Cell Isolation and Culture
Human iliac and renal arteries were obtained from organ donors (approved by the local ethics committee). Human arterial endothelial cells (HAECs) were isolated and passaged according to techniques described previously.22
Chemicals obtained from Sigma included a water-soluble cholesterol preparation (polyoxyethanyl-cholesteryl sebacate, No. C1145), 7-ketocholesterol (7-KC; 5-cholesten-3β-ol-7-one, No. C-2394), cholesterol 5α,6α-epoxide (No. C-2773), cholesterol 5β,6β-epoxide (β-CE; No. C-2648), 7β-hydroxycholesterol (7β-OH-C; No. H-6891), 25-hydroxycholesterol (25-OH-C; No. H-1015), cholestan-3β,5α,6β-triol (No. C-2523), 1,4-dioxane (No. D-9553), tert-butanol (No. B-2138), and aurintricarboxylic acid (ATA; No. A-1895). 7α-Hydroxycholesterol (No. C-6420) was obtained from Steraloids Inc. For radioactive uptake studies, 1α,2α[N]-3H-cholesterol (No. NET-139) and 25-[26,27-3H]-hydroxycholesterol (No. NET-674) from New England Nuclear were used.
Bromodeoxyuridine (BrdU) ELISA was performed in 96-well plates. For further details, please see http://www.atvb.ahajournals.org.
MTS Viability Assay and Measurement of Cell Death
A CellTiter-96 AQueous nonradioactive cell viability assay (Promega) was used to assess cell viability and proliferation. For further details, please see http://www.atvb.ahajournals.org.
Flow Cytometry for Measurement of Annexin V and Propidium Iodide Uptake
Double staining for annexin V and propidium iodide was performed as previously described.23 For further details, please see http://www.atvb.ahajournals.org.
Whole-Cell Extracts and Determination of CPP32 Activity
Cells were washed 3 times in cold PBS and then lysed for 30 minutes at 4°C in lysis buffer containing 50 mmol/L Tris-HCl (pH 8.0), 2 mmol/L EDTA (pH 8.0), 150 mmol/L NaCl, 0.5% Nonidet P-40, and the following protease inhibitors: 0.5 mmol/L PMSF, 1 μg/mL aprotinin, 1 μg/mL leupeptin, and 0.5 μg/mL pepstatin A. After centrifugation at high speed, the supernatant was collected and the protein content of all samples determined by using the Bio-Rad protein assay with γ-globulin as the standard. A commercial apopain assay kit (Bio-Rad) was used to measure CPP32 activity in whole-cell extracts. Seventy-five micrograms of protein extracts was used and subsequently diluted in 1000 μL of 1× reaction buffer from the kit before 10 μL of the fluorogenic substrate (Ac-DEVD-AFC) was added. Fluorescence was measured in a Bio-Rad VersaFluor fluorometer with a filter setting of 390 nm (excitation) and 550 nm (emission) at various time points. The increase in fluorescence was linear over 3 hours and was standardized with free AFC.
Determination of [Ca2+]i With Fluo 3-AM
[Ca2+]i was determined by using the cell-permeable calcium probe fluo 3-AM. Endothelial cells were incubated for 4 to 12 hours with the indicated oxysterols. The culture medium was then replaced by plain growth medium containing 1 μmol/L fluo 3-AM, and the cells were incubated for an additional 45 minutes at 37°C. After washing the cells 3 times in PBS, 1 mL of HEPES-buffered PBS (10 mmol/L HEPES) was added to the intact cell layer. Then the cells were carefully scraped off the plate into a 1-mL cuvette and fluorescence was recorded. [Ca2+]i determination was performed at the excitation wavelength of 520 nm and emission at 510 nm. To calculate [Ca2+]i, a standard curve was generated with different concentrations of free calcium and fluo 3.
Uptake of Radioactive Cholesterol and 25-OH-C
Cells (50 000 per well) were plated in 6-well culture dishes. 1α,2α[N]-3H-cholesterol and 25-[26,27-3H]-hydroxycholesterol were divided into aliquots in 1.5-mL Eppendorf tubes. The solvent, which contained alcohol, was completely evaporated by centrifugation in a speed vac, and radioactive cholesterol was redissolved in endothelial growth medium. Where indicated, 0.5% ethanol was added for comparison of uptake. Cells were incubated with a defined amount of the indicated [3H]cholesterol derivative (80 000 counts per minute per well) over 1 to 20 hours. The concentration of the labeled compounds, as calculated from their specific activities, was ≈5 ng/mL. At the end of the incubation period, the cells were washed in PBS, lysed in 450 μL of 0.5 mol/L NaOH, and transferred to scintillation vials. Measurement was conducted in a scintillation counter (4 minutes per vial) with a tritium-sensitive channel.
All values are expressed as mean±SEM, whether in text or graphically. Differences between groups were assessed by an unpaired t test (2 groups) or 1-way ANOVA (>2 groups). Subsequent multiple comparisons for ≥3 groups were performed only when the 1-way ANOVA reached statistical significance (P<0.05) by using the Student-Newman-Keuls test to compare all pairs or Dunnett’s post test to compare each group against controls. The level of statistical significance is indicated in the figures: NS indicates not significant, *P<0.05, #P<0.01, and §P<0.001. Statistical calculations were carried out with GraphPad Prism version 3.00 for Windows (GraphPad Software).
Alcohol Enhances Oxysterol-Induced Toxicity in HAECs
To investigate the influence of alcohol on oxysterol-induced death in endothelial cells, we dissolved all cholesterol oxides in cell culture medium with addition of 0.04% acetone. The same results were achieved when acetone was replaced by dioxane as the solvent. Addition of as little as 0.02% ethanol was sufficient to raise the rate of cell death induced by 12 μg/mL β-CE from 8±3% to 62±2% (P<0.01; Figure 1A⇓). Lower oxysterol concentrations required higher ethanol concentrations to reach maximum cell death, indicating an “additive” mechanism. To exclude the possibility that alcohol could shift the redox balance in the cell, all experiments were repeated with tert-butanol, which cannot be further oxidized. Addition of 0.01% tert-butanol to the medium also augmented cell death ≈4-fold (Figure 1B⇓), whereas tert-butanol up to a concentration of 1% was perfectly tolerated by the cells. Ethanol 0.05% also potentiated cell death induced by 7-KC or 7β-OH-C (Figures IA and IB; please see http://www.atvb.ahajournals.org). Red wine, methanol, and isopropanol showed a similar trend (data not shown). Interestingly, the toxic effect of 25-OH-C, which is oxidized only at the C25 atom as part of the aliphatic side chain, could not be augmented by 0.05% ethanol (Figure⇓ IC; please see http://www.atvb.ahajournals.org). Finally, the antiproliferative effect of nontoxic concentrations of oxysterols was evaluated by measuring BrdU incorporation (Figure⇓ II; please see http://www.atvb.ahajournals.org). Again, 0.05% ethanol augmented the antiproliferative effect of steran ring–oxidized oxysterols only.
Oxysterols Induce Apoptosis in HAECs by Induction of CPP32
Programmed cell death, or apoptosis, requires initiation of a specific signaling cascade in the cell, leading to downstream activation of caspases. CPP32 is an important downstream effector in apoptosis and has been shown to mediate oxidized LDL–induced death of human endothelial cells.24 Flow-cytometric analysis revealed no significant increase in propidium iodide uptake as a sign of increased cytoplasmic membrane permeability (Figure⇑ IIIA; please see http://www.atvb.ahajournals.org), whereas annexin V–positive particles increased from 1% to 9% (P<0.05) as a sign of programmed cell death (apoptosis, Figure⇑ IIIB; please see http://www.atvb.ahajournals.org). Furthermore, we found that treatment of HAECs with both 7-KC and 7β-OH-C led to induction of CCP32 activity (Figure⇑ IVA; please see http://www.atvb.ahajournals.org). Caspase-8, which is involved in Fas/Fas ligand-mediated apoptosis, was not activated (data not shown). To investigate whether increased apoptosis with addition of alcohol was correlated with CCP32 activity, we treated endothelial cells for 16 hours with 7-KC at different concentrations, with or without 0.05% ethanol. As expected, CCP32 activity was significantly higher when ethanol was added to the cells (Figure⇑ IVB; please see http://www.atvb.ahajournals.org). Ethanol itself did not have any influence on caspase activity. The increase in CCP32 activity with addition of ethanol was even more apparent when concentrations of 7β-OH-C alone (without ethanol) were subtoxic (from 0% to 97% of control, P<0.01; Figure 2⇓).
Kinetics of Alcohol Effect
To evaluate the critical time frame for the potentiation of apoptotic cell death by alcohol, cells were treated with 12 μg/mL β-CE, and the medium was replaced by normal growth medium at the indicated time points (Figure⇑ VA; please see http://www.atvb.ahajournals.org). The results suggest that alcohol-enhanced apoptosis cannot be reversed after a few hours, because cell death at 24 hours reaches >90% of the respective cell death toxicity even when an oxysterol-containing medium is replaced after 4 hours with fresh growth medium. To further characterize the mechanism by which alcohol potentiates oxysterol-induced apoptosis, we looked at a possible impact of alcohol on the kinetics of oxysterol uptake. First we confirmed a concentration-dependent uptake of oxysterols into the cell by using 3H-labeled cholesterol and 25-OH-C. Addition of cold cholesterol competed with 3H-labeled cholesterol or 25-OH-C and reduced their uptake significantly (Figure⇑ VB; please see http://www.atvb.ahajournals.org). Addition of 50 μg/mL cold cholesterol also reduced apoptosis induced by either 12 μg/mL β-CE or 18 μg/mL 7-KC ≈10-fold, independent of the presence or absence of 0.05% ethanol (Figure 3A⇓). To test the possibility that ethanol increased membrane permeability for oxysterols, uptake of 3H-labeled cholesterol and 25-OH-C was measured in the absence or presence of 0.05% ethanol. At no time point could a significant increase in cholesterol or oxysterol uptake under the influence of alcohol be found (Figure 3B⇓).
Inhibition of Calcium Influx Abrogates Potentiation of Oxysterol-Induced Apoptosis by Alcohol
All of the experiments shown so far point to a mechanism for alcohol-induced enhancement of oxysterol toxicity that is somehow initiated within 4 hours of oxysterol application. Calcium, which has long been known as a rapid-messenger molecule in intracellular signaling, has recently been shown to play an important role in mediating apoptosis induced by oxidized LDL.18 In HAECs treated with 7-KC, we found a substantial increase (+50%) in [Ca2+]i levels after only 4 hours (Figure 4A⇓), reaching a maximum after 8 to 10 hours. Ethanol by itself (0.05% concentration) led to an increase of only 10% in [Ca2+]i but potentiated calcium influx in cells treated with subtoxic levels of oxysterols (Figure 4B⇓). The dramatic rise in calcium influx generated by combined treatment of oxysterols and alcohol was significantly inhibited when ATA, a calcium channel inhibitor, was added simultaneously (32% vs 139%, P<0.001; Figure 4B⇓). EGTA, a calcium chelator, reduced the amount of calcium influx by only 42% (81% vs 139%, P<0.001; Figure 4B⇓). Finally, inhibition of calcium influx by cotreatment with ATA abrogated alcohol-enhanced cell death induced by 7-KC (7% vs 24%, P<0.01) or β-CE (2% vs 13%; Figure 4C⇓). Again, EGTA led to a significant but less dramatic inhibition of cell death (28% vs 39%, P<0.05).
Oxysterols and Apoptosis in Endothelial Cells
There is increasing evidence that cholesterol oxidation products are far more damaging to vascular health than is native cholesterol itself.25 Cholesterol oxides can be exogenously obtained from the diet as well as endogenously generated in vivo through free radical or enzyme reactions.26 Statistically, elevated serum cholesterol is associated with an increased risk of coronary artery disease. However, numerous exceptions occur at both ends of the spectrum, suggesting cholesterol oxides as the missing piece of the puzzle. They can also be found in human atherosclerotic plaques.2 In cell culture, it has long been known that cholesterol oxides, here referred to as oxysterols, exert a toxic effect on human endothelial cells.5 7 Recent studies have demonstrated that oxidized LDL causes endothelial cell apoptosis through induction of CPP32.15 16
We previously established an in vitro model for endothelial injury by using primary cultures of human endothelial cells.27 28 29 Here, we tested a variety of physiologically occurring oxysterols in HAECs for their potential to cause endothelial injury. Initially we found all oxysterols to induce programmed cell death, or apoptosis, characterized by nuclear condensation, loss of mitochondrial activity, and subsequent activation of CPP32. Although receptors for oxidized LDL (LOX-1 and -2) have been cloned recently,30 a putative oxysterol receptor has not yet been found.
Calcium-Mediated Enhancement of Apoptosis by Alcohol
Because many studies claim a protective effect of alcohol on the process of atherogenesis, we next investigated its influence on oxysterol-induced apoptosis in cultured endothelial cells. Oxysterols are preferably dissolved in ethanol, which prevents analysis of a possible synergistic effect of the 2 substances. Using nonalcoholic solvents, we systematically examined the influence of various alcohols on oxysterol-mediated cell death. Remarkably, methanol, ethanol, isopropanol, and red wine all led to a significant augmentation of oxysterol toxicity. The alcohol effect was dose dependent and eventually reached a plateau. Fluorescence-activated cell sorting analysis showed increased annexin V staining with addition of alcohol, and measurement of CPP32 activity revealed substantial induction when alcohol was added to oxysterols. Both results strongly suggest the enhancement of apoptosis by alcohol. Using radioactively labeled cholesterol derivatives, we have shown that oxysterol uptake is not altered by alcohol. Competitive inhibition of oxysterol uptake with cold cholesterol prevented cell death, thus excluding acute alcohol toxicity. tert-Butanol, which cannot be oxidized further, gave results similar to those of the other alcohols, revealing that alcohol itself rather than any of its metabolites is responsible for the potentiation of cell death. Surprisingly, alcohol did not enhance the apoptotic effect of 25-OH-C, an oxysterol with oxidation of the aliphatic side chain. This side chain somewhat resembles tert-butanol, suggesting a “built-in” alcohol effect.
Apoptosis is known to be mediated by liberation of calcium from intracellular stores.14 18 Negre-Salvayre and Salvayre31 and Escargueil-Blanc et al32 first described the calcium dependence of apoptosis induced by oxidized LDL, as well as the protective effect of calcium channel blockers and calcium chelators. Given our results, it seems most likely that oxysterols are the bioactive compounds of oxidized LDL, which initiate the calcium signaling. Chu et al33 showed that ethanol-induced, calcium-activated potassium channels reconstituted into planar lipid bilayers. In cultured human endothelial cells, Li et al34 showed that ethanol significantly increased the open-state probability of calcium-activated potassium channels. Our experiments with the calcium channel blocker ATA as well as the calcium chelator EGTA suggest that alcohol leads to enhanced conductance of membrane-bound calcium channels in human endothelial cells, thus facilitating oxysterol-induced calcium influx and subsequent potentiation of cell death.
Alcohol and Atherosclerosis
The importance of oxysterols in human atherosclerosis is documented by the data from Zieden et al,35 showing that increased plasma 7β-OH-C concentrations alone identified a population with high risk for cardiovascular disease. Kiechl et al21 investigated for the Bruneck study group the effects of alcohol consumption on the incidence and progression of atherosclerosis over a 5-year period. They found that intake of 51 to 99 g of alcohol per day was already sufficient to double the odds ratio for a progression of carotid atherosclerosis from 1.0 to 2.04. The deciding factor was the amount of alcohol rather than the type of alcoholic beverage. Finally, high LDL cholesterol was a severe risk condition in abstainers, moderate drinkers, and heavy drinkers but not in consumers of low amounts of alcohol (<50 g/d). Thus, it seems fair to assume a protective effect of alcohol in small quantities, ie, through antithrombotic and antioxidative mechanisms. Our in vitro model suggests that cholesterol derivatives, when already oxidized, exert a more toxic effect on the endothelium when they occur in conjunction with alcohol of any source. Because addition of the phenolic compounds resveratrol and quercetin did not abrogate cell death in our model (data not shown), one obviously has to distinguish between already oxidized cholesterol products (oxysterols) and not-yet-oxidized LDL to discuss the beneficial effects of alcohol. Extrapolation of this “alcohol” effect to clinical conditions could explain the extremely adverse influence of moderate to high alcohol intake combined with high LDL levels (and thus, higher amounts of oxysterols). Further clinical studies are needed to differentiate more accurately among the many components of LDL, such as oxysterols, to evaluate the exact source of endothelial cytotoxicity.
This work was supported by grants DFG Sp-502/2 of the Deutsche Forschungsgesellschaft (to I.S.), the University of Tübingen (fortüne Sp-504/98 to I.S.), and the Karl-Kuhn-Stiftung (to I.S.). The assistance of Heike Runge for isolating and subculturing human endothelial cells and Kerida Shook for proofreading of the manuscript are greatly appreciated.
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