Cerivastatin Does Not Prevent Oxidative Injury of Human Aortic Endothelial Cells

Abstract

The anti-atherogenic properties of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) have been well established in several circulatory beds. Increasing evidence suggests that statins may help attenuate ischemia–reperfusion injury, a beneficial effect that may be related to the antioxidant capabilities of statins; however, this remains controversial. We performed this study to determine whether the HMG-CoA reductase inhibitor cerivastatin can prevent oxidative stress-induced injury in cultured human aortic endothelial cells (HAEC).

The HAEC were subjected to oxidative stress in the absence and presence of increasing concentrations of cerivastatin (50 nM–1,000 nM). Oxidative stress was induced by increasing concentrations of hydrogen peroxide or endogenous superoxide anions generated by the inhibition of superoxide dismutase using diethylthiocarbamate (10 mM). Cell viability and mitochondrial activity were measured by mitochondria-dependent 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) conversion. Cell morphology was also examined using light microscopy.

Exposing HAEC to cerivastatin for 24 hours had no effect on cell viability using both cell morphology and MTT conversion: the HAEC incubated in 100 nM cerivastatin had 90% ± 2.2% viability of the control. As expected, hydrogen peroxide produced a concentration-dependent decrease in cell viability. Varying concentrations of cerivastatin pretreatment for ≤18 hours showed no protection of HAEC against hydrogen peroxide-induced injury. As a positive control, the prototype antioxidant N-acetyl-L-cysteine was cytoprotective even with the highest hydrogen peroxide concentration. Neither cerivastatin nor N-acetyl-L-cysteine protected HAEC against diethylthiocarbamate-induced oxidative injury at any concentration.

In this study, cerivastatin did not protect cultured HAEC against oxidative stress induced by hydrogen peroxide or diethylthiocarbamate.

Clinical and experimental evidence shows that 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors reduce the incidence of ischemic stroke. These agents have been shown to reduce infarct size in experimental animal models of stroke and myocardial infarction. ^1,2 These beneficial effects may be at least partially mediated by mechanisms independent of the lipid-lowering properties of HMG-CoA reductase inhibitors.

The HMG-CoA reductase inhibitors inhibit inducible nitric oxide synthase (iNOS) and improve endothelium-dependent relaxation under hypoxemic conditions by increasing endothelium-dependent nitric oxide synthase (eNOS) expression and preventing hypoxia-mediated down-regulation of eNOS activity in cultured human endothelial cells. ^3,4 In addition, HMG-CoA reductase inhibitors may attenuate the inflammatory cytokine responses that accompany ischemia, modulate leukocyte activity and adhesiveness, ⁵ inhibit vascular smooth-muscle-cell proliferation, ⁶ reduce the synthesis of chemokines (monocyte chemotactic protein-1), ⁷ and reduce the synthesis of adhesion molecules, such as E-selectin. ⁸ Moreover, HMG-CoA reductase inhibitors possess antioxidant properties that may ameliorate ischemic oxidative stress. ⁹ Despite these findings, the direct protective effect of HMG-CoA reductase inhibitors on human vascular cells against oxidative stress is not yet fully understood.

It has been suggested that reactive oxygen species such as superoxide anion, hydrogen peroxide, and hydroxyl radicals are primary mediators of the ischemia– reperfusion-induced damage. ⁹ In addition to oxygen-centered free radicals and oxidants, nitric oxide appears to play a variable role in ischemia–reperfusion and to modulate the biological effects of reactive oxygen species. ¹⁰ Nitric oxide undergoes radical-to-radical reactions with superoxide at near diffusion-limited rates to yield peroxynitrite, which is a potent oxidizing agent to lipids, aromatic amino acid residues, protein sulfhydryls, and DNA. Peroxynitrite initiates lipid peroxidation in biological membranes at rates that are 1,000-fold higher than they are for hydrogen peroxide. However, nitric oxide displays a dual action with lipids: in addition to pro-oxidant characteristics through peroxynitrite-mediated oxidation reactions, ¹¹ it has a potent capability to inhibit lipid radical chain propagation.

These multiple reactions between oxidative stress and nitric oxide, in addition to the increased nitric oxide activity in HMG-CoA reductase inhibitor-treated cells, led us to think that HMG-CoA reductase inhibitors might modulate oxidative stress-induced injury. We performed the present study to investigate the effect of cerivastatin administration on oxidative stress-induced injury in cultured human aortic endothelial cells (HAEC).

Materials and Methods

Cell Culture

Human aortic endothelial cells were harvested from aortas of human donor heart allografts with use of Type II collagenase as described previously. ¹² Cells were grown to confluence at 37 °C in a humidified incubator with 5% carbon dioxide in a culture medium containing Medium 199, 20-mM HEPES buffer, 50 μg/mL endothelial cell growth factor, 100 μg/mL heparin sulfate, supplemented with 2 mM glutamine, penicillin-streptomycin (100 U/mL and 100 μg/mL, respectively), and 10% fetal bovine serum. Experiments were performed on HAEC that were derived from multiple donors and split in <12 passages. The HAEC were incubated with cerivastatin either in varying concentrations or for varying durations.

Experimental Design

The protective effect of cerivastatin was investigated with use of HAEC in a model of oxidative stress that represented events in ischemia– reperfusion injury. Oxidative injury was induced by 2 mechanisms: first, the HAEC were treated with hydrogen peroxide. Concentrated hydrogen peroxide stock (30%) was diluted by phosphate-buffered solution to create decreasing concentrations, and the concentration was determined by the absorbance at 230 nm as previously described. ¹³ Second, oxidative injury was induced by inhibition of the endogenous antioxidant enzyme superoxide dismutase, using 10 mM diethylthiocarbamate (DETC). The DETC was administered after 30 minutes of treatment with the protein synthesis inhibitor cycloheximide (10 μM).

Monolayers of HAEC were divided into the following groups:

• Group 1. Monolayer cultures were incubated with and without cerivastatin, 100 nM, for 18 hours and then were exposed to increasing hydrogen peroxide concentrations ranging from 100 μM to 1,000 μM for 2 hours in order to obtain a dose-dependent response.

• Group 2. Monolayer cultures were incubated with and without N-acetyl-L-cysteine, 10 mM, for 30 minutes and then were exposed to increasing hydrogen peroxide concentrations ranging from 100 μM to 1,000 μM for 2 hours in order to obtain a dose-dependent response.

• Group 3. Monolayer cultures were incubated with increasing concentrations of cerivastatin ranging from 50 nM to 1,000 nM for 18 hours and then were either exposed or not exposed to a constant concentration of hydrogen peroxide, 200 μM, for 2 hours.

• Group 4. Monolayer cultures were incubated with a constant concentration of cerivastatin, 1,000 nM, for increasing durations (from 3 to 18 hours) and then either exposed or not exposed to a constant concentration of hydrogen peroxide, 200 μM, for 2 hours.

• Group 5. Monolayer cultures were incubated with DETC, 10 mM, with and without a constant concentration of cerivastatin 1,000 nM, after 30 minutes of pretreatment with protein synthesis inhibitor cycloheximide, 10 μM, for increasing durations (from 3 to 18 hours).

MTT and Lactate Dehydrogenase Assay

For MTT assay, cells were treated for 2 hours with hydrogen peroxide or DETC, washed with phosphate buffered solution, incubated in a conditioned medium for 1 hour with 2 μg/mL MTT, and then were lysed. Absorbance was measured at 570 nm using a spectrophotometric microplate reader (Multiskan EX, Labsystems; Helsinki, Finland). ¹⁴ Values were converted to MTT reduction using a standard curve generated by known numbers of viable cells. The MTT reduction for treated samples was then normalized to nontreated control samples and was reported as a percentage viability of the control. Lactate dehydrogenase activity released from damaged cells was determined using the LDH Cytotoxicity Detection Kit (Boehringer Mannheim; Mannheim, Germany) and the same microplate reader.

Materials and Reagents

Cerivastatin (BAY w 6228) was obtained from Bayer AG; Leverkusen, Germany. Medium 199, Dulbecco's Modified Eagles Medium, fetal bovine serum, L-glutamine, penicillin-streptomycin solution, trypsin-EDTA solution, phosphate buffered solution (×10), N-acetyl-L-cysteine, DETC, 30% hydrogen peroxide, MTT, and isopropanol were all obtained from Sigma Chemical Co.; Poole, UK. All tissue culture plastics were from Helena Biosciences (Tyne and Wear, UK).

Data Analysis

Data are summarized by group and expressed as means ± SEM of the indicated sample size or displayed as representative observations of at least 3 separate experiments. Statistical comparisons among groups were performed using ANOVA and appropriate post hoc tests. Statistical significance was accepted at P ≤0.05.

Results

The HAEC exposed to 100 nM cerivastatin did not show any significant change in cell viability during 24 hours as determined by cell morphology and MTT conversion. The cell viability was 90% ± 2.2% of the viability of the control.

In Group 1, HAEC that were not incubated with cerivastatin and were exposed to increasing concentrations of hydrogen peroxide for 2 hours showed a significant concentration-dependent decrease in cell viability. Compared with control, the percentages of viability were 93.72% ± 0.95%, 101.72% ± 2.23%, 57.4% ± 1.8%, 7.66% ± 0.6%, and 4.65% ± 0.17% with use of 100, 250, 500, 750, and 1,000 μM of hydrogen peroxide, respectively.

Cerivastatin pretreatment, 100 nM, for 18 hours did not make any significant difference in the decrease of cell viability compared with the non-cerivastatin-incubated monolayer cultures. The percentages of viability were 95.86% ± 0.34%, 95.18% ± 2.24%, 43.96% ± 2.32%, 6.88% ± 1.03%, and 5.34% ± 0.17% of the viability of the control with use of 100, 250, 500, 750, and 1,000 μM of hydrogen peroxide, respectively (Fig. 1).

Fig. 1 The results in Group 1.

CTR = control; H₂O₂ = hydrogen peroxide; MTT = mitochondria-dependent 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide; w/o = without

In Group 2, the HAEC that were not incubated with N-acetyl-L-cysteine and were subjected to increasing concentrations of hydrogen peroxide for 2 hours showed a significant decrease in cell viability similar to that in Group 1. Compared with control, the percentages of viability were 80.26% ± 3.24%, 81.07% ± 1.13%, 50.42% ± 0.34%, 5.42% ± 0.15%, and 4% ± 0.11% with use of 100, 250, 500, 750, and 1,000 μM of hydrogen peroxide, respectively. However, HAEC incubated with the prototype antioxidant N-acetyl-L-cysteine, 10 mM, before exposure to hydrogen peroxide had preserved cell viabilities, in comparison with control, of 68.38% ± 2.94%, 77.11% ± 1.2%, 75.81% ± 2.43%, 69.27% ± 2.76%, and 70.42% ± 2.86% at 100, 250, 500, 750, and 1,000 μM of hydrogen peroxide, respectively. The cytoprotective effect of N-acetyl-L-cysteine was significant at hydrogen peroxide concentrations of 750 and 1,000 μM, P < 0.05 (Fig. 2).

Fig. 2 The results in Group 2.

CTR = control; H₂O₂ = hydrogen peroxide; MTT = mitochondria-dependent 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide; NAC = N-acetyl-L-cysteine; w/o = without

In Group 3, cerivastatin in increasing concentrations up to 1,000 nM did not show any protective effect against a constant 200 μM of hydrogen peroxide-induced oxidative injury. Compared with control, the percentages of viability were 50.04% ± 4.85%, 48.52% ± 3.02%, 41.61% ± 3.1%, 44.87% ± 1.11%, and 33.97% ± 2.3% with use of 50, 100, 250, 500, and 1,000 nM of cerivastatin, respectively (Fig. 3).

Fig. 3 The results in Group 3.

H₂O₂ = hydrogen peroxide; MTT = mitochondria-dependent 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide; w = with; w/o = without

In Group 4, cerivastatin, 1,000 nM, in increasing durations of incubation up to 18 hours did not show any protective effect against a constant 200 μM of hydrogen peroxide-induced oxidative injury. Compared with control, the percentages of viability were 63.33% ± 7.25%, 64.63% ± 2.44%, 71.73% ± 5.12%, 67.99% ± 0.1%, 61.04% ± 0.99%, and 44.54% ± 0.53% at 0, 3, 6, 9, 12, and 18 hours, respectively (Fig. 4).

Fig. 4 The results in Group 4.

CRT= control; H₂O₂ = hydrogen peroxide; MTT = mitochondria-dependent 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide; w = with; w/o = without

In Group 5, DETC, 10 mM, in the presence of protein synthesis inhibitor cycloheximide, 10 μM, produced a significant decrease in cell viability. In comparison with control, the percentages of viability were 100% ± 1.11%, 75.17% ± 1.49%, 53.9% ± 2.06%, 53.22% ± 2.8%, 48.03% ± 1.87%, and 41.22% ± 0.44% at 0, 3, 6, 9, 12, and 18 hours, respectively. However, in the presence of 1,000 nM of cerivastatin, DETC-mediated cytotoxicity showed less of a decrease in cell viability, but not significant. The percentages of viability were 100% ± 3.22%, 89.96% ± 2.69%, 68.16% ± 3.86%, 65.07% ± 3.95%, 61.62% ± 3.97%, and 58.12% ± 3.36% at 0, 3, 6, 9, 12, and 18 hours, respectively (NS) (Fig. 5).

Fig. 5 The results in Group 5.

Ceriv = cerivastatin; CX = cycloheximide; DETC = diethylthiocarbamate; MTT = mitochondria-dependent 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide

Lactate Dehydrogenase Activity

Comparisons between the controls and each of the 5 groups showed no significant difference in the released lactate dehydrogenase activity.

Discussion

In this study, cerivastatin, in different concentrations and for different durations of incubation, did not offer any protective effect for HAEC against the oxidative stress induced by hydrogen peroxide. On the other hand, cerivastatin offered a slight but not significant protective effect for HAEC against the oxidative stress induced by DETC.

Clinical studies have shown that statins reduce the incidence of ischemic stroke and possess important neuroprotective properties that attenuate the effects of ischemia on the brain vasculature. ¹⁵ The effect of statins on stroke has been well established in experimental animal models. Statins not only up-regulate eNOS and down-regulate iNOS, ¹⁶ but they may also attenuate inflammatory cytokine responses that accompany ischemia. In addition, statins possess antioxidant properties that most likely ameliorate ischemic oxidative stress in the brain. Oxidative stress-induced vascular injury has been implicated in the entire spectrum of atherosclerotic lesion formation. ^17,18 In the pathogenesis of ischemic heart disease, it is suspected that an imbalance between the production of superoxide anions and nitric oxide in the vessel wall may be an important factor. Statins have been shown to effectively lower the superoxide-forming capacity of native endothelial cells. ¹⁹

Recently, it has been suggested that acute administration of simvastatin may modulate nitric oxide synthase messenger-RNA expression and reduce apoptosis after ischemia–reperfusion in a cell culture model. ²⁰ In our study, we used the recently developed HMG-CoA reductase inhibitor cerivastatin, which had demonstrated its efficacy in the nanogram range. The concentration of cerivastatin used in our in vitro experiments is equivalent to the maximal plasma concentrations of 2.27 to 2.88 μg/L found in healthy male volunteers; ²¹ therefore, the findings of this study could have many clinical implications. However, further in vitro and in vivo studies are required to confirm these findings.

This study showed that there is no cytoprotective effect of cerivastatin on human aortic endothelial cells under ischemia–reperfusion conditions. We found that cerivastatin does not have a direct antioxidative effect. The possible beneficial effect of cerivastatin against vascular damage may be due to the effect of other compounds, which have derived from the synthetic pathway of cholesterol metabolism. ²² Further studies are required to determine how statins achieve beneficial effects in patients with ischemic heart disease and stroke.