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Protective Effect of Ginkgo Biloba Extract on Endothelial Cell Against Damage Induced by Oxidative Stress

Ren, De Cheng; Du, Guan-Hua; Zhang, Jun Tian

Journal of Cardiovascular Pharmacology: December 2002 - Volume 40 - Issue 6 - p 809-814
Original Articles
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The viability of bovine aortic endothelial cells (BAECs) treated with 0.1 m M H2O2 was decreased by 39.8%, and 100 mg/l EGb761 increased the viability by 20.6%. Exposure BAECs to H2O2 for 6 min resulted in a significant elevation in the intracellular free Ca2+. Pretreatment of BAECs with 10 mg/l and 100 mg/l EGb761 for 10 min showed a decrease in the intracellular free Ca2+, 4.5% and 20.6%, respectively. The apoptotic rate of BAECs measured by propidium iodide (PI) staining was (38.1 ± 2%) after 18 h of treatment with H2O2. Pretreatment of BAECs with 100 mg/l EGb761 for 1 h reduced the apoptotic rate to 27 ± 1%. In addition, there were about 5–7% of cells stained positive measured by TUNEL assay. When BAECs were exposed to 0.1 m M H2O2 for 18 h, the number of TUNEL-positive cells increased to 37–44%. When 10 mg/l EGb761 and 100 mg/l EGb761 were used, the TUNEL-positive cells decreased to 26.5 ± 3.1% and 17.5 ± 1.7%, respectively. Furthermore, EGb761 also inhibited caspase-3 activity induced by H2O2. It is concluded that EGb761 has protective effect on bovine vascular endothelial cells against damage induced by H2O2. Further studies are needed to clarify the mechanisms of action of EGb761.

Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China

Received September 17, 2001; accepted May 13, 2002.

Address correspondence and reprint requests to Dr. Du Guan-Hua at the National Center for Pharmaceutical Screening, Institute of Materia Medica, Chinese Academy of Medical Science and Peking Union Medical College, Beijing 100050, China. E-mail: dugh@imm.ac.cn

This work was supported by the National Natural Science Foundation of China, No. 3001161940.

Endothelial cells have a critical physiological role in maintaining normal vessel and organ function. Damage to endothelial cells may cause the alteration of endothelial permeability barrier and vascular tone. Endothelial cell injury induced by oxidative stress is very common. Therefore, the protection of endothelial cells against damage caused by oxidative stress is an important therapeutic strategy.

Ginkgo biloba extract (EGb761) is known to act on cardiac, cerebral, and pulmonary disorders, which is a standardized product of the amount of 24% ginkgo-flavone glycosides and 6% terpenoid (1). One of the mechanisms of the beneficial pharmacologic effects of EGb761 is its antioxidant action. EGb761 has the ability to scavenge free radicals such as superoxide anion, hydroxyl and peroxyl radicals, and nitric oxide (1).

Recent studies have shown that EGb761 had protective effects on myocyte, neuronal cells, and vascular endothelial cells (2–5). EGb761 inhibited monocyte and neutrophil adhesion to bovine cerebral microvascular endothelial cell (6), relaxed porcine basilar artery, and enhanced the TNS-induced relaxation via a NO pathway (7).

To study the effects of EGb761 on endothelial cells induced by oxidative stress, the protective effects of EGb761 on bovine aortic endothelial cells (BAECs) against damage induced by H2O2 were investigated.

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MATERIALS AND METHODS

Reagents

The fura 2-AM, propidium iodide (PI), and sulforhodamine B (SRB) were obtained from Sigma Chemical (St. Louis, MO, U.S.A.). An in situ cell apoptosis detection kit was purchased from Sino-America Biotechnology Company (Shanghai, China). Hydrogen peroxide (H2O2) and other agents were purchased from Beijing Chemical Reagents Company (Beijing, China) at AR grade. EGb761 was provided by the IPSEN Institute (Paris, France) and dissolved in redistilled water.

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Cell culture

The BAECs were isolated from bovine artery and cultured in Dulbecco modified Eagle medium containing 15% bovine serum, benzylpenicillin 100 kU/l and streptomycin sulfate 100 mg/l at 37°C in humidified 5% CO2 of air. After confluence, BAECs were passaged every 2–3 days. Cells from passage 6–10 were used for the experiments.

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Endothelial cell survival test

Cultured confluent BAECs were washed twice with PBS and incubated with 10 and 100 mg/l EGb761 for 1 h at 37 C, then 0.1 m M H2O2 was added and incubation was continued for 8 h. The survival of BAECs was measured by the method of SRB described by Liu and Jan (8) with slight modification. Briefly, after treatment with EGb761 for 8 h, cells were washed twice with phosphate-buffer saline (PBS) and fixed with 10% trichloroacetic acid in Hanks solution at 4°C for 1. Following fixation, cells were washed with tap water and stained with 0.4% SBR for 30 min. After removal of the SRB solution, the cells were washed three times with 1% acetic acid and air dried. The SRB dye was extracted with 200 μl of 10 m M tris(hydroxymethyl)-aminomethane buffer (pH 10.5) for measuring the absorbance at 565 nm with a spectrophotometer from FLUOstar Galaxy (BMG LabTechnologies GmbH, Offenburg, Germany).

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Determination of apoptosis

After the BAECs had been incubated with 100 mg/l EGb761 at 37°C for 1 h, H2O2 was added at the final concentration of 0.1 m M and incubation was continued for 18 h. Apoptosis of BAECs was evaluated by the DNA propidium iodide staining method described by Ding et al. (9) and TUNEL assay according to the apoptosis kit (Sino-American Biotechnology).

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Caspase-3-enzyme activity

Caspase-3-enzyme activity was performed according to Hermann et al. (10). Briefly, BAECs (1 × 106 cells) were lysed in buffer (1% Triton X-100, 0.32 mol/l sucrose, 5 m M ethylenediaminetetraaceticacid (EDTA), 1 m M phenylmethylsulfonyl fluoride, 1 mg/l aprotinin, 1 mg/l leupeptin, 2 m M dithiothreitol, and 10 m M Tris-HCl, pH 8) for 15 min at 4°C followed by centrifugation (20,000 g, 10 min). Capase-3 activity was detected in the resulting supernatants by measuring the proteolytic cleavage of the fluorogenic substrate N-acetyl-Asp-Glu-Val-Asp-7-amino-4-methylcoumarin (Ac-DEVD-AMC). The H2O2 increased caspase-3 activity by 175.5% using an excitation wavelength of 380 nm and an emission wavelength of 460 nm.

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Measurement of Ca2+i

The Ca2+i was measured as follows (11). After confluence, BAECs were collected and loaded by fura 2-AM in Hanks solution at 37°C in the dark for 30 min. Hanks solution contains NaCL 137 m M, KCL 5 m M, CaCL2 1.3 m M, MgSO4 · 7H2O 0.8 m M, Na2HPO4 0.6 m M, KH2PO4 0.4 m M, NaHCO3 3 m M, glucose 5.6 m M, pH 7.4. The final concentration of fura 2-AM was 5 μM. After loading with fura 2-AM, the cells were centrifuged at 200 g for 5 min twice and resuspended in Hanks solution containing 0.2% bovine serum albumin at 109 cell/l. The fura 2-AM loaded cells were incubated with EGb761 at 37°C for 10 min. Then 0.1 m M H2O2 was added and mixed for 6 min. The intracellular concentration of Ca2+i was measured with the Fluostar Galaxy (Germany) at λex 340 nm and 380 nm, λem 520 nm. The Ca2+i was calculated with Kd of 224 nmol/l by the following formula (12):EQUATION

where R indicates the rate of F340/F380, and Fmin and Fmax indicate the density of fluorescence with EDTA and triton X-100.

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Statistical analysis

Data are expressed as mean ± SD. The statistical analysis was evaluated by Student t test. A value of p < 0.05 indicated statistical significance.

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RESULTS

Effect of EGb761 on survival of BAECs

The survival of BAECs was significantly decreased after treatment with 0.1 m M H2O2 for 8 h, and the absorption value of SRB in the control group was much lower than that of the black group (p < 0.01). Findings showed that 10 mg/l and 100 mg/l EGb761 could improve the survival of BAECs and increase the absorption value by 2.9% and 20.6% (p < 0.01), respectively (Table 1). The EGb761 treatment alone did not affect cell viability (not shown).

TABLE 1

TABLE 1

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Effect of EGb761 on apoptosis of BAECs induced by H2O2

In normal cultured BAECs, the apoptotic rate was 7.1 ± 0.7% in current experiment. After treatment with 0.1 m M H2O2 for 18 h, the apoptotic rate increased to 38.1 ± 2%, as tested by the PI staining. The 100 mg/l EGb761 significantly reduced the apoptotic rate induced by H2O2 to 27 ± 1% (Fig. 1).

FIG. 1.

FIG. 1.

There were about 5% to 7% of cells stained positive in the normal BAEC group (black group) when tested by the method of TUNEL. BAECs cultured with 0.1 m M H2O2 for 18 h showed an increase in the number of apoptotic cells to 37%–44%. After pretreatment with 10 mg/l and 100 mg/l EGb761, the number of apoptotic cells markedly decreased to 26.5 ± 3.1% and 17.5 ± 1.7%, respectively (Fig. 2).

FIG. 2.

FIG. 2.

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Influence of EGb761 on caspase-3

The H2O2 increased caspase-3 activity by 175.5 ± 13.8%. Findings showed that 10 mg/l and 100 mg/l EGb761 reduced the H2O2 -induced increase of caspase-3 activity by 7 ± 1.6% and 33.2 ± 5.9%, respectively (Fig. 3).

FIG. 3.

FIG. 3.

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Effect of EGb761 on the intracellular Ca2+i increase induced by H2O2 in BAECs

The resting Ca2+i in BAECs was 111.14 ± 12.19 nM in Hanks solution (Fig. 4). It was found that 0.1 m M H2O2 increased the Ca2+i by 52.13% (p < 0.001). Findings also showed that 10 mg/l and 100 mg/l EGb761 inhibited the H2O2-induced Ca2+i elevation by 4.5% and 20.6% (p < 0.01), respectively, after BAECs were preincubated with EGb761 for 10 min.

FIG. 4.

FIG. 4.

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DISCUSSION

Some studies have already demonstrated the neuroprotective potency of EGb761 (13). But the protective effect of EGb761 on endothelial cells is less known. In the current experiments, the results showed that EGb761 was able to protect endothelial cells against H2O2-induced endothelial cell injury.

Our results showed that H2O2 decreased the survival rate of BAECs and increased the apoptotic rate of BAECs. In light of the lack of specificity of the SRB assay to differentiate between necrosis and apoptosis, we reasoned that not only necrosis but also apoptosis was involved in the H2O2-induced endothelial cell death. Several reports have shown that endothelial permeability increase, lipid peroxidation of cellular membrane, and denaturation of protein and nucleic acids induced by oxygen free radicals were involved in the pathways leading to irreversible injury (14–17). Moreover, lipid peroxidation may alter plasma membrane fluidity, leading to altered transport and enzyme properties. In particular, it has been hypothesized that the alternation in cytosolic free Ca2+i plays an important role in oxidative stress–induced endothelial cell injury (18–21). In addition, oxygen free radicals could induce cell apoptosis in various models (22). It has been shown that the overexpression of the endogenous antioxidant system or the protooncogene bcl-2 prevented apoptosis (23,24). Taken together, oxygen free radicals could result in necrosis and apoptosis through different pathways.

In our results, EGb761 increased the survival of endothelial cells and decreased the apoptotic rate of endothelial cells. Although the mechanisms by which EGb761 prevents endothelial cell damage induced by H2O2 were not fully clarified, the antioxidant property of EGb761 might be one of the protective mechanisms. EGb761 contains 24% ginkgo-flavone glycosides and 6% terpenoid. The flavonoid glycosides have been shown to scavenge hydroxyl radicals (25), superoxide anions (26), and lipid peroxides. The terpene lactones scavenge superoxide anions (27). Heron et al. (28) indicated that suppression of the production of reactive oxygen species reduced lipid peroxidation and membrane viscosity and enhanced membrane fluidity. Therefore, the radical scavenging capacity of EGb761 might play a critical role in inhibiting H2O2-induced endothelial cell injury.

Ca2+ is a widely used second messenger that regulates a variety of biologic processes including gene expression, neurotransmission, cell motility, and cell growth. The elevation of the intracellular level of Ca2+ could activate a lot of cellular proteases such as PKC and Ca2+-calmodulin kinases (29). Moreover, Ca2+ influx was a signal that initiated platelet-activating factor (PAF) synthesis (30), stimulated NOS activity (31), and appeared to serve as a common early signal for the initiation of apoptosis (32,33). Some investigations have indicated that H2O2 caused an increase in Ca2+I due to the influx of Ca2+ from the extracellular medium (34). Therefore, the intracellular Ca2+ elevation might in part be responsible for the necrosis and apoptosis of BAECs induced by H2O2. In the current study, we also found that EGb761 depressed the intracellular free Ca2+i elevation induced by H2O2. Thus, EGb761 might inhibit Ca2+-activated pathways through suppressing the elevation of intracellular Ca2+, which was beneficial in the protection of endothelial cells against oxidative injury.

In addition, our data also showed that EGb761 inhibited caspase-3 activity, which could in part explain the antiapoptotic action of EGb761. It is well known that the cysteine protease family of caspases plays an important role in apoptotic signal transduction. Caspase-3 is one of the best-characterized caspases and has been termed the “central executioner” of apoptosis. Once activated, caspase-3 can cleave numerous proteins involved in cell structure, signaling, and repair, and is essential for DNA fragmentation (35). Our studies confirmed the observation of other investigators that caspase-3 is activated during endothelial cell apoptosis induced by H2O2 (36). Besides inhibiting caspase-3 activity, the effect of EGb761 on other components of the apoptotic pathway, including up- and downstream caspases, remains undetermined. This awaits further investigation.

Mitochondria participate in the initiation of apoptotic programs either by releasing cytochrome c or by opening mitochondrial membrane transitions. These two events are upstream of caspase-3 activation. Mitochondrial dysfunction due to oxidative stress has been reported to be suppressed by EGb (13). Our previous studies also showed that EGb761 protected mitochondria against damage induced by anoxia–reoxygenation (1). These results suggest that protecting mitochondria against reactive oxygen species–induced dysfunction also might be involved in the protective actions of EGb761.

The PAF was a potent phospholipid autocoid implicated in a number of pathophysiologic conditions including inflammation, ischemia–reperfusion, and shock (37). Recent reports have indicated that PAF is a critical factor contributing to oxidant-mediated endothelial dysfunction. H2O2 has been shown to stimulate the production and release of PAF in endothelial cells (31). EGb761 was able to suppress the PAF-induced generation of reactive oxygen species and other actions of PAF due to its PAF antagonistic properties (38,39).

In summary, the results of the current study suggest that EGb761 is able to protect cultured endothelial cells against damage induced by H2O2. These effects of EGb761 are, at least in part, attributable to its antioxidant property and suppression of the intracellular Ca2+i elevation and caspases-3 activity. Certainly, further studies are needed to clarify the mechanisms of action of EGb761.

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REFERENCES

1. Du GH, Willet K, Mickalad AM, et al. Egb761 protects liver mitochondria against injury induced by in vitro anoxia/reoxygenation. Free Radic Biol Med 1999; 27:596–604.
2. Chen JX, Zeng H, Chen X, et al. Induction of heme oxygenase-1 by Ginkgo biloba extract but not its terpenoids partially mediated its protective effect against lysophosphatidylcholine (LPC)-induced damage. Pharmacol Res 2001; 43:63–9.
3. Yao Z, Drieu K, Papadopoulos V. The Ginkgo biloba extract Egb761 rescues the PC12 neuronal cells from beta-amyloid–induced cell death by inhibiting the formation of beta-amyloid–derived diffusible neurotoxic soluble ligands. Brain Res 2001; 889:181–90.
4. Ahlemeyer B, Junker V, Huhne R, et al. Neuroprotective effects of NV-31, a bilobalide-derived compound: evidence for an antioxidative mechanism. Brain Res 2001; 890:338–42.
5. Liebgott T, Miollan M, Berchadsky Y, et al. Complementary cardioprotective effects of flavonoid metabolites and terpenoid constituents of Ginkgo biloba extract (Egb761) during ischemia and reperfusion. Basic Res Cardiol 2000; 95:368–77.
6. Xu JP, Rui YCh, Li TJ. Antagonistic effects of Ginkgo biloba extract on adhesion of monocytes and neutrophils to cultured cerebral microvascular endothelial cells. Acta Pharmacol Sin 1999; 20:423–5.
7. Chen X, Liu LY, Li ZF. Cardiovascular protective effects and NO-mediated cerebrovasorelaxant effects on extract of Ginkgo biloba leaves. Natl Med J China 1998; 78:692–5.
8. Liu F, Jan KY. DNA damage in arsenite- and cadmium-treated bovine aortic endothelial cells. Free Radic Biol Med 2000; 28:55–63.
9. Ding X, Kuszynski CA, El-Metwally TH, et al. Lipoxygenase inhibition–induced apoptosis, morphologic changes, and carbonic anhydrase expression in human pancreatic cancer cells. Biochem Biophys Res Commun 1999; 266:392–9.
10. Hermann C, Zeiher AM, Dimmeler S. Shear stress inhibits H2O2-induced apoptosis of human endothelial cells by modulation of the glutathione redox cycle and nitric oxide synthase. Arterioscler Thromb Vasc Biol 1997; 17:3588–92.
11. Li XT, Wang YL. Effects of tetrandrine on cytosolic free calcium in cultured rat myocardial cells. Acta Pharmacol Sin 1996; 17:55–8.
12. Grynkiewicz G, Poenie M, Tsien RY. A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem 1985; 260:3440–50.
13. Ahlemeyer B, Mowes A, Krieglstein J. Inhibition of serum deprivation- and staurosporine-induced neuronal apoptosis by Ginkgo biloba extract and some of its constituents. Eur J Pharmacol 1999; 367:423–30.
14. Ager A, Gordon JL. Differential effects of hydrogen peroxide on indices of endothelial cell function. J Exp Med 1984; 159:592–603.
15. Ono DP, Yang WD. Exposure to low concentration of hydrogen peroxide causes delayed endothelial cell death and inhibits proliferation of surviving cell. Atherosclerosis 1995; 114:235–45.
16. Henning B, Chow CR. Lipid peroxidation and endothelial cell injury. Implications Atheroscler 1988; 4:99–106.
17. Nicotera P, Thor H, Orrenius S. Cytosolic free Ca2+ and cell killing in hepatoma1c1c7 cells exposed to chemical anoxia. FASEB J 1989; 3:59–64.
18. Franceschi D, Graham D, Sarasua M, et al. Mechanisms of oxygen-free radical-induced calcium overload in endothelial cells. Surg St. Louis 1990; 108:292–7.
19. Shasby DM, Lind SE, Shasby SS, et al. Reversible oxidant–induced increases in albumin transfer across cultured endothelium: alterations in cell shape and calcium homeostasis. Blood 1985; 65:605–14.
20. Lawrie AM, Zolle O, Simpson AWM. Modulation of mitochondrial Ca2+ in ECV304 endothelial cells by agents which elevate cAMP. Cell Calcium 1997; 22:229–34.
21. Hyslop PA, Hinshaw DB, Schraufstatter IU, et al. Intracellular calcium homeostasis during hydrogen peroxide injury to cultured P399D1 cells. J Cell Physiol 1986; 129:356–66.
22. Jabs T. Reactive oxygen intermediates as mediators of programmed cell death in plants and animals. Biochem Pharmacol 1999; 57:231–45.
23. Coyle JT, Puttfarchen P. Oxidative stress, glutamate, and neurodegenerative disorders. Science 1993; 262:689–95.
24. Hockenberry DM, Oltvai ZN, Yin XM, et al. Bcl-2 functions in an antioxidant pathway to prevent apoptosis. Cell 1993; 75:241–51.
25. Husain SR, Cillard J, Cillard P. Hydroxyl radical scavenging activity of flavonoids. Phytochemistry 1987; 26:2489–91.
26. Robak J, Gryglewski RJ. Flavonoids are scavengers of superoxide anions. Biochem Pharmacol 1988; 37:837–41.
27. Marcocci L, Packer L, Droy-Lefaix MT, et al. Antioxidant action of Ginkgo biloba extract EGb761. Methods Enzymol 1994; 234:462–75.
28. Heron DS, Schinitzky M, Hershkowitz M, et al. Lipid fluidity markedly modulates the binding of serotonin to mouse brain membrane. Proc Natl Acad Sci U S A 1980; 77:7463–7.
29. Kunsch C, Medford RM. Oxidative stress as a regulator of gene expression in the vasculature. Circ Res 1999; 85:753–66.
30. Lewis MS, Whatley RE, Cain P, et al. Hydrogen peroxide stimulates the synthesis of platelet-activating factor by endothelium and induces endothelial cell–dependent neutrophil adhesion. J Clin Invest 1988; 82:2045–55.
31. Shimizu S, Nomoto M, Naito S, et al. Stimulation of nitric oxide synthase during oxidative endothelial cell injury. Biochem Pharmacol 1998; 55:77–83.
32. Cohen JJ. Duck RC. Glucocorticoid activation of a calcium-dependent endonuclease in thymocyte nuclei leads to cell death. J Immunol 1984; 132:38–42.
33. Ueda N, Shah SV. Role of intracellular calcium in hydrogen peroxide–induced renal tubular cell injury. Am J Physiol 1992; 263:F214–21.
34. Geerawrts MD, Ronveraux-Dupal MF, Lemasters JJ, et al. Cytosolic free Ca2+ and proteolysis in lethal oxidative injury in endothelial cells. Am J Physiol 1991; 261:C889–96.
35. Troy CM, Stefanis L, Prochiantz A, et al. The contrasting roles of ICE family proteases and interleukin-1β in apoptosis induced by trophic factor withdrawal and by copper/zinc superoxide dismutase downregulation. Proc Natl Acad Sci U S A 1996; 93:5635–40.
36. Granville DJ, Shaw JR, Leong S, et al. Release of cytochrome c, Bax migration, Bid cleavage, and activation of caspases 2, 3, 6, 7, 8, and 9 during endothelial cell apoptosis. Am J Pathol 1999; 155:1021–5.
37. Lum H, Roebuck KA. Oxidant stress and endothelial cell dysfunction. Am J Physiol Cell Physiol 2001; 280:C719–41.
38. Smith PF, Maclennan K, Darlington CL. The neuroprotective properties of the Ginkgo biloba leaf: a review of the possible relationship to platelet-activating factor (PAF). J Ethnopharmacol 1996; 50:131–9.
39. Whatley RE, Nelson P, Zimmerman GA, et al. The regulation of platelet-activating factor production in endothelial cells. J Biol Chem 1989; 264:6325–33.
Keywords:

Apoptosis; Ca2+; Caspase-3; EGb761; Endothelial cell; H2O2

© 2002 Lippincott Williams & Wilkins, Inc.