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Inhibitions of vascular endothelial growth factor expression and foam cell formation by EGb 761, a special extract of Ginkgo biloba, in oxidatively modified low-density lipoprotein-induced human THP-1 monocytes cells.

Abstract

It has been reported that oxidatively modified low-density lipoprotein (Ox-LDL) involvement with vascular endothelial growth factor (VEGF) and foam cell formation play an important role in atherosclerosis (AS). Protective effects of Ginkgo biloba extract (EGb 761) have been identified for some cardiovascular and neurological disorders. The aim of this study was to investigate whether Ox-LDL regulates VEGF expression in human THP-1 monocytes, as well as the effect of EGb 761 on VEGF expression and the formation of foam cells. After exposure to Ox-LDL alone or in combination with EGb 761 for up to 48 h, cell viability was measured using the MTT assay. VEGF protein content in the supernatant was analyzed by enzyme-linked immunosorbent assay (ELISA). VEGF mRNA was determined by real-time PCR. To determine the effect of EGb 761 on foam cell formation, an Ox-LDL-induced foam cell model was used. Ox-LDL inhibited the growth of THP-1 cells and EGb 761 increased the cell survival rate. Ox-LDL markedly increased VEGF expression in THP-1 cells in a time- and concentration-dependent manner, which was significantly suppressed by EGb 761. EGb 761 also inhibited monocyte/macrophage-derived foam cell formation. These results suggest that Ox-LDL is involved in the development of human AS through VEGF induction in monocytes, and that EGb 761 prevents in vitro atherogenesis, probably via downregulation of VEGF expression in monocytes and inhibition of monocyte/macrophage-derived foam cell formation. The findings suggest a mechanism for the in vivo anti-AS effect of EGb 761 and support its potential clinical use in AS.

[C]2008 Elsevier GmbH. All rights reserved.

Keywords: Vascular endothelial growth factor; Ginkgo biloba extract; Atherosclerosis; Oxidized low-density lipoprotein; Foam cells

Introduction

Ginkgo biloba extract EGb 761 is an extract from leaves of the ancient tree Ginkgo biloba. Recently, EGb 761 has been used as a therapeutic agent for some cardiovascular and neurological disorders (Liu et al., 2007; Ramassamy et al., 2007). EGb 761 is a potent scavenger of several reactive oxygen species, such as singlet oxygen, superoxide anions, and hydroxyl radicals (Eckert et al., 2003; Lippi et al., 2007). EGb 761 protects the heart against ischemia reperfusion damage and endothelial dysfunction evoked by oxygen free radicals (Eckert et al., 2003; Akiba et al., 2004; Fan et al., 2006). EGb 761 reduced the atherosclerotic nanoplaque formation and size in cardiovascular high-risk patients (Rodriguez et al., 2007). Although the exact mechanism is not known, accumulating in vitro and in vivo evidence demonstrates the protective effects of EGb 761 in cardiovascular and cerebrovascular diseases (Rodriguez et al., 2007; Siegel et al., 2007).

Atherosclerosis (AS) is an inflammatory disease characterized by lipid accumulation and foam cell formation by monocyte-derived macrophages and smooth muscle cells (Tabata et al., 2003; Deng et al., 2005). Recent findings indicate that oxidized low-density lipoprotein (Ox-LDL) may play a key role in the AS process (Shashkin et al., 2005). Ox-LDL is avidly taken up by macrophages and leads to the formation of foam cells (Kunjathoor et al., 2002; Shashkin et al., 2005). The development of macrophage-derived foam cells that contain massive amounts of cholesteryl esters is a hallmark of the early stage of AS lesions (Libby, 2002; Yang et al., 2004).

During the atherogenesis process, vascular endothelial growth factor (VEGF), also known as vascular permeability factor, is released from macrophages within lesions (Ramos et al., 1998; Matsumoto and Mugishima, 2006). VEGF is a heparin-binding homo-dimeric glycoprotein ([M.sub.r] = 46,000) belonging to the platelet-derived growth factor family. There are four different VEGF isoforms (of 121, 165, 189, and 206 amino acids in humans) that are generated by alternative splicing of a single pre-mRNA species. VEGF is an important regulator of vasculogenensis, angiogenesis, and vascular permeability in vivo (Matsumoto and Mugishima, 2006). It has also been suggested that VEGF plays an important role in atherogenesis.

Although EGb 761 exhibits antioxidant effects both in vivo and in vitro, it is not clear if it can directly inhibit atherogenesis. In the present study, we tested the hypothesis that EGb 761 can downregulate Ox-LDL-induced VEGF expression in THP-1 cells and inhibit foam cell formation. The results provide a rationale for the potential use of EGb 761 in clinical AS.

Materials and methods

Chemicals and reagents

RPMI 1640 medium and Hepes were purchased from Gibco. Fetal bovine serum was purchased from Bio International. LDL and Oil red O were from Sigma. MMLV and reverse transcription-polymerase chain reaction (RT-PCR) kits were purchased from Promega. SYBR Green PCR Master Mix was from Takara (Japan). The Human Vascular Endothelial Growth Factor (Hu VEGF) ELISA kit was purchased from Invitrogen. EGb 761 purchased from Dr. Willmar Schwabe Pharmaceutical Co., Karlsruhe, Germany, a total ginkgo flavonoid content of 24% and a ginkgo lactone content of 6%. EGb 761 was diluted in RPMI 1640 medium at different concentration prior to addition to cell cultures.

Ox-LDL preparation

LDL (d = 1.019-1.063 kg/l) was sterilized by filtration through 0.45-um Millipore membranes and stored at 4 [degrees]C as previously described (Yang et al., 2003). After EDTA removal by dialysis, the LDL was oxidized by incubation in 10 [micro]mol/l [CuSO.sub.4] at 37 [degrees]C for 16 h, and then dialyzed in phosphate-buffered saline containing 0.1 mmol/l EDTA disodium salt at 4 [degrees]C for 24 h. Oxidation was monitored by measuring the amount of thiobarbituric acid-reactive substances (10.7 nmol/mg protein) produced. Owing to their higher negative charge, Ox-LDL showed greater mobility on agarose gel electrophoresis compared to LDL.

Cell culture

The human THP-1 monocyte line (passage 20, Shanghai Institute of Cell Biology, Chinese Academy of Sciences) was grown in RPMI 1640 medium or Dulbecco's modified Eagle's medium containing 10% (v/v) fetal bovine serum, 2 mmol/l L-glutamine, 0.45% (w/v) glucose, 10 mmol/l Hepes, 1 mM sodium pyruvate, 1 x [10.sup.-5] mol/l [beta]-mercaptoethanol, 100U/ml penicillin, and 100 [micro]g/ml streptomycin in a humidified 5% [CO.sub.2] atmosphere at 37 [degrees]C. The medium was replaced every 3 days during culture. For experiments, THP-1 cells were pretreated with serum-free RPIM 1640 for 12 h. The medium was then replaced with fresh serum-free medium containing EGb 761 (0, 0.001, 0.01, and 0.1 mg/l) with or without Ox-LDL (0, 10, 20, 40 mg/l) and the cells were incubated for a further 24 or 48 h.

MTT assay for cell viability

After exposure to Ox-LDL or EGb 761 for 48 h, cell viability was measured using the MTT assay (Jiang et al., 2007). In brief, cells were incubated with 1 g/1 MTT for 4h at 37 [degrees]C. MTT/formazan was extracted by overnight incubation at 37 [degrees]C with l00 [micro]l of extraction buffer (20% sodium dodecyl sulfate, 50% formamide adjusted to pH 4.7 with 0.02% acetic acid and 0.025 N HC1). The optical density was measured at 570 nm using extraction buffer as a blank. Statistically significant differences between groups in MTT assay were determined by ANOVA, followed by post-hoc Dunnett's t-tests. The probability of < 0.05 was considered to be statistically significant.

RNA extraction and real-time quantitative RT-PCR analysis

RNA was collected using Trizol reagent and cDNA was reverse-transcribed using MMLV. The expression level of VEGF mRNA was analyzed by real-time quantitative RT-PCR using a LightCycler instrument. cDNA was used for real-time quantitative RT-PCR using SYBR Green PCR Master Mix and VEGF forward (5'-AGC AAG GCC CAC AGG GAT TT-3') and reverse (5'-CCC TGA GAT CGA GTA CAT CTT-3') primers. The values obtained were normalized against expression levels of human actin (forward, 5'-GTG GAC ATC CGC AAA GAC-3'; reverse, 5'-AAA GGG TGT AAC GCA ACT A-3'). All real-time RT-PCR results are expressed as the fold change in mRNA expression with respect to control cells. Expression of the target gene was normalized to that of the housekeeping gene 18S for each sample. Data were analyzed using the [2.sup.-[delta][delta]CT] method (Livak and Schmittgen, 2001). All reactions were performed in triplicate. The efficacy of the four target sequences was evaluated and the best was chosen for subsequent experiments.

VEGF protein quantification

THP-1 cells were pretreated with serum-free RPIM 1640 for 12 h. The medium was then replaced with fresh serum-free medium containing EGb 761 (0, 0.01, 0.1, and 1 mg/1) with or without Ox-LDL (0, 10, 20, 40 mg/1), and cells were incubated for a further 48 h at 37 [degrees]C in a 5% [CO.sub.2]/95% ambient air environment. After incubation, the conditioned medium was collected and centrifuged at 1000 rpm for 5 min, and the VEGF protein content in the supernatant was analyzed using a Hu VEGF ELISA kit according to the manufacturer's protocol.

Oil red O-stain analysis

For Ox-LDL uptaking experiments, THP-1 cells were differentiated into adherent macrophages by treatment with 160 nM phorbol ester (PMA) for 24 h (Shashkin et al., 2005). Foam cell models derived from macrophages were established 48 h after adding 0.1 mg/1 EGb 761 with or without 40 mg/1 Ox-LDL into an RPMI 1640 medium of macrophages. At defined time points, the cells were fixed in phosphate buffered saline (PBS)-buffered 2% paraformaldehyde solution for 15 min, and 0.5% Oil red O (in 60% isopropanol) staining was done for 20 min essentially as described (Shashkin et al., 2005). Cell nuclei were then stained in haematoxylin for a few second. All procedures were performed at room temperature. Lipid droplet accumulation in the foam cells was evaluated by a microscope (Leica, German). In essence, foam cells were defined as macrophages in which the entire cytoplasm was filled with Oil red O-stainable lipid droplets.

Statistical analysis

Each experiment was performed at least three times and data are reported as mean [+ or -] SD where applicable. Statistically significant differences between groups in each assay were determined using a Student's t-test (two-sided). A probability of p < 0.05 was considered to be significant.

Results

MTT assay for cell viability

After exposure to Ox-LDL or EGb 761 for 48 h, cell viability was measured using the MTT assay. Ox-LDL inhibited the growth of THP-1 cells in a concentration-dependent manner. The rate of inhibition was 50.44% for 80 mg/1 Ox-LDL. After co-incubation with 0.1 mg/1 EGb 761, the cell survival rate increased (Fig. 1).

[FIGURE 1 OMITTED]

Ox-LDL induces VEGF mRNA expression in THP-1 cells

The expression of VEGF mRNA in THP-1 cells with or without Ox-LDL (0, 10, 20, 40 mg/1) for 24 h or 40 mg/1 Ox-LDL for different times (0, 6, 12, 24 h) was determined. Ox-LDL increased VEGF mRNA expression in THP-1 cells in a concentration-dependent (Fig. 2A) and time-dependent manner (Fig. 2B). VEGF mRNA induction was increased 4.36-fold by treatment with 40 mg/1 Ox-LDL for 24 h.

[FIGURE 2 OMITTED]

Ox-LDL induces VEGF production in a conditioned medium

After incubation of the THP-1 cells with serum-free RPIM 1640 for 12 h, the THP-1 cells were incubated with or without Ox-LDL for 48 h. The conditioned medium was collected and detected using hVEGF ELISA kit. VEGF production from THP-1 cells was increased with Ox-LDL stimulated in a concentration-dependent manner (Fig. 3).

[FIGURE 3 OMITTED]

Effect of EGb 761 on VEGF expression in THP-1 cells

To examine whether EGb 761 has an effect on VEGF expression in THP-1 cells, changes in VEGF mRNA and protein expression were determined. For THP-1 cells stimulated with 40 mg/1 Ox-LDL for 24 or 48 h, 1 h of pretreatment with 0.1 mg/1 EGb 761 significantly suppressed VEGF mRNA and protein expression compared to Ox-LDL stimulation alone (Fig. 4).

[FIGURE 4 OMITTED]

Effect of EGb 761 on foam cell formation

To determine whether EGb 761 has an effect on foam cell formation, we use an Ox-LDL-induced foam cell model. Normal THP-1 macrophages contain few neutral lipids and exhibit weak staining with Oil red O, a dye specific for neutral lipids. After 48-h incubation with 40 mg/1 Ox-LDL, a number of THP-1-derived foam cells filled with large cytoplasmic lipid droplets were observed (Fig. 5B). However, cells pretreated with 0.1 mg/1 EGb 761 and then incubated with 40 mg/1 Ox-LDL for 48 h showed a remarkable decrease in lipid staining compared to Ox-LDL stimulation alone (Fig. 5C).

[FIGURE 5 OMITTED]

Discussion

The results of the present study show that (1) Ox-LDL induces VEGF expression in a time- and concentration-dependent manner in THP-1 cells; (2) EGb 761 inhibits Ox-LDL-induced VEGF upregulation in a concentration-dependent manner in THP-1 cells; and (3) EGb 761 inhibits Ox-LDL uptake in macrophages. Together, these findings indicate that EGb 761 can directly inhibit foam cell formation in vitro by modulating VEGF expression.

During AS progression, a number of growth regulatory molecules and cytokines may be formed and released from cells within lesions, including endothelial cells, smooth muscle cells, macrophages, and T-lymphocytes (Hansson et al., 2006). The presence and the distribution of VEGF have been demonstrated in human AS arteries (Ramos et al., 1998; Khurana et al., 2005). It has been suggested that VEGF plays an important role in AS. In early AS lesions, VEGF staining was frequently observed in subendothelial macrophage-rich regions. However, in non-AS arteries only weak positive staining for VEGF was observed in the intima of some sections. VEGF expression has been demonstrated in mouse RAW 264 macrophages, human smooth muscle cells, and endothelial cells in response to Ox-LDL (Ramos et al., 1998; Inoue et al., 2001). In our previous studies, we showed that lysophosphatidylcholine, which is increased in the plasma of hypercholesterolemic patients and is a component of Ox-LDL, upregulated VEGF mRNA expression in endothelial cells and stimulated VEGF protein secretion from the cells in a time- and concentration-dependent manner (Zhang et al., 2002). In the current study we demonstrated that Ox-LDL induced VEGF mRNA and protein expression in a time- and concentration-dependent manner in THP-1 monocytes. The results indicate that monocytes are possibly a source of VEGF.

EGb 761 is an extract of the leaves of the G. biloba tree (Chinese name, Pai-kuo or Yin-hsing), a member of the family Ginkgoaceae that has been cultivated in China since the mid-1700s. EGb 761 has been used as a traditional Chinese preparation for some cardiovascular and neurological disorders for over 1000 years (Liu et al., 2007; Ramassamy et al., 2007). In recent years, a standardized clinical preparation of EGb 761, EGb 761, which contains 240mg/g fiavonoids (ginkgoflavone glycosides) and 60mg/g terpenoids (ginkgolides and bilobalides), has been used to treat cardiovascular and cerebrovascular diseases such as coronary heart disease and stroke (Rodriguez et al., 2007). According to a recent prospective community-based cohort study (Dartigues et al., 2007) and a randomized, placebo-controlled, double-blind clinical trial (Napryeyenko and Borzenko, 2007), treatment with EGb 761 increased the probability of survival in an elderly population and was efficacious in patients aged [greater than or equal to] 50 years with Alzheimer's disease or vascular dementia.

Since EGb 761 contains many compounds, there is considerable controversy regarding its mechanism of action. Previous studies showed that EGb 761 can directly scavenge superoxide anions, hydroxyl radicals, peroxyl radical species, and nitric oxide (Eckert et al., 2003; Akiba et al., 2004; Fan et al., 2006). Pretreatment with ginkgolides protected against A[beta] 1-42-induced neuronal death (Jiang et al., 2007) and synapse damage (Bate et al., 2008). EGb 761 increased the expression of hepatic metabolic enzymes, especially CYP2B1/2 mRNA expression and activity, and caused potential interactions with anti-hypertensive and anti-diabetic agents in rats (Tada et al., 2008). Our previous studies revealed inhibitory effects of EGb 761 on VEGF-induced permeability of bovine coronary endothelial cells, indicating that EGb 761 has the potential to protect against AS (Qiu et al., 2004). Another of our studies showed that EGb 761 inhibits the production of the pro-inflammatory cytokines IL-lb and TNF-[alpha] and upregulates the production of anti-inflammatory cytokines IL-10 and IL-10R in brain of atherosclerotic rats and in U937 cells, which might be related to its anti-AS actions (Jiao et al., 2005; Jiao et al., 2007). EGb 761 inhibited TNF-[alpha]-induced reactive oxygen species generation, transcription factor activation, and cell adhesion molecule expression in human aortic endothelial cells (Chen et al., 2003). Akiba et al. (2007) found that EGb 761 suppressed the Ox-LDL- and 4-hydroxynonenal-induced production of MMP-1 through the inhibition of PDGFR-[beta] activation in human coronary smooth muscle cells. In the present study, EGb 761 inhibited VEGF mRNA expression and protein production induced by Ox-LDL in THP-1 cells, suggesting that the mechanism of action of EGb 761 in AS involves inhibition of VEGF expression.

Intense VEGF staining was observed in AS plaques, and VEGF was localized mainly in macrophage-derived foam cells (Ramos et al., 1998), suggesting the involvement of Ox-LDL with VEGF in AS. In this study, we demonstrated that EGb 761 inhibited macrophage-derived foam cell formation induced by Ox-LDL.

In summary, we have demonstrated that Ox-LDL induces VEGF expression in human THP-1 cells and that EGb 761 downregulated this effect in vitro. We have also shown the protective effect of EGb 761 on monocyte/macrophage-derived foam cell formation. Our findings suggest the possible involvement of Ox-LDL in the development of human AS through VEGF induction in monocytes and that EGb 761 prevents in vitro atherogenesis probably via downregulation of VEGF expression in monocytes and inhibition of monocyte/macrophage-derived foam cell formation. These findings suggest a mechanism for the in vivo anti-AS effect of EGb 761 and support its potential clinical use in AS.

Acknowledgements

This project was supported by the National Natural Science Foundation of China (No. 30500615), the Young Scholars Foundation of Shanghai Public Health Bureau (No. 2004-55-94), and the Young Scholars Foundation of SMMU (No. 2003-SQ-11).

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HJ. Liu (a),(1), X.L. Wang (b),(1), L. Zhang (c), Y. Qiu (d), T.J. Li (b), R. Li (c), M.C. Wu (c), L.X. Wei (c), Y.C. Rui (b),*

(a) Changhai Hospital, Secondary Military Medical University, 174 Changhai Road, Shanghai 200433, China

(b) Second Military Medical University, 800 Xiangyin Road, Shanghai 200433, China

(c) Tumor Immunology and Gene Therapy Center, Eastern Hepatobiliary Surgery Hospital, Second Military Medical University, 225 Changhai Road, Shanghai 200438, China

(d) Department of Pharmacy, the 454th Hospital of PLA, 1 Malu Street, Nanjing 210002, China

Abbreviations: VEGF, vascular endothelial growth factor; EGb 761, Ginkgo biloba extract; AS, atherosclerosis; Ox-LDL, oxidatively modified low-density lipoprotein; EC, endothelial cells; SMC, smooth muscle cells.

* Corresponding author. Tel./fax: +8621 25074471. E-mail address: rui_yc@yahoo.cn (Y.C. Rui).

(1) The authors share the first authorship.
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Title Annotation:human acute monocytic leukemia cells
Author:Liu, H.J.; Wang, X.L.; Zhang, L.; Qiu, Y.; Li, T.J.; Li, R.; Wu, M.C.; Wei, L.X.; Rui, Y.C.
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Geographic Code:9CHIN
Date:Mar 1, 2009
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