Isolation and Cultivation of EPCs
EPCs were cultured according to previously described techniques. 1,9 Briefly, total mononuclear cells (MNCs) were isolated from blood of healthy young human volunteers by Ficoll density gradient centrifugation. Cells were plated on culture dishes coated with human fibronectin (Chemicon) and maintained in Medium 199 (Sigma) supplemented with 20% fetal-calf serum, VEGF (10 ng/mL, Chemicon), penicillin (100 U/mL), and streptomycin (100 μg/mL). After 4 days in culture, nonadherent cells were removed by washing with PBS, new media were applied, and the culture was maintained through day 7. Informed consent was obtained from all volunteers, and all of the procedures were done in accordance with national and international laws and policies.
Fluorescent chemical detection of EPCs was performed on attached MNCs after 7 days in culture. Direct fluorescent staining was used to detect dual binding of FITC-labeled Ulex europaeus agglutinin (UEA)-1 (Sigma) and 1,1-dioctadecyl-3, 3, 3,3-tetramethylindocarbocyanine (DiI)-labeled acetylated low-density lipoprotein (acLDL; Molecular Probe). Cells were first incubated with acLDL at 37°C and later fixed with 2% paraformaldehyde for 10 minutes. After washing, the cells were reacted with UEA-1 (10 μg/mL) for 1 hour. After the staining, samples were viewed with an inverted fluorescent microscope (Leica) and further demonstrated by laser scanning confocal microscope (Leica). Cells demonstrating double-positive fluorescence were identified as differentiating EPCs. 8,9 Two or three independent investigators evaluated the number of EPCs per well by counting 15 randomly selected high-power fields (×200) with an inverted fluorescent microscope.
Flow Cytometry Analysis
Fluorescence-activated cell sorting detection of EPCs was performed on attached MNCs after 7 days in culture. Mononuclear cells were detached with 0.25% trypsin followed by repeated gentle flushing through a pipette tip. Cells (2 × 105) were incubated for 30 minutes at 4°C with anti–vascular endothelium (VE)-cadherin (Chemicon) phycoerythrin-conjugated monoclonal antibodies against kinase insert domain-containing receptor (KDR, R&D), CD34, AC133 (Miltenyi Biotec). A FITC-conjugated anti-mouse antibody (Vector) was added for staining with VE-cadherin. Isotype-identical antibodies served as controls. After treatment, the cells were fixed in 1% paraformaldehyde. Quantitative FACS was performed on a FACStar flow cytometer (Coulter). 9,10
Cells were serum depleted for 24 hours before experiments. To demonstrate a concentration-dependent effect of Ginkgo biloba extract (Ginkgo biloba extract used in this study was obtained from Zhejiang Conba Pharmaceutical Co., Ltd., China. The extract contained 24% flavonoids and 6% terpene lactones). On EPCs, cells were incubated with 10 mg/L, 25 mg/L, and 50 mg/L Ginkgo biloba extract for 24 hours, respectively. To determine reaction time course, cells were treated with 25 mg/L Ginkgo biloba extract for 6, 12, 24, and 48 hours. The vehicle used in this study was Medium 199 supplemented with 20% fetal calf serum.
EPC migration was evaluated by using a modified Boyden chamber assay. In brief, isolated EPCs were detached using 0.25% trypsin, harvested by centrifugation, resuspended in 500 μL M199, and counted; then 2 × 104 EPCs were placed in the upper chamber of a modified Boyden chamber. VEGF in serum-free M199 media was placed in the lower compartment of the chamber. After 24 hours incubation at 37°C, the lower side of the filter was washed with PBS and fixed with 2% paraformaldehyde. For quantification, cells were stained with Giemsa solution. Cells migrating into the lower chamber were counted manually in 3 random microscopic fields. 8
Cell Adhesion Assay
After 24 hours of incubation with Ginkgo biloba extract, human EPCs were washed with PBS and gently detached with 0.25% trypsin. After centrifugation and resuspension in M199, 5% FBS, identical cell numbers were replated onto fibronectin-coated culture dishes and incubated for 30 minutes at 37°C. Adherent cells were counted by independent blinded investigators. 2
EPC Proliferation Assay
The effect of Ginkgo biloba extract on EPCs proliferation was determined by MTT assay. After being cultured for 7 days, EPCs were digested with 0.25% trypsin and then cultured in serum-free medium in 96-well culture plate (200 μL per well), to which was added Ginkgo biloba extract (to make a series of final concentrations: 10 mg/L, 25 mg/L, 50 mg/L). Each concentration included 6 wells, while the serum-free medium served as a control. After being cultured for 24 hours, EPCs were supplemented with 10 μL MTT (5 g/ L, Fluka Co.) and incubated for another 6 hours. Then the supernatant was discarded by aspiration and the EPC preparation was shaken with 200 μL dimethyl sulfoxide for 10 minutes, before the OD value was measured at 490 nm.
In Vitro Vasculogenesis Assay
In vitro vasculogenesis assay was performed with the In Vitro Angiogenesis Assay Kit (Chemicon). The protocol was according to the manufacturer's instructions. Briefly, ECMatrix™ solution was thawed on ice overnight, then mixed with 10 × ECMatrix™ Diluent and placed in a 96-well tissue culture plate at 37°C for 1 hour to allow the matrix solution to solidify. EPCs were harvested as described above and replated (10,000 cells per well) on top of the solidified matrix solution. Cells were grown with Ginkgo biloba extract or vehicle control, and incubated at 37°C for 12 hours. Tubule formation was inspected under an inverted light microscope at 200× magnification. Tubule formation was defined as a structure exhibiting a length 4 times its width. 10,11 Five independent fields were assessed for each well, and the average number of tubules/200 × field was determined.
All data are presented as mean ± standard deviation. Differences between group means were assessed by an unpaired Student's t test for single comparisons and by ANOVA for multiple comparisons. Values of P < 0.05 were considered significant.
Characterization of Human EPCs
Total MNCs isolated and cultured for 7 days resulted in a spindle-shaped, endothelial cell–like morphology (Fig. 1). EPCs were characterized as adherent cells double positive for DiLDL-uptake and lectin binding by using LSCM (Fig. 2). They were further documented by demonstrating the expression of VE-cadherin (76 ± 8.6%), KDR (78 ± 7.8%), CD34 (28.7 ± 6.9%), and AC133 (17.1 ± 8.1%) by flow cytometry.
Ginkgo Biloba Extract Increased EPCs In Vitro
Incubation of isolated human MNCs with Ginkgo biloba extract increased the number of differentiated, adherent EPCs in a concentration-dependent manner, which became apparent at 10 mg/L, with a plateau at 25 mg/L (about 1-fold increase, Figs. 2 and 3A).In time-course experiments performed with a Ginkgo biloba extract concentration of 25 mg/L, increase of EPCs number became apparent at 12 hours and reached the maximum at 24 hours (about 1-fold increase, Fig. 3B).
Effect of Ginkgo Biloba Extract on EPCs Proliferation
The effect of Ginkgo biloba extract on EPCs proliferation was assayed using a MTT assay (Fig. 4). Ginkgo biloba extract improved EPC proliferative activity, maximal at 25 mg/L Ginkgo biloba extract (control versus 25 mg/L Ginkgo biloba extract: 0.513 ± 0.069 vs 0.702 ± 0.074,490 nm light absorbance, P < 0.01). In time-course experiments performed with a Ginkgo biloba extract concentration of 25 mg/L, increase of EPCs proliferative activity became apparent at 12 hours (P < 0.05) and reached the maximum at 24 hours (P < 0.01).
Effect of Ginkgo Biloba Extract on Human EPCs Migration in Response to VEGF
The effects of Ginkgo biloba extract on EPCs migration were analyzed in a modified Boyden chamber assay (Fig. 5). Ginkgo biloba extract profoundly enhanced cell migration, maximal at 25 mg/L Ginkgo biloba extract (control vs 25 mg/L Ginkgo biloba extract, 12.6 ±4.0 vs 28.8 ± 6.1, cells per high-powered field, P < 0.01). Ginkgo biloba extract (25 mg/L) also time-dependently enhanced EPCs migratory activity, which became apparent at 12 hours (P < 0.05) and reached the maximum at 24 hours (P < 0.01).
Effect of Ginkgo Biloba Extract on EPCs Adhesiveness
To study the possibility that Ginkgo biloba extract alters adhesiveness of cultured human EPCs, EPCs were incubated with Ginkgo biloba extract for 24 hours. After replating on fibronectin-coated dishes, EPCs preexposed to Ginkgo biloba extract exhibited a significant increase in the number of adhesiveness cells at 30 minutes (Fig. 6). The increase in the number of adhesiveness cells occurred dose dependently, with a maximal effect achieved at 25 mg/L. In addition, Ginkgo biloba extract time-dependently increased EPCs adhesive activity, which became apparent at 6 hours (P < 0.05) and reached the maximum at 24 hours (P < 0.01).
Effects of Ginkgo Biloba Extract on EPCs Vasculogenesis
Recent studies have demonstrated that circulating EPCs home into sites of neovascularization and differentiate into endothelial cells in situ 3,12 in a manner consistent with a process termed vasculogenesis. In vitro vasculogenesis assay was to simulate this process, and here was used to investigate the ability of EPCs to participate in neovascularization, which is the most important activity of EPCs. The response of the EPCs to Ginkgo biloba extract is depicted in Figure 7. Tubule number increased in a dose-response to Ginkgo biloba extract concentrations at 24 hours of incubation, with peak production at 25 mg/L Ginkgo biloba extract. Moreover, tubules in the Ginkgo biloba extract wells were qualitatively different and more complex than those in the control wells.
Ginkgo biloba extract, an extract prepared from the leaves of Ginkgo biloba, contains 24% flavonoid glycosides, 6% ginkgolides, and 70% of other substances. In the cardiovascular system, the extract has been shown to have complex and multiple effects. 13–16 It increases peripheral and cerebral blood flow and microcirculation and reduces capillary permeability. Ginkgo biloba extract also improves blood rheology and has antithrombotic effects similar to those of aspirin in an experimental model of thrombosis. Moreover, Ginkgo biloba extract has recently been shown to have protective effects on endothelial cells injury or endothelial dysfunction induced by lipid peroxide and chemical hypoxia.
However, endothelial dysfunction ultimately loses a balance between the magnitude of injury and the capacity for repair. Local migration and proliferation of endothelial cells adjacent to the site of injury had been regarded as the principal mechanism of endothelial repair until Asahara and colleagues, in 1997, described circulating EPCs. 3 These cells are bone marrow–derived and have the capacity to home in on sites of endothelial injury. Here they incorporate into the endothelium and thereby repair the defects. 17 More recently, 2 groups have documented in animals and human subjects that EPCs contribute up to 25% of endothelial cells in newly formed vessels. 18,19 Moreover, Vasa et al have recently reported that patients with CAD revealed reduced levels and functional impairment of EPCs, which correlated with risk factors for CAD. Therefore, the augmentation of EPCs numbers by pharmacological modulation may be a novel strategy to improve neovascularization after ischemia. Statins, VEGF, and GM-CSF have been reported to increase EPC numbers and functions, but they exhibited some side effects, which might limit their application. Therefore, it is necessary to find a new therapeutic method to improve EPCs numbers and functions.
The results of the present study demonstrated for the first time that Ginkgo biloba extract could augment EPCs number, promote EPCs proliferation, migration, adhesion, and in vitro vasculogenesis capacity. Given the well-established role of EPCs participating in neovascularization and reendothelialization, stimulation of EPCs by Ginkgo biloba extract may contribute to the clinical benefit of Ginkgo biloba extract therapy in patients with CAD. Thus, our results might suggest a novel functional effect of Ginkgo biloba extract: namely, Ginkgo biloba extract not only directly protects endothelial cells but also increases EPCs numbers and functions at the same time, thus accelerating endothelial repair process, which contributes to protective effects on endothelial cells and improves the clinical symptoms and prognosis of patients with CAD. Since Ginkgo biloba extract have the merits of low toxicity and rare complications, it might be a promising medication to improve postnatal neovascularization and reendothelialization in patients with CAD.
The mechanisms by which Ginkgo biloba extract increases EPCs numbers and activity remain to be determined. There are several possible scenarios by which Ginkgo biloba extract could increase the number of circulating EPCs. One explanation might be decreased apoptosis of premature progenitor cells. Another explanation is that Ginkgo biloba extract may interfere with the signaling pathways regulating EPCs differentiation or mobilization.
The results of the present study may define a novel functional effect of Ginkgo biloba extract: the augmentation of EPCs with enhanced functional activity.
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