Despite accumulating evidence demonstrating that endothelial progenitor cells (EPC) play an important role in endothelium maintenance, being implicated in both reendothelialization and neovascularization,1-6 the factors that influence circulating EPC number and function are just beginning to be identified. Recently, we have documented that Ginkgo biloba extract dose and time dependently increased EPC numbers and enhanced cell proliferation, migration, adhesion, and in vitro vasculogenesis capacity.7 However, the precise mechanisms by which Ginkgo biloba extract increased EPC numbers and activity remain to be determined.
Studies have demonstrated that EPC senescence was associated with EPC numbers and activity.8-10 Cellular aging or senescence is characterized by cell-cycle arrest and can be triggered by different pathways.11 Loss of telomerase activity has been suggested to constitute the molecular clock that triggers cellular senescence.12 Interestingly, Murasawa et al13 have demonstrated that overexpression of human telomerase reverse transcriptase (hTERT) by adenovirus-mediated gene delivery could result in a delay of senescence and a recovery/enhancement of the regenerative properties of EPCs.
On the basis of these considerations, we investigated whether Ginkgo biloba extract would prevent the onset of EPC senescence through telomerase activation, leading to potentiation of cellular activity. In the present study, we have demonstrated that Ginkgo biloba extract prevented the onset of EPC senescence, leading to potentiation of cellular activity. Moreover, Ginkgo biloba extract significantly increased telomerase activity and serine/threonine kinase Akt phosphorylation.
MATERIALS AND METHODS
Isolation, Cultivation, and Characterization of Circulating EPCs
EPCs were isolated, cultured, and characterized according to previously described techniques.1,3,7,14 Briefly, mononuclear cells (MNCs) were isolated from peripheral blood of healthy human volunteers by Ficoll density gradient centrifugation and cultured on fibronectin (Chemicon)-coated dishes in Medium 199 (Sigma) supplemented with 20% fetal-calf serum and VEGF (50 ng/mL, Chemicon). EPCs were characterized as adherent cells double positive for DiLDL uptake and lectin binding under a laser scanning confocal microscope. They were further documented by demonstrating the expression of VE-cadherin, KDR, CD34, and AC133 by flow cytometry (data not shown).
After 4 days of culture, adherent cells were gently detached with EDTA. Cells (1 × 105) were seeded in methylcellulose plates (Methocult GF H4434, CellSystems, Germany) with 100 ng/mL of human recombinant VEGF. Plates were studied under phase-contrast microscopy, and colonies were counted after 10 days of incubation by two independent investigators.8
EPC Proliferation Assay
EPC proliferation was determined by MTT assay. EPCs were digested with EDTA and were then cultured in serum-free medium in a 96-well culture plate (200 μL per well). EPCs were supplemented with 10 μL of MTT (5 g/L) and incubated for another 6 hours. Then, the supernatant was discarded by aspiration and the EPC preparation was shaken with 200 μL of DMSO for 10 minutes before the OD value was measured at 490 nm.7
Senescence-Associated β-Galactosidase Activity Assay
Senescence-associated β-galactosidase (SA-β-gal) activity was measured with β-Galactosidase Staining Kit (BioVision). The protocol was conducted according to the manufacturer's instructions. Briefly, EPCs were washed in PBS, fixed for 10 to 15 minutes at room temperature with 0.5 mL of fixative solution, washed, and incubated overnight at 37°C with the staining solution mix. Cells were observed under a microscope for development of blue color (200 × total magnification).8,9
Telomeric Repeat-Amplification Protocol Assay
To investigate the effect of Ginkgo biloba extract on telomerase activity, EPCs at day 4 were treated with a different Ginkgo biloba extract (10, 25, or 50 mg/L) for 24 hours, with or without pretreatment with LY294002 (10 μM). For quantitative analyses of telomerase activity, the telomeric repeat-amplification protocol (TRAP) assay, in which telomerase reaction product is amplified by polymerase chain reaction (PCR),15 was performed using the TeloTAGGG PCR ELISAPLUS kit (Roche Molecular Biochemicals) according to the manufacturer's instructions.
The EPCs were treated with various agents of Ginkgo biloba extract for 24 hours. They were then washed twice with phosphate-buffered saline and lysed in ice-cold HNTG buffer. The lysates were centrifuged at 12,000g (4°C) for 20 minutes, and the protein concentrations of the supernatants were determined using the Bio-Rad protein assay. Equal amounts of proteins (50 μg) were separated on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) gels, as described previously.16 After the separation, proteins were transferred to a nitrocellulose membrane (Amersham). Membranes were blocked by incubation in tris-buffered saline (pH 7.5) containing 0.1% (v/v) Tween 20 and 5% (v/v) nonfat dry milk for 2 hours, followed by 2 hours of incubation at room temperature with rabbit polyclonal anti-phospho-Akt-Ser473 or anti-Akt antibodies (Cell Signalling Technology). The filters were washed extensively in tris-buffered saline containing 0.1% (v/v) Tween 20 before incubation for 1 hour with a secondary antirabbit or antimouse antibody conjugated to horseradish peroxidase. Membranes were then washed and developed using the EZ-ECL Detection Kit (Biological Industries).
Data are expressed as means ± SD from at least three independent experiments. Statistical analysis was performed by the two-tailed t test or ANOVA for multiple comparisons.
Ginkgo Biloba Extract Prevented EPC Senescence
EPCs were generated from peripheral blood mononuclear cells as previously described.1,3,7,14 To assess the onset of senescence, acidic β-galactosidase was detected as a biochemical marker for acidification typical for the onset of cellular senescence.11,17 Cultivation of EPCs resulted in an increase in SA-β-gal-positive cells after prolonged cultivation. Coincubation with Ginkgo biloba extract significantly inhibited the increase in SA-β-gal-positive cells. The inhibition of EPC senescence occurred dose dependently, with a plateau at 25 mg/L of Ginkgo biloba extract (Fig. 1).
Effects of Ginkgo Biloba Extract on Proliferation of EPCs
Having demonstrated that Ginkgo biloba extract prevented the onset of senescence, we examined whether that would translate into an increase of proliferation. An MTT assay demonstrated that the mitogenic potential of EPCs treated with Ginkgo biloba extract exceeded that in untreated (control) EPCs-an effect that was dose dependent (Fig. 2).
Effects of Ginkgo Biloba Extract on Clonal Expansion in EPCs
To investigate the clonal expansion potential of the cultivated EPCs, we performed an outgrowth assay. For this purpose, EPCs were cultivated for 4 days. Then, cells were detached and 1 × 105 EPCs were seeded in methylcellulose plates in the presence or absence of Ginkgo biloba extract. As shown in Figure 3, the number of colonies was significantly higher in EPCs that had been pretreated with Ginkgo biloba extract.
Effects of Ginkgo Biloba Extract on Telomerase Activity in EPCs
Cellular senescence is critically influenced by the telomerase, which elongates telomeres, thereby counteracting telomere length reduction induced by each cell division. Therefore, we measured telomerase activity in EPCs using the TeloTAGGG PCR ELISAPLUS kit. As demonstrated in Figure 4, Ginkgo biloba extract dose-dependently increased telomerase activity, with a maximal increase at 25 mg/L.
Ginkgo Biloba Extract Induces Telomerase Activity via the PI3K/Akt Cascade
Recent studies have demonstrated that Akt plays an essential role in regulating cell senescence and telomerase activity.18,19 Moreover, Akt has been shown to play an important role in atorvastatin-mediated prevention of EPC senescence.8 To examine whether the PI3K/Akt cascade is involved in the Ginkgo biloba extract-induced telomerase activation, EPCs were pretreated with or without LY294002 (10 μM) before incubation with Ginkgo biloba extract and were then subjected to assess the telomerase activity. Interestingly, LY294002 clearly attenuated the increase in Telomerase activity by Ginkgo biloba extract (Fig. 4).
We next investigated whether Ginkgo biloba extract would induce the phosphorylation of Akt, a downstream effector of PI3K. EPCs were stimulated with different Ginkgo biloba extracts for 24 hours, and immunoblots were performed with anti-phospho-Akt (Ser473) or anti-Akt antibody. As shown in Figure 5, stimulation with Ginkgo biloba extract led to dose-dependent phosphorylation of Akt, although it did not affect the total amount of Akt.
Recent studies have provided increasing evidence that the functional regeneration of ischemic tissue by improved neovascularization and possibly tissue repairs are critically dependent on the mobilization and integration of EPCs into the ischemic tissue. Moreover, infusions of EPCs expanded ex vivo can limit the extension of scar tissue in the ischemic myocardium,6 improve the recovery of contractility, and, thereby, may be useful as a novel therapeutic approach.20 However, several studies have indicated that aging or senescence may limit the ability of EPCs to sustain neovascularization and repair of ischemic tissue,1,14,21,22 highlighted a potentially relevant feature of the endogenous repair process, and posed an interesting question as to the value of therapies based on expansion of endogenous EPCs in either elderly patients or patients with atherosclerotic risk factors. With respect to unresolved problems, we have shown for the first time that Ginkgo biloba extract prevented the onset of EPC senescence, which was associated with a high proliferative capacity and profoundly increased clonal expansion potential. However, Ginkgo biloba extract did not decrease apoptosis of EPCs (data not shown).
Recently, Assmus et al8 have shown that EPC senescence was associated with the reduction of numbers and impairment of activity. In addition, Imanishi et al9 have documented that proatherosclerotic risk factor ox-LDL accelerated the onset of EPC senescence, leading to cellular dysfunction. More recently, estrogen has been shown to reduce EPC senescence through augmentation of telomerase activity, leading to the potentiation of proliferative activity and network formation.10 Keeping these findings in mind, we speculated that EPC senescence, not apoptosis of EPCs, might partly account for the mechanisms by which Ginkgo biloba extract increases EPC numbers and activity.
The mechanisms by which Ginkgo biloba extract prevents the onset of cellular senescence and increases the proliferative capacity of EPCs seem to involve telomerase activity. Telomerase, an RNA-directed DNA polymerase, extends telomeres of eukaryotic chromosomes and delays the development of senescence. Recently, Murasawa et al13 have revealed that overexpression of hTERT by adenovirus-mediated gene delivery could result in a delay in senescence and recovery/enhancement of the regenerative properties of EPCs. In addition, estrogen has been shown to significantly increase telomerase activity, which was considered to be associated with EPC senescence induced by estrogen.9 Our data demonstrated that Ginkgo biloba extract dose-dependently increased telomerase activity. Thus, it was suggested Ginkgo biloba extract prevented the onset of EPC senescence, most likely through activation of telomerase.
However, the molecular mechanisms by which Ginkgo biloba extract increased telomerase activity remains to be determined. In this study, we have shown that Ginkgo biloba extract significantly increased Akt phosphorylation. Recent studies have demonstrated that the serine/threonine kinase Akt, also named protein kinase B, enhanced telomerase activity through phosphorylation of TERT in human umbilical cord endothelial cells and melanoma cells.18,19 Besides the direct phosphorylation of TERT, Akt might also act by increasing the activity of the endothelial NO synthase (eNOS), because NO has been demonstrated to activate telomerase and delays endothelial-cell senescence.23 However, inhibition of the NO synthase by NG-mono-methyl-L-arginine did not reverse the inhibitory effect of Ginkgo biloba extract (data not shown), suggesting that the regulation of EPC senescence is independent of NO, which is in line with what Assmus et al8 have reported. Taken together, our data indicated that Ginkgo biloba extract increased Akt phosphorylation in EPCs, which might lead to increased phosphorylation of TERT. Increased phosphorylation of TERT might enhance telomerase activity and, thereby, prevent the onset of EPC senescence. Further studies are required to elucidate our speculation.
In conclusion, the results of the present study demonstrate that Ginkgo biloba extract delay the onset of EPC senescence, which may be related to activation of telomerase and Akt phosphorylation. The inhibition of EPC senescence by Ginkgo biloba extract in vitro may improve the functional activity of EPCs in a way that is important for potential cell therapy.
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