Effects of Resveratrol on Endothelial Progenitor Cells and Their Contributions to Reendothelialization in Intima-injured Rats

J, Gu*; CQ, Wang*; HH, Fan; HY, Ding*; XL, Xie*; YM, Xu*; BY, Wang*; DJ, Huang*

Journal of Cardiovascular Pharmacology: May 2006 - Volume 47 - Issue 5 - pp 711-721
doi: 10.1097/01.fjc.0000211764.52012.e3
Original Articles

The aim of this study was to investigate the effects of resveratrol on endothelial progenitor cell (EPC) activities in vitro and on the mobilization of circulating EPCs, and reendothelialization in balloon-injured aorta of rats. After being isolated, cultured, and characterized, human EPCs were stimulated with resveratrol. We found that a low concentration of resveratrol (1 μM) led to significant enhanced activities of proliferation, migration, and adhesion, as well as promoting endothelial nitric acid synthetase (eNOS) expression in EPCs, whereas a high concentration (60 μM) inhibited the aforementioned functions and eNOS expression. In a rat model of injured aorta, a low dosage of resveratrol (10 mg/kg) increased the amount of EPCs in rat circulation as compared with placebo, whereas the result of a high dosage (50 mg/kg) did not reach statistical difference. In addition, 10 mg/kg of resveratrol both accelerated reendothelialization and inhibited neointimal formation; however, 50 mg/kg only reduced neointimal formation, which was not as effective as the previous one. eNOS expression in injured arteries was potently enhanced in the 10 mg/kg group, but not in the 50 mg/kg group.These findings suggest that a low dosage of resveratrol could markedly raise the proliferative, migrative, and adhesive activities of EPCs and upgrade eNOS expression in vitro as well as increase EPC mobilization, enhance eNOS expression, and accelerate the repair of injured artery; however, a high dosage cannot.

*Department of Cardiology, Renji Hospital, affiliated to Shanghai Jiao-Tong University School of Medicine

Laboratory of Blood Engineering, Shanghai Blood Center, Shanghai, China

Supported by a grant from the National Natural Science Foundation of China (No. 30170365).

Reprints: ChangQian Wang, Department of Cardiology, Renji Hospital affiliated to Jiao-Tong University School of Medicine, Shanghai, 200001, China (e-mail: wcqian@yahoo.com.cn).

Received for publication December 10, 2005; accepted February 13, 2006

Article Outline

Vascular injury leads to pathologic repair and remodeling that involve vascular smooth muscle cell migration and proliferation, resulting in neointimal hyperplasia.1 Endothelial cell (EC) loss is a major contributing factor to the pathologic repair of the injured blood vessel.2 The disruption of endothelial integrity leads to a concomitant reduction in the production of vasculoprotective mediators and increased vasoconstrictor and growth-promoting substances,3,4 resulting in elevated vascular tone, platelet adhesion, enhanced inflammation, and medial smooth muscle cell proliferation.3,4 The resultant neointimal hyperplasia is the pathologic basis for restenosis after revascularization procedures such as angioplasty, stenting, and bypass grafting.1,2

Because EC loss plays a pivotal role in the pathogenesis of intimal hyperplasia after vascular injury, a therapeutic strategy that promotes early reendothelialization of the injured vessels would inhibit intimal lesion development, facilitate vascular repair, and improve long-term vessel patency. Endothelial progenitor cells (EPCs) originating from the bone marrow have previously been isolated from the mononuclear cell (MNC) fraction of peripheral blood.5,6 These cells have high proliferative potential5 and under specific growth conditions, differentiate into ECs,7 suggesting that they may be suitable as a substrate for the reendothelialization of damaged vessels. Studies have shown that transplantation of circulating EPCs onto denuded arteries led to rapid reendothelialization of the injured artery.8,9

Epidemiologic studies have demonstrated that the incidence of coronary artery disease in France is strinkingly lower as compared with other western countries with a fat-containing diet. This so-called “French paradox” has been attributed to moderate consumption of red wine in France. Resveratrol (trans-3,5,4′-trihydroxystilbene), a polyphenol compound found in grapes and red wine in significant amounts, has been designated as the responsible agent of the French paradox. It has been found that resveratrol could inhibit platelet aggregation and/or adhesion, lower oxidative stress in platelets, protect against low-density lipoprotein oxidation, suppress proliferation or hypertrophy of smooth muscle cells, and increase high-density lipoprotein cholesterol.10 In particular, resveratrol have been shown to decrease neointimal thickening in animal models of injured artery.11 More recently, studies showed that resveratrol could protect heart cells from ischemia/reperfusion,12 which is ascribed to its ability to enhance endothelial nitric acid synthetase (eNOS) expression. In addition, resveratrol also promotes eNOS expression in ECs in vitro13,14 and eNOS has been approved to be at least in part responsible for the mobilization of circulating EPCs in vivo15 and its angiogenic functions in vitro.16–18 So we hypothesized that resveratrol may have a modulating effect for the mobilization of EPCs and their functions in vitro.

In the present study, we evaluated the effects of resveratrol on the angiogenic activities and eNOS expression of isolated human EPCs in vitro and the effects of resveratrol on the mobilization of EPCs from bone marrow, reendothelialization, neointimal hyperplasia, and eNOS expression in injured arteries.

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Isolation and Cultivation of Human EPCs

Total MNCs were isolated from peripheral blood of healthy human volunteers by Ficoll density gradient centrifugation (1.077, Amersham Bioscience). A total of 106 MNCs/cm2 were plated on fibronectin-coated (2 μg/cm2, Chemicon) dishes and maintained in Medium 199 (Gibco) supplemented with 20% fetal calf serum (Dingguo, Beijing), 12 μg/mL of bovine brain extract (Sigma), 10 ng/mL of vascular endothelial growth factor (VEGF) (Cytolab), 2 ng/mL of basic fibroblast growth factor (bFGF) (Cytolab), penicillin (100 U/mL), and streptomycin (100 μg/mL). After 4 days in culture, nonadherent cells were removed by washing with phosphate-buffered saline (PBS), new media was applied, and the culture was maintained through day 7.

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Human EPC Characterization

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 fluorescein isothiocyanate (FITC)-UEA-I (Sigma) and DiI-acLDL (Molecular Probe) of the cells. Briefly, attached cells were first incubated with DiI-acLDL (10 μg/mL) at 37°C for 4 hours and later fixed with 2% paraformaldehyde for 10 minutes. After washing, the cells were reacted with FITC-UEA-I (10 μg/mL) at 37°C for 1 hour. After the staining, samples were demonstrated by laser scanning confocal microscope (LSM510, Zeiss). Cells demonstrating double-positive fluorescence were identified as differentiating EPCs.19,20 Attached MNCs were further identified by flow cytometry analysis. MNCs were detached with 1 mM ethylenediaminetetraacetate (EDTA) followed by repeated gentle flushing through a pipette tip. Cells (2×105) were incubated for 30 minutes at 4°C with PE-conjugated antihuman CD34 (Immunotech), kinase insert domain containing receptor (VEGFR-2) antibodies (R&D), and FITC-conjugated antihuman CD31 antibody (Caltag). Isotype-identical antibodies served as negative controls. Quantitative FACS was performed on a FACStar flow cytometer (Coulter).

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Resveratrol Therapy

After 7 days in culture and identification of EPCs, the cells were serum depleted for 24 hours before experiments. Then cells were treated with different concentrations of resverarol (control, 1, 5, 15, and 60 μM) for 72 hours. Resveratrol (Sigma) was dissolved in 10% dimethyl sulfoxide (Sigma) and 10% dimethyl sulfoxide was used as negative control.

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EPC Proliferation Assay

The effect of resveratrol on EPC proliferation was determined by MTT assay (colorimetric viability test using the 3-[4,5 dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide). After 72 hours of resveratrol pretreatment, EPCs were digested with 1 mM EDTA and then cultured in 10% FCS supplemented-medium in 96-well culture plates. EPCs were supplemented with 20 μL MTT (5 g/L, Ameresco) and incubated for another 4 hours. Then the supernatant was discarded by aspiration and the EPC preparation was shaken with 150 μL dimethyl sulfoxide for 10 minutes, before the optical density value was measured at 570 nm.

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EPC Migration Assay

EPC migration20 was evaluated by using a transwell chamber assay (R&D). In brief, isolated EPCs were detached using 1 mM EDTA, then 5×104 EPCs in 100 μL M199 were placed in the upper chamber of a transwell chamber. VEGF (50 ng/mL) and bFGF (10 ng/mL) in 600 μL 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 0.2% crystal violet solution. Cells migrating into the lower chamber were counted manually in 5 random microscopic fields (×200).

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EPC Adhesion Assay

After resveratrol therapy, EPCs were detached with 1 mM EDTA. Then identical cell numbers were replated onto fibronectin-coated culture dishes and incubated for 30 minutes at 37°C. Adherent cells were counted manually in 5 random microscopic fields (×200).21

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eNOS ELISA for Human EPCs

The concentration of eNOS in EPC lysates was measured in triplicate using a Quantikine human eNOS kit (R&D) according to the manufacturer's instructions.22 Values were normalized to cell protein content determined by the Bradford method (Shenergy Biocolor, Shanghai).

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Animal Preparations

Fifty-four healthy male Sprague-Dawley rats (weighing 250 to 350 g, purchased from Shanghai Laboratory Animal Center, Chinese Academy of Science) were randomized into 4 groups: sham operation (n=9), placebo-treated group (n=15), 10 mg/kg/d (n=15), and 50 mg/kg/d (n=15) of resveratrol-treated group. The resveratrol-treated rats began to be gavaged with resveratrol (purity: 99.9%, Shanghai Nabio) in 10% ethanol using a stomach needle (1.2 mm diameter) 2 weeks before operation until being killed (at 1, 2, and 4 weeks after operation). The sham and placebo-treated groups were administered with equivalent volumes of 10% ethanol. The investigation conformed with the Guide for the Care and Use of Laboratory Animals published in China.

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Aortic Balloon Injury

Injury to the aorta of the rat was induced by means of an inflated balloon catheter as previously described.23 In brief, after undergoing pentobarbital anesthesia (40 mg/kg body weight, intraperitoneal), rats underwent balloon injury of the thoracic and abdominal aortas. A 2F Fogarty balloon catheter (Edward) was introduced via the left common carotid artery (LCCA) into the aorta and then inflated and withdrawn 3 times. Finally, the LCCA was ligated. Noninjured control rats were subjected to the same operation, but the catheter was not introduced into the LCCA.

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Rat EPC Isolation and Culture

After pentobarbital anesthesia, 5 to 10 mL of peripheral blood was withdrawn from posterior vena cava. Rat peripheral blood MNCs were isolated by density-gradient centrifugation with Histopaque-10831 (Sigma). Cells were resuspended in M199 medium supplemented with 20% FCS, 12 μg/mL of bovine brain extract, 100 U/mL of penicillin, 100 μg/mL of streptomycin, 10 ng/mL of VEGF, and 2 ng/mL of bFGF, then plated on the fibronectin-coated (2 μg/cm2) coverslips in 6-well plates. After 4 days in culture, nonadherent cells were removed by washing with PBS, new media were added, and the culture was maintained through 7 days.

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Rat EPC Characterization and Counting

Cytochemical analysis: After being cultured for 7 days in vitro, cells were incubated with the rabbit polyclonal Flk-1 (VEGFR-2) and eNOS antibody (both Santa Crutz, 1:100). The sections were then incubated with the HRP-conjugated secondary antibody (GeneTech), developed with DAB, and counterstained with hematoxylin. Fluorescent staining was also used to detect dual binding of FITC-UEA-I and DiI-acLDL as described in the “Human EPC Characterization” section. Two or 3 independent investigators evaluated EPC number per well by counting 15 randomly selected high-power fields (×200) with an inverted fluorescent microscope.

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Semiquantitative Reverse Transcription-Polymerase Chain Reaction

Expression of eNOS in both injured arteries and human EPCs after resveratrol pretreatment was evaluated by reverse transcription-polymerase chain reaction (RT-PCR). Total RNA was extracted by using the Trizol reagent (Invitrogen) according to the manufacturer's instructions. Total RNA 0.5 μg was subjected to RT-PCR by using the 1-step AccessQuick RT-PCR system (Promega). The rat eNOS and β-actin primers were as follows: eNOS (189 bp) sense: 5′-TGC ACC CTT CCG GGG ATT CTG GCA-3′; eNOS antisense: 5′-GGA TCC CTG GAA AAG GCG GTG AGG-3′; β-actin (314 bp) sense: 5′-CGT AAA GAC CTC TAT GCC AA-3′; β-actin antisense: 5′-AGC CAT GCC AAA TGT GTC AT-3′. The human eNOS and β-actin primers were as follows: eNOS (160 bp) sense: 5′-ATG TTT GTC TGC GGC GAT GT-3′; antisense: 5′-AGC GTG AGC CCG AAA ATG TC-3′; β-actin (230 bp) sense: 5′-GGG TCA GAA GGA TTC CTG TG-3′; antisense: 5′-GGT CTC AAA CAT GAT CTG GG-3′. The intensity of the eNOS bands was assessed relative to their respective β-actin bands with Bandscan version 5.0 (Glyko).

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Evans Blue Staining

To measure the reendothelialized area at 2 weeks after operation, 1.5 ml Evans blue solution (Sinopharm Chemical Reagent Company, 2.5% in saline) was injected into rats via the femoral vein as previously described.24 Thirty minutes after the injection, the aortas were dissected out and opened longitudinally to observe the Evans blue uptake macroscopically. The reendothelialized area was defined macroscopically as the area not stained with Evans blue dye. Planimetric analysis was performed in Image Pro Plus Version 5.0. Then, the aforementioned specimens were fixed in 4% paraformaldehyde for hematoxylin and eosin (H&E) staining so as to observe the neointimal formation and immunohistochemistry as described in the “Neointimal Formation” section.

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Neointimal Formation and Immunohistochemistry

Animals were killed with excessive anesthesia at the different end points after denudation. Every injured vessel was cut into 2 portions and fixed with 4% paraformaldehyde for histologic analysis, dehydrated by ethanol, and embedded in paraffin. Then, serial three 6-μm cross sections were cut from both portions of each specimen, and stained with H&E to observe the intimal hyperplasia. Immunohistochemical detections of eNOS and factor VIII were performed on 6-μm thick paraffin-embedded cross sections. Slides were incubated with rabbit polyclonal antibody for eNOS (Santa Crutz, 1:200) and factor VIII (Signet, 1:40), respectively. After washing with PBS, the sections were incubated with HRP-conjugated secondary antibody (GeneTech), developed with DAB, and counterstained with hematoxylin. Negative controls consisted of omission of primary antibody. For quantification, the intima-to-media (I/M) ratio and intensity optical density of factor VIII and eNOS staining were calculated with Image Pro Plus Version 5.0.

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Statistical Analysis

Data were expressed as mean±standard deviation (SD) and were compared by analysis of variance and Student t test with SPSS 11.5 software. A P value less than 0.05 was considered statistically significant.

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Characterization of Human EPC

Total MNCs isolated and cultured for 7 days resulted in a spindle-shaped, endothelial cell-like morphology. EPCs were characterized as adherent cells double positive for DiLDL uptake and lectin binding by using LSM510. They were further documented by demonstrating the expression of CD34 (25.8±6.2%), KDR (35.1±7.5%), and CD31 (55.2±11.3%) by flow cytometry (Fig. 1).

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Effect of Resveratrol on Human EPC Proliferation

MTT assay showed that a low dosage of resveratrol improved EPC proliferative activity (control vs. 1 μM: 0.22±0.07 vs. 0.33±0.04, P<0.05), whereas a high dosage decreased the proliferative activity (control vs. 60 μM: 0.22±0.07 vs. 0.09±0.05, P<0.05) (Fig. 2).

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Effect of Resveratrol on Human EPC Migration

Transwell chamber assay demonstrated that a low dosage of resveratrol profoundly enhanced cell migration (control vs. 1 μM: 30±6 vs. 40±7, P<0.05), whereas a high dosage decreased migratory activity of the cells (control vs. 60 μM: 30±6 vs. 16±6, P<0.05) (Fig. 2).

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Effect of Resveratrol on Human EPC Adhesiveness

After replating on fibronectin-coated dishes, EPCs preexposed to a low dosage of resveratrol exhibited a significant increase in the number of adhesive cells at 30 minutes (control vs. 1 μM: 33±8 vs. 44±9, P<0.05), but a high dosage attenuated the adhesive activity of human EPCs (control vs. 60 μM: 33±8 vs. 16±5, P<0.05) (Fig. 2).

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Effect of Resveratrol on eNOS Expression in Human EPCs

To evaluate resveratrol modulation of eNOS mRNA expression, we performed semiquantitative RT-PCR. The results showed that a low dosage of resveratrol (1 μM) induced up-regulation of eNOS; however, a high dosage (60 μM) down-regulated it (Fig. 3). An enzyme linked immunosorbent assay (ELISA) assay also suggested that a higher cellular concentration of eNOS in a low dosage of resveratrol-treated cells (1 μM vs. control: 1489.43±113.68 vs. 950.14±214.73 pg/mg, P<0.05) and a lower cellular concentration of eNOS in a high dosage of resveratrol-treated cells (15 and 60 μM vs. control: 422.49±125.10 and 249.80±68.40 pg/mg vs. 950.14±214.73 pg/mg, P<0.05 and P<0.01) (Fig. 3).

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Circulating EPC can be Isolated from Rat Peripheral Blood

Individual data showed no statistical differences among different rat groups in weight and surgical time (data not shown). Rat PBMCs isolated and cultured for 7 days resulted in a spindle-shaped, endothelial cell-like morphology. To confirm their differentiation into cells of the endothelial lineage, rat PBMC-derived cells were analyzed by immunohistochemistry. At day 7 of culture, the cells showed expression of EC-specific markers such as Flk-1 and eNOS. The cells could also take up DiI-acLDL particles from the media and stained positive for FITC-UEA-1 (Fig. 4).

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Effect of Resveratrol on Rat CEPC Mobilization

To demonstrate the effect of resveratrol on the number of circulating EPCs, rat EPCs were defined as costaining with DiI-acLDL and FITC-UEA-I and counted in an inverted fluorescent microscope. At 1 week after balloon denudation, placebo-treated rats showed a higher number of circulating EPCs compared with the sham group (21.58±3.69 vs. 14.63±2.10, P<0.05). Administration of a small dosage of resveratrol (10 mg/kg) led to a 1.5-fold increase of EPCs at 1 week after operation compared with the placebo-treated group (32.46±6.52 vs. 21.58±3.69, P<0.05); however, a large dosage of resveratrol (50 mg/kg) did not increase the circulating EPCs (22.48±6.89 vs. 21.58±3.69, P>0.05) (Fig. 5).

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Effect of Resveratrol on eNOS Expression in Injured Artery

RT-PCR and immunohistochemical staining were used to determine the eNOS mRNA and protein expression induced by resveratrol, respectively. As shown in Figures 6 and 7, and Table 1, 10 mg/kg resveratrol markedly up-regulated the expression of eNOS mRNA and protein in the injured artery (P<0.05), whereas 50 mg/kg resveratrol did not cause any change in the expression of eNOS mRNA and protein compared with the placebo-treated group (P>0.05).

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Effect of Resveratrol on Reendothelialization

Planimetric analysis of rat aorta specimens after Evans blue staining at 2 weeks documented that 10 mg/kg of resveratrol treatment produced accelerated reendothelialization of the balloon-injured arterial segments (Fig. 8); the reendothelialized area of 10 mg/kg resveratrol-treated rats was 80.8±7.7% of the total denuded area. In contrast, the reendothelialized ratio in the placebo-treated group and 50 mg/kg resveratrol-treated group was 58.4±5.8% (P<0.01) and 63.8±10.2% (P<0.05), respectively. There was no statistical difference between the placebo-treated group and 50 mg/kg resveratrol-treated group (P>0.05). At 1 week, immnunohistochemical staining of factor VIII also showed clear differences (Fig. 9, Table 1) between 10 mg/kg of resveratrol-treated rats and placebo-treated rats (P<0.05) as well as 50 mg/kg of resveratrol-treated rats (P<0.05). The results of the 50 mg/kg resveratrol-treated group was similar to those of the placebo-treated group (P>0.05).

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Effect of Resveratrol on Neointimal Formation

The impact of resveratrol therapy on neointimal thickening was studied at 2 and 4 weeks after aorta injury. In placebo-treated rats, the neointimal thickness (I/M) ratio increased markedly at 2 weeks (I/M ratio 0.23±0.06) and at 4 weeks (0.34±0.04). Compared with placebo, however, 10 mg/kg resveratrol therapy, resulted in a statistically significant reduction in neointimal thickening at all time points. I/M ratios at 2 and 4 weeks of animals treated with 10 mg/kg resveratrol were 0.09±0.04 and 0.15±0.07, respectively (P<0.05 and P<0.01 vs. placebo-treated animals). Animals treated with the higher dose of resveratrol (50 mg/kg) only demonstrated a reduction in the I/M ratio at 4 weeks (0.24±0.05, P<0.05 vs. controls), which was not effective as the lower dose of resveratrol (P=0.046) (Fig. 10).

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Reendothelialization at sites of spontaneous or iatrogenic disruption has classically been thought to be the result of the migration and proliferation of adjacent endothelial cells within the vessel wall. Neighboring endothelial cells, however, may not constitute the exclusive source of endothelial cells for repair. Recently, a series of investigations has suggested that EPCs derived from the bone marrow are present in peripheral blood and that these cells can be recruited to denuded areas and incorporated into nascent endothelium for accelerating reendothelialization and attenuating neointimal formation.8,9,25,26 Therefore, mobilization of EPCs may have efficacy for promoting endothelial regeneration in injured artery. In our study, we found that the amount of EPCs in peripheral blood of placebo-treated rats was higher than that of sham-operated rats, indicating that endothelium denudation may also mobilized EPCs from bone marrow. However, a critical limitation, so far, for the therapeutic application of postnatal EPCs is their low number in the circulation (only 0.2% of PBMCs). Moreover, the ability to obtain a sufficient number of cells may be limited by the onset of cell senescence.27,28

Other approaches to overcome this problem may be by using cytokines, growth factors, and drugs to mobilize the EPCs in vivo or expand them in vitro. A large body of literature has shown that some drugs have the ability of mobilizing EPCs in vivo or promoting their angiogenic activity in vitro, which include statin,29 peroxisome-proliferator activated receptor (PPAR-γ) agonists,30 estrogen,31 puerarin, Ginkgo Biloba Extract, etc. Moreover, it has been found that eNOS is involved in the aforementioned mobilizing course of EPCs and their activities in vitro.29–31 Aicher and colleagues15 demonstrated that mice deficient in eNOS displayed impaired ischemia-induced angiogenesis and reduced EPC mobilization; in in vitro studies, eNOS also has a direct effect on promoting EPCs functions. Statins increase EPCs number via a phosphatidylinositol-3 kinase-dependent pathway that is related to the release of NO.16,17 CRP inhibits EPCs differentiation, survival, and function, which occurs in part via an effect of CRP to reduce eNOS expression in EPCs; furthermore, the PPARr agonist rosiglitazone inhibits the effects of CRP on EPCs differentiation and promotes EPCs survival and function by restoring eNOS expression.18 For resveratrol, increasing evidences have testified that it could up-regulate eNOS expression both in vivo and vitro.12–14,32 Klinge et al14 demonstrated that the ability of resveratrol to enhance expession of eNOS was dependent on its estrogen property. In addition, resveratrol can activate PPAR-α and PPAR-γ ligands in ECs,33 so PPAR-γ activation may be also involved in the mechanism by which resveratrol upgraded the expression of eNOS due to the ability of PPAR-γ to activate eNOS.30

In the present study, we used resveratrol pretreatment as a strategy to stimulate both EPC angiogenic functions in vitro and the mobilization of circulating EPCs to enhance rapid reendothelization of balloon-injured vessels in vivo. In previous in vitro studies, both small (10 to 100 nM)14 and large (10 to 100μM)13 doses of resveratrol could up-grade the expression of eNOS, but the dose to enhance eNOS expression in vivo was limited to a low dosage (2.5 to 10 mg/kg);12,32 whether a higher dosage of resveratrol could induce eNOS expression in vivo has not been reported. In the present study, we used resveratrol at different dosages both in vivo (10 and 50 mg/kg/d) and in vitro (1 to 60 μM) to observe its influence on eNOS expression as well as EPC mobilization and angiogenic functions.

In a rat model of an injured aorta, our results showed that a low dose of resveratrol (10 mg/kg) increased the abundance of circulating EPCs and accelerated the rate of reendothelialization. More significantly, a low dose inhibited neointimal thickening in balloon-injured arteries. This appears to be at least in part owing to the increased availability of circulating EPCs capable of inducing rapid reendothelialization of the injured vessel. In addition, a low dose could increase eNOS expression in injured arteries. The above results provide us 2 possible mechanisms: first, resveratrol may have the ability to promote the mobilization of EPCs from bone marrow via the eNOS pathway like the aforementioned drugs,29–31 which was at least in part responsible for the repair of the injured vessel. Second, resveratrol up-regulated eNOS expression and mobilized the EPCs, to accelerate the repair of injured artery, and mobilization of EPCs was independent of the up-reguated expression of eNOS. The actual mechanism needs to be further elucidated.

As for a large dose of resveratrol (50 mg/kg), we could not find its effect on eNOS expression, EPCs number, and reendothelialization of denudated artery; it only inhibited neointimal formation at 4 weeks after angioplasty, which was not as effective as the small dose. Therefore, we presumed that only a lower dose of reveratrol could enhance expression of eNOS in vivo, which was consistent with the fact that low concentration of resveratrol exerted protective role in human body.34 Why a large dosage could not enhance eNOS expression in vivo requires further investigations. Concerning the mechanisms by which resveratrol reduced the intima hyperplasia independent of eNOS, former researches have confirmed that resveratrol could down-regulate the expression of endothelin-1 and inhibit the activation of extracellular signal-regulated kinase,35 which in turn reduce the proliferation of smooth muscle cells. Resveratrol also can suppress cell proliferation and induce apoptosis, accompanied by cell cycle arrest at specific points in S and G2 via the induction of tumor-suppressor gene protein p53 and cyclin-dependent kinase inhibitor p21.36

In accordance with studies in vivo, we demonstrated that only a low concentration of resveratrol had promoting effects on proliferative, migrative, and adhesive functions of EPCs as well as eNOS expression in vitro; the high concentration caused a significant decrease of the aforementioned activities and eNOS expression. So we hypothesize that eNOS may contribute to the effect of resveratrol on the EPCs activities in vitro. Moreover, it is important to note the diverse effects on eNOS expression of mature ECs and EPCs. Resveratrol could up-regulate eNOS expression at both low and high concentrations in ECs,12,13 but for EPCs, it exerts the promoting effect at low concentration and inhibitory effect at high concentration. This may be related to the different characteristics between mature cells and stem/progenitor cells, which need further investigations. Furthermore, the above results were consistent with the fact that low plasma concentration (10 to 40 nmol/L after moderate resveratrol consumption) exerted a protective role in the human body.34

In in vitro studies, we have shown that the effect of resveratrol was not dose dependent or reaching plateau, but rather attenuated at high dose, suggesting a toxic effect for EPCs. Therefore, we presumed that lower, more physiologic concentrations may have a stronger effect on functional properties of isolated EPCs. Additionally, the fact that a large dose of resveratrol could not reach the same effect as a low dose in animal studies may be related to its antiangiogenic effect at high dose in vitro.36

In conclusion, we found that a low concentration of resveratrol could promote EPC activities and eNOS expression in vitro, but a high concentration exerta an adverse effect. In vivo, a low dose of resveratrol could accelerate the mobilization of EPCs, boost reendothelialization, attenuate the neointimal formation, and up-regulate the expression of eNOS after balloon injury, whereas a large dosage of resveratrol only inhibited the intima hyperplasia at 4 weeks. The above conclusions may be one of the mechanisms by which resveratrol protects the cardiovascular system. This needs to be further investigated.

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resveratrol; endothelium denudation; endothelial progenitor cells; eNOS

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