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Published as doi: 10.1096/fj.06-7491com.
 (The FASEB Journal. 2007;21:3845-3852.)
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Moderate consumption of red wine (cabernet sauvignon) improves ischemia-induced neovascularization in ApoE-deficient mice: effect on endothelial progenitor cells and nitric oxide

Jonas Lefèvre, Sophie-Élise Michaud, Paola Haddad, Sylvie Dussault, Catherine Ménard, Jessika Groleau, Julie Turgeon and Alain Rivard1

Department of Cardiovascular Research, Centre Hospitalier de l’Université de Montréal, Montréal, Québec, Canada

1Correspondence: Centre Hospitalier de l’Université de Montréal, 1560 Sherbrooke Est, Montreal, QC, H2L 4M1 Canada. E-mail: rivardal{at}total.net


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Moderate consumption of red wine is associated with a decreased incidence of cardiovascular diseases in populations with relatively high amount of fat in the diet. However, the mechanisms involved in this protective effect are not completely understood. Here we show that moderate consumption of red wine (equivalent to 2 glasses/day in humans) but not ethanol only, improves blood flow recovery by 32% after hindlimb ischemia in hypercholesterolemic ApoE-deficient mice. In ischemic tissues, red wine consumption reduces oxidative stress and increases capillary density by 46%. Endothelial progenitor cells (EPCs) have been shown to have an important role in postnatal neovascularization. We found that the number of EPCs is increased by 60% in ApoE mice exposed to red wine. Moreover, the migratory capacity of EPCs is significantly improved in red wine-drinking mice. The wine used in our study is a cabernet sauvignon from Languedoc-Roussillon, France, which contains a relatively high concentration (4–6 mg/L) of the polyphenolic antioxidant resveratrol. We demonstrate that resveratrol can rescue oxidized low-density lipoprotein (oxLDL)-induced impairment of in vitro angiogenic activities in human umbilical vein endothelial cells (HUVECs). Resveratrol exposure is also associated with increased activation of Akt/eNOS together with a restoration of nitric oxide production in HUVECs exposed to oxLDL. Our study suggests that moderate consumption of red wine improves ischemia-induced neovascularization in high-cholesterol conditions by increasing the number and the functional activities of EPCs and by restoring the Akt-eNOS-NO pathway.—Lefèvre, J., Michaud, S.-E., Haddad, P., Dussault, S., Ménard, C., Groleau, J., Turgeon, J., Rivard, A. Moderate consumption of red wine (cabernet sauvignon) improves ischemia-induced neovascularization in ApoE-deficient mice: effect on endothelial progenitor cells and nitric oxide.


Key Words: hypercholesterolemia • resveratrol


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
IN POPULATIONS WITH HIGH INTAKE of dietary cholesterol and saturated fat, moderate and regular consumption of wine, in particular red wine, is associated with a decreased incidence of cardiovascular diseases (1 , 2) . This phenomenon has been described as "The French Paradox" (3) . The precise mechanisms by which red wine can prevent cardiovascular events are not completely understood but might be related to its antioxidant properties. Red wine and red wine polyphenols such as resveratrol have been shown to reduce the oxidation of low-density lipoproteins (LDL; refs. 4 , 5 ), reduce platelet aggregation (6 7 8) , and inhibit smooth muscle proliferation (9) and migration (10) . Moreover, the exposure to red wine or red wine polyphenols is associated with increased bioactivity of nitric oxide (NO) and improved endothelial function in animal models and in humans (11 12 13) . Taken together, these properties could contribute to reduce atheroclerosis and prevent acute cardiovascular events in patients exposed to red wine. However, whether moderate consumption of red wine might also have a positive effect on the response to ischemia, and more specifically on ischemia-induced neovascularization, is currently unknown.

The ability of the organism to spontaneously develop new blood vessels constitutes an important response to vascular occlusive disease that determines in large part the severity of residual tissue ischemia (14) . Postnatal neovascularization necessitates the activation, migration, and proliferation of mature endothelial cells (angiogenesis; ref. 15 ). Vascular endothelial growth factor (VEGF), an endothelial cell specific mitogen, has been shown to be a critical limiting factor for the induction of angiogenesis (16) . The importance of NO for endothelial cell migration and VEGF-induced angiogenesis was also recently demonstrated (17 , 18) . However, increasing evidence suggests that postnatal neovascularization relies not exclusively on sprouting of preexisting vessels but also involves the contribution of bone marrow-derived circulating endothelial progenitor cells (EPCs; ref. 19 ). For instance, it has been demonstrated that circulating EPCs in adults can home to ischemic tissues and contribute to the formation of new blood vessels (20) .

Interestingly, the same risks factors that promote the development of atherosclerotic diseases are also associated with reduced neovascularization in response to ischemia (21 22 23 24) . Hypercholesterolemia, an important cardiovascular risk factor, has been shown to be associated with impaired blood flow recuperation and angiogenic response in different animal models (23 , 25) . A common feature of all cardiovascular risk factors (including hypercholesterolemia) is increased oxidative stress (26) . Excessive oxidative stress has also been associated with impaired angiogenesis in different models (27 , 28) . However, whether postnatal neovascularization in clinical situations of increased oxidative stress can be significantly improved with antioxidants remains to be determined.

In the present study, we tested the hypothesis that regular red wine consumption can improve ischemia-induced neovascularization and blood flow recuperation in hypercholesterolemic conditions. We also investigated potential mechanisms involved in this physiopathology including the role of oxidative stress, NO, and EPCs.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Murine ischemic hindlimb model and monitoring of blood flow
The protocol was approved by the Comité Institutionnel de Protection des Animaux (CIPA) of the Centre Hospitalier de l’Université de Montréal (CHUM). Six- to eight-week-old ApoE–/– mice on a C57Bl/6 background were purchased from Jackson Laboratory (Jackson Mice, Bar Harbor, ME, USA) and housed in a pathogen-free isolation facility with a light/dark cycle of 12/12 h. Mice were maintained on ad libitum chow and received drinking water only (controls) or water containing either 6% ethanol or 50% red wine (final concentration of 6% ethanol). The red wine used in this study was "Réserve Maison Nicolas" (12% ethanol), a cabernet sauvignon from Languedoc-Roussillon, France, that contains a relatively high concentration of resveratrol (4–6 mg/L) (29) . Since each mouse drank around 4 ml of liquid per day, we calculated that red wine-treated mice consumed an average of 8 g of ethanol/kg/day (7% of the total daily energy intake), which is equivalent to moderate consumption (two 5-oz glasses per day) in adult humans (30) . After 2 wk of treatment, unilateral hindlimb ischemia was surgically induced and hindlimb blood flow was monitored with a laser Doppler perfusion imager (LDPI) system (24 , 31) .

Tissue preparation and immunohistochemistry
For immunohistochemistry, whole ischemic hindlimbs were immediately fixed in tissue-fix overnight. After bones had been carefully removed, 3 µm thick tissue transverse sections of the hindlimbs were cut at the level of the gastrocnemius muscle and paraffin-embedded so that the whole leg could be analyzed on each section. Identification of endothelial cells was performed by immunostaining for platelet endothelial cells adhesion molecule-1 (PECAM-1 or CD31) with a rat monoclonal antibody directed against mouse CD31 (Pharmigen, San Diego, CA, USA), and capillary density was determined as described previously (24) . To evaluate local oxidative stress levels in ischemic muscles, an antibody against nitrotyrosine (Upstate, Lake Placid, NY, USA) was used. Intensities of fluorescence were measured and analyzed using computer-based analysis (Metamorph) with the same threshold for all sections under x200. The specificity of the test was confirmed by preincubating the antibody with 10 mM nitrotyrosine (data not shown).

Cell culture
Human umbilical vein endothelial cells (HUVECs) were purchased from Cascade Biologics (Portland, OR, USA) and cultured in medium 200 supplemented with 10% fetal bovine serum (FBS), 3 ng/ml basic fibroblast growth factor (bFGF), 10 µg/ml heparin, 1 µg/ml hydrocortisone, and 10 ng/ml EGF. Cells were grown at 37°C and 5% CO2. HUVECs were passaged when they reached 80% confluence and passages 2–5 were used for all experiments.

EPCs isolation and characterization
Seven days after ischemia, mouse bone marrow mononuclear cells were isolated from the femora and tibiae by flushing the bone marrow cavities using culture medium. After red blood cell lysis and being washed, bone marrow mononuclear cells were plated on 0.005% fibronectin (Sigma, St. Louis, MO, USA) and cultured in complete medium 200 containing 20% FBS. After 4 days in culture, nonadherent cells were removed by thorough washing with PBS. Adherent cells were stained with 1,1'-dictadecyl-3,3,3',3' tetramethyllindocarbocyanine perchlorate-acetylated low-density lipoprotein (DiI-acLDL, 2.5 µg/ml for 1 h, Invitrogen, OR, USA) and FITC-labeled lectin BS-1 (Bandeiraea simplicifolia, 10 µg/ml for 1 h, Sigma). Bone marrow EPCs were characterized as adherent cells that were positive for both DiI-acLDL uptake and lectin binding and were quantified by examination of random microscopic fields.

HUVEC capillary-like tube formation on matrigel
HUVECs were plated in 96-well plates that had been precoated with 50 µl of growth factor reduced Matrigel Matrix (Becton Dickinson Labware, Bedford, MA, USA) and cultured at 37° for 8 h with either 0.1% FBS (control) or 50 ng/ml VEGF. To determine the effect of oxidized LDL (oxLDL) and resveratrol on VEGF-induced tube formation, cells were treated or not with oxLDL (Biomedical Technologies, Stoughton, MA, USA), resveratrol, or ethanol (Sigma). The doses used were based on preliminary experiments showing maximal inhibition of tube formation without induction of apoptosis with 50 µg/ml oxLDL and optimal rescue of tube formation with 50 µM resveratrol in these conditions (data not shown). After the different treatments, capillary-like tubes were photographed under a dissecting microscope and all side branches in a well were counted by a single investigator in a blinded manner. Each experiment was performed in duplicate for each condition. A branch was defined as a straight cellular segment connecting two cell masses (nodes).

HUVEC and EPC migration assay
Cell migration was assessed using a modified Boyden chamber assay as described previously (28) . Inserts were placed in a 24-well plate containing medium 200 with 50 ng/ml VEGF. Cells (15,000) were added to the upper chamber of the inserts in medium 200. Cells were allowed to migrate from the upper to the lower chamber for 6 h at 37°C. Nonmigratory cells were removed from the upper chamber by wiping the upper surface with an absorbant tip. Cells that had migrated to the lower side of the Transwell insert were fixed for 10 min with 3.7% formaldehyde and stained with hematoxilin. After extensive PBS washing to remove excess hematoxilin, the number of cells that had migrated was counted in three different representative high power (x200) fields per insert (2 inserts per condition). In some experiments, HUVECs were treated (or not) with oxLDL and resveratrol as described above.

HUVEC MTS assay
HUVEC proliferation was assessed using the MTS Celltiter 96 aqueous nonradioactive cell proliferation assay (Promega, Madison, WI, USA; ref. 28 ).

HUVEC NO release
NO production was determined indirectly by measuring the concentration of the stable end products nitrate and nitrite using a commercial kit (R&D Systems, Minneapolis, MN, USA) based on the Griess reaction. HUVECs were exposed to oxLDL and resveratrol for 16 h. After treatment, NO concentrations were measured in fresh media after 6 h of VEGF stimulation (50 ng/ml).

Measurements of intracellular reactive oxygen species (ROS) in HUVECs
Intracellular ROS generation was assessed using 2', 7'-DCF diacetate (DCFH-DA, Sigma). ROS in the cells oxidize DCFH-DA, yielding the fluorescent compound 2',7'-DCF (DCF). HUVECs were plated at a density of 105/well in a 24-well plate in complete Medium 200, 10% FBS. Adherent cells were washed with warm PBS and treated for 16 h in the presence or absence of oxLDL and resveratrol. Cells were washed, and DCF-DA (10 µmol/L) was added for 30 min. The media were removed, and cells were washed and quickly analyzed for fluorescence (excitation at 498 nm and emission at 522 nm). The specificity of the test was confirmed using 500 µM H2O2 and 5 mM Tiron as positive and negative controls, respectively (data not shown). Dihydroethidium (DHE, Calbiochem, San Diego, CA, USA) was used to evaluate the presence of superoxide anion oxygen radicals. In the presence of O2–, DHE is oxidized to ethidium bromide, which binds to DNA. The fluorescence reaction was carried out by incubating HUVECs with DHE (2 µmol/L) for 20 min. DHE fluorescence was detected with a 585 nm filter. The specificity of the test was confirmed using 50 ng/ml TNF-{alpha} and 5 mM Tiron as positive and negative controls, respectively (data not shown).

Western blot analysis
Western blotting of p44/p42 MAPK (Thr202/Tyr204), eNOS (Ser-1177), and Akt (Ser-473 was performed using phospho-specific antibodies as previsouly described; ref. 28 ).

Statistical analysis
All results are mean ± SE. Statistical significance was evaluated by ANOVA. A value of P < 0.05 was interpreted to denote statistical significance.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Moderate consumption of red wine improves ischemia-induced neovascularization in ApoE-deficient mice
Mice in our study consumed an amount of red wine that is equivalent to moderate consumption (two 5-oz glasses per day) in adult humans (30) . This regimen did not significantly modify cholesterol concentrations and was well tolerated by hypercholesterolemic ApoE–/– mice, without any sign of hepatic toxicity (Table 1 ). Hindlimb perfusion was evaluated postoperatively by serial LDPI studies. As shown on Fig. 1 , similar low levels of blood flow were documented in the different groups immediately after surgery (day 0). However, at day 21 after surgery, the Doppler flow ratio was significantly improved in mice exposed to red wine (0.71±0.04) compared to control mice (0.54±0.04; P<0.05) or mice exposed to ethanol only (0.55±0.03; P<0.05). At the microvascular level, tissue sections from the ischemic hindlimb muscles were examined histologically to determine capillary density at day 21 after surgery. As shown on Fig. 2 A, CD31 immunostaining demonstrated a significant increase in capillary density in mice exposed to red wine (244±17 capillaries/mm2) compared to control mice (167±15 capillaries/mm2; P<0.05) or mice exposed to ethanol only (189±16 capillaries/mm2; P<0.05). This increased capillary density was associated with a significant reduction of oxidative stress levels in ischemic muscles of ApoE–/– mice exposed to red wine, as assessed by nitrotyrosine staining (Fig. 2B ).


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  		Table 1. Biochemical analysis of the different treatment groups


Figure 1
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  		Figure 1. Effect of red wine and ethanol consumption on blood flow recuperation after hindlimb ischemia in hypercholesterolemic ApoE–/– mice. Representative results of laser Doppler measurements 21 days after surgery in control (H2O, n=13), ethanol (ETOH, n=15) and red wine-drinking mice (n=14). A color scale illustrates blood flow variations from minimal (dark blue) to maximal (red) values. Arrow indicates ischemic (left) limb. Right panel represents laser Doppler perfusion ratios (ischemic/nonischemic hindlimb) over time in the different groups. Data are mean ± SE. *P < 0.05 vs. H2O and ETOH groups.


Figure 2
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  		Figure 2. Effect of red wine and ethanol consumption on capillary density and oxidative stress in ischemic muscles. CD31 (A, n=9–11 per group) and nitrotyrosine (B, n=4 per group) immunostaining of ischemic muscles extracted at day 21 in H2O, ETOH and red wine-drinking mice. Arrows indicate positive staining. Data are mean ± SE. *P < 0.05 vs. H2O and ETOH mice; @P < 0.05 vs. H2O mice.

Consumption of red wine increases EPC number and migratory activity in ApoE-deficient mice
To identify potential mechanisms involved in the beneficial effect of red wine on postnatal neovascularization, we isolated and characterized EPCs from the bone marrow of ApoE-deficient mice exposed or not to red wine. EPCs were defined as adherent cells that were positive for both DiI-acLDL uptake and lectin binding (Fig. 3 A). As shown on Fig. 3B , the number of EPCs was significantly increased in mice exposed to red wine compared to controls (59±6 vs. 37±4 cells per field; P<0.01). Moreover, the migratory capacity (Fig. 3C ) of EPCs isolated from red wine-drinking mice was also significantly improved compared to controls (23±4 vs. 11±3 cells per field; P<0.05).


Figure 3
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  		Figure 3. Effect of red wine consumption on EPC number and migratory activity in ApoE–/– mice. A, B) EPC number was assessed using DiI-acLDL, lectin-FITC, and DAPI stains. Cells positive for all 3 dyes were counted as EPCs. EPCs from 6 different mice/group were quantified. C) Cell migration was assessed using a modified Boyden chamber assay. EPCs from 5 different mice/group were tested in duplicate. Data are mean ± SE. *P < 0.05 vs. H2O EPCs.

Resveratrol, a potent antioxidant in red wine, restores the angiogenic properties of endothelial cells exposed to oxLDL
In HUVECs, VEGF-induced capillary-like tube formation is completely abolished in the presence of oxLDL. However, resveratrol (but not ethanol) can rescue the angiogenic potential of HUVECs exposed to oxLDL (Fig. 4 A). In addition, VEGF-induced proliferation and migration are impaired in HUVECs exposed to oxLDL but can be rescued by resveratrol treatment (Fig. 4B, C ).


Figure 4
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  		Figure 4. Effect of resveratrol on the angiogenic properties of endothelial cells. A) Evaluation of tube formation was performed using a Matrigel assay in HUVECs exposed or not to VEGF (50 ng/ml), oxLDL (50 µg/ml), resveratrol (50 uM), and ethanol (1%). One representative experiment of four is shown. Lower panel represents statistical analysis of the number (left) and the total length (right) of tubes formed by HUVECs. B) Cell proliferation was assessed using the MTS assay in HUVECs exposed to different conditions. Results of 6 independent experiments performed in triplicates are presented. C) Cell migration was assessed using a modified Boyden chamber assay in HUVECs exposed to different conditions. Results of 4 independent experiments are presented. Data are mean ± SE. *P < 0.05 vs. CTL; @P < 0.05 vs. VEGF; #P < 0.05 vs. VEGF oxLDL; &P < 0.05 vs. VEGF oxLDL RESV.

Resveratrol reduces oxidative stress in HUVECs exposed to oxLDL
ROS and superoxide anions generation in HUVECs were measured using DCFH-DA and DHE staining, respectively. As seen in Fig. 5 A, ROS (DCFH-DA in green) and superoxide (DHE in red) are significantly increased in HUVECs exposed to oxLDL. However, treatment with resveratrol can prevent the increase of oxidative stress in HUVECs exposed to oxLDL (Fig. 5A-C ).


Figure 5
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  		Figure 5. A) ROS generation and superoxide formation in HUVECs exposed or not to oxLDL and resveratrol. Intracellular ROS generation was assessed using DCF-DA. Intracellular superoxide formation was assessed using DHE. B) Quantification of DCF-DA pictures from four different experiments. C) Quantification of DHE pictures from four different experiments. Data are mean ± SE. *P < 0.01 vs. CTL; @P < 0.01 vs. oxLDL.

Resveratrol restores the Akt/eNOS/NO pathway in HUVECs exposed to oxLDL
We also investigated the effect of oxLDL and resveratrol exposure on the induction of angiogenic transduction pathways in HUVECs. As shown on Fig. 6 A, VEGF-induced activation of phosphorylated p44p42 MAPK, Akt, and eNOS is significantly impaired in HUVECs exposed to oxLDL but can be rescued by resveratrol treatment. In addition, NO release by HUVECs after VEGF exposure is impaired in the presence of oxLDL but can be rescued by resveratrol (Fig. 6B ).


Figure 6
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  		Figure 6. A) p44/p42, Akt and eNOS activation was evaluated by phospho-specific Western blots in HUVECs stimulated with VEGF (0, 15, 30, or 120 min) and exposed to different conditions. B) NO production was determined indirectly by measuring concentration of stable end products nitrites/nitrates (Griess reaction) in HUVECs exposed to different conditions. Data are mean ± SE. *P < 0.01 vs. CTL; @P < 0.01 vs. VEGF; #P < 0.001 vs. VEGF oxLDL.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
To our knowledge, the present study is the first one to document the favorable effect of moderate red wine consumption on ischemia-induced neovascularization. Our experiments were performed in ApoE deficient mice, a hypercholesterolemic model of atherosclerosis that has also been shown to present impaired neovascularization in response to ischemia (23) . We demonstrate that after hindlimb ischemia, ApoE-deficient mice exposed to red wine present improved blood flow recuperation and increased capillary density in ischemic tissues. Interestingly, these effects were not seen when the mice were exposed to ethanol only.

The mechanisms by which red wine can improve ischemia-induced neovascularization are potentially diverse. It has been proposed that the cardiovascular benefits of moderate wine consumption are due, at least in part, to the antioxidant properties of red wine (5) . Oxidative stress is associated with the development of atherosclerotic diseases but has also been shown to impair endothelial cell growth and angiogenesis (27 , 28) . Here we demonstrate that in hypercholesterolemic conditions moderate red wine consumption is associated with reduced oxidative stress in ischemic tissues, as assessed by nitrotyrosine staining. Because excessive oxidative stress has been shown to impair the effects of angiogenic growth factors such as VEGF (28) , it is conceivable that red wine might improve neovascularization by restoring VEGF angiogenic activities through its antioxidant properties.

The precise constituents of red wine that are responsible for its beneficial effects are not completely known. The fact that we did not observe any positive effect of ethanol only on ischemia-induced neovacularization in hypercholesterolemic conditions suggests that other specific components of red wine must be involved. Resveratrol is a major polyphenolic antioxidant obtained primarily from red wine in most human diets. Recent evidence suggests that resveratrol might be at least partly responsible for the beneficial cardiovascular effects of red wine and the so-called French paradox (32 , 33) . The red wine used in our in vivo study is a cabernet sauvignon from Languedoc-Roussillon, France, that contains relatively high levels of resveratrol compared to similar red wines from Italy, California, or Australia (29) . Resveratrol can be detected in human LDL particles after consumption of red wine (34) . It can prevent LDL oxidation (5) and oxLDL-induced cytotoxicity in endothelial cells in vitro (35) . Resveratrol can also enhance vasorelaxation, prevent platelet aggregation in vitro and in vivo, and protect against ischemia/reperfusion injury in isolated hearts (33) . Globally, these effects could contribute to reduce the development of atherosclerotic diseases and prevent acute ischemic events.

The present study suggests that resveratrol might also have a beneficial effect on VEGF-dependent angiogenesis. VEGF is the most potent activator of endothelial cells. It is induced by hypoxia in ischemic tissues, where it has been shown to have a critical role for the induction of angiogenesis (16) . Here we found that VEGF angiogenic activities (endothelial cell proliferation, migration and tube formation) are completely abrogated in the presence of oxLDL. However, treatment with resveratrol can rescue VEGF angiogenic activities in endothelial cells exposed to oxLDL. The mechanism by which resveratrol improves VEGF-dependent angiogenesis could certainly be related to its antioxidant potential. We and others have previously shown that situations of excessive oxidative stress can impair VEGF-induced angiogenesis in endothelial cells (27 , 28) . In the present study, we show that endothelial cells exposed to oxLDL exhibit high levels of oxidative stress, but that resveratrol treatment can prevent this increase of oxidative stress. We also demonstrate that in endothelial cells exposed to oxLDL resveratrol can rescue the expression of several important transduction pathways that are involved in VEGF angiogenic activities such as p44p42MAPK, Akt, and eNOS (36) . Importantly, we found that resveratrol can also rescue NO release in endothelial cells that are exposed to oxLDL. Recent studies have shown that NO is an essential mediator of endothelial cell migration and VEGF-induced angiogenesis (17 , 18) . Globally, our results indicate that resveratrol can reduce oxidative stress and rescue VEGF-dependent angiogenesis in endothelial cells exposed to oxLDL.

Recent studies (19 , 20) suggest that postnatal neovascularization relies not exclusively on sprouting of preexisting vessels (angiogenesis) but also involves the contribution of bone marrow-derived circulating EPCs. Interestingly, cardiovascular risk factors involving increased oxidative stress are associated with impaired number and functional activity of EPCs in patients with coronary artery disease (CAD) (37) . Even in the absence of clinically apparent CAD, hypercholesterolemia by itself was recently shown to reduce the number and the function of EPCs (38) . Taken together, these results suggest that impaired number and/or function of EPCs could contribute to impair postnatal neovascularization in hypercholesterolemic conditions. In the present study, we found that a moderate consumption of red wine is associated with an important increase of bone marrow EPC number in hypercholesterolemic ApoE mice. Our findings are limited to bone marrow EPCs and might not always directly correlate with EPCs that are mobilized into the peripheral blood under different conditions. Nevertheless, these results suggest that hypecholesterolemic mice exposed to red wine possess a larger central pool of EPCs that could be recruited for postnatal neovascularization. In addition, we also demonstrated that the migratory activity of EPCs is significantly improved in ApoE mice exposed to red wine. The precise mechanisms by which red wine increases EPC number and functional activity remain to be determined. EPCs have been shown to express higher levels of antioxidative enzymes and enhanced protection against oxidative stress compared to mature endothelial cells (39) . However, the functional activities of EPCs are impaired in conditions of increased oxidative stress (40 , 41) . Moreover, the number and the functional activity of EPCs directly correlate with serum antioxidant capacity (41) . Therefore, the antioxidant potential of red wine could contribute to restore EPC number and function in situations of increased oxidative stress such as hypercholesterolemia.

In summary, our study indicates that moderate red wine consumption improves neovascularization and blood flow recovery after ischemia in hypercholesterolemic conditions. We propose that red wine polyphenols such as resveratrol are at least partly responsible for these positive effects by reducing oxidative stress and restoring the angiogenic Akt/eNOS/NO pathway. Together with the positive effect on EPC number and functional activity, this could contribute to explain the beneficial effect of moderate red wine consumption on the incidence of cardiovascular diseases. Moreover, the results of this study could lead to the development of novel strategies to prevent the complications of ischemic vascular diseases.


   ACKNOWLEDGMENTS
 
This study was supported by a grant from the CIHR to A.R. (#74687). A.R. is a scholar from the Fédération de Recherche en Santé du Québec (FRSQ). S.-E.M. is a doctoral research awardee from the Heart and Stroke Foundation of Canada. J.G. is a doctoral research awardee from the CIHR.

Received for publication March 26, 2007. Accepted for publication June 21, 2007.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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