Intake of Red Wine Increases the Number and Functional Capacity of Circulating Endothelial Progenitor Cells by Enhancing Nitric Oxide Bioavailability
Objective— Red wine (RW) consumption has been associated with a reduction of cardiovascular events, but limited data are available on potential mediating mechanisms. This study tested the hypothesis that intake of RW may promote the circulating endothelial progenitor cell (EPC) level and function through enhancement of nitric oxide bioavailability.
Methods and Results— Eighty healthy, young subjects were randomized and assigned to consume water (100 mL), RW (100 mL), beer (250 mL), or vodka (30 mL) daily for 3 weeks. Flow cytometry was used to quantify circulating EPC numbers, and in vitro assays were used to evaluate EPC functions. After RW ingestion, endothelial function determined by flow-mediated vasodilation was significantly enhanced; however, it remained unchanged after water, beer, or vodka intake. There were significantly increased numbers of circulating EPC (defined as KDR+CD133+, CD34+CD133+, CD34+KDR+) and EPC colony-forming units only in the RW group (all P<0.05). Only RW ingestion significantly enhanced plasma levels of nitric oxide and decreased asymmetrical dimethylarginine (both P<0.01). Incubation of EPC with RW (but not beer or ethanol) and resveratrol in vitro attenuated tumor necrosis factor-α–induced EPC senescence and improved tumor necrosis factor-α–suppressed EPC functions and tube formation. Incubation with nitric oxide donor sodium nitroprusside significantly ameliorated the inhibition of tumor necrosis factor-α on EPC proliferation, but incubation with endothelial nitric oxide synthase inhibitor l-NAME and PI3K inhibitor markedly attenuated the effect of RW on EPC proliferation.
Conclusion— The intake of RW significantly enhanced circulating EPC levels and improved EPC functions by modifying nitric oxide bioavailability. These findings may help explain the beneficial effects of RW on the cardiovascular system. This study demonstrated that a moderate intake of RW can enhance circulating levels of EPC in healthy subjects by increasing nitric oxide availability. Direct incubation of EPC with RW and resveratrol can modify the functions of EPC, including attenuation of senescence and promotion of EPC adhesion, migration, and tube formation. These data suggest that RW ingestion may alter the biology of EPC, and these alterations may contribute to its unique cardiovascular-protective effect.
Epidemiological evidence confirms that light to moderate alcohol consumption reduces the risk of morbidity and mortality from cardiovascular disease.1,2 Regular consumption of any type of alcoholic beverage appears to confer healthy benefits, but red wine (RW), with its abundant antioxidant contents, seems to offer additional healthy benefits.3,4 Recent studies indicated that the beneficial effects of RW are derived from increased endothelium-derived nitric oxide (NO),5 implying that enhanced NO bioavailability may mediate the cardiovascular protection provided by RW.
During the past decades, endothelial function has been shown to play an important role in the clinical manifestations of established atherosclerotic lesions.6 Endothelial dysfunction is characterized by a reduction of the bioavailability of vasodilators, particularly NO, and can predict future cardiovascular events.7 Increasing evidence suggests that the injured endothelial monolayer is regenerated by circulating bone marrow-derived endothelial progenitor cells (EPC), which accelerate reendothelialization and protect against the initiation and progression of atherosclerosis.8,9 Levels of circulating EPC reflect vascular repair capacity and have been shown to be associated with endothelial function.10 Moreover, EPC are significantly downregulated in patients with coronary artery disease or risk factors for coronary artery disease.11 Clinical evidence showed that the number of circulating EPC predicts the occurrence of cardiovascular events and death from cardiovascular causes and may help to identify patients at increased cardiovascular risk.12 A progressive reduction in circulating EPC when risk factors are elevated may contribute to a vicious cycle, resulting in endothelial dysfunction and the rapid progression of atherosclerosis.13 Although many epidemiological studies have indicated that light to moderate consumption of RW can reduce the incidence of coronary artery disease, the direct effect of RW consumption on circulating EPC and their functions remains unclear. Therefore, we designed this study to investigate whether intake of RW could enhance the number and function of EPC through increasing NO bioavailability.
Subjects and Methods
Human Study Protocol
The human study was a prospective, randomized, diet-controlled, open-label trial in which 80 young, healthy subjects were enrolled (please see Supplemental material available online at http://atvb.ahajournals.org).
Endothelium-Dependent Flow-Mediated Vasodilation
Endothelium-dependent flow-mediated vasodilation was assessed using a 7.5-MHz linear array transducer (Hewlett-Packard Sonos 5500) as previously described (available online at http://atvb.ahajournals.org).14
After 8 hours of overnight fasting, all subjects had a venous blood sample taken for measurement of high-sensitivity C-reactive protein, tumor necrosis factor-α (TNF-α), NO, asymmetrical dimethylarginine, adiponectin, and oxidized low-density lipoproteins (please see http://atvb.ahajournal.org).
A volume of 100 μL peripheral blood was incubated for 15 minutes in the dark with monoclonal antibodies against human KDR (R&D) followed by PE-conjugated secondary antibody, with the FITC-labeled monoclonal antibodies against human CD45 (Becton Dickinson), with the PE-conjugated monoclonal antibody against human CD133 (Miltenyi Biotec, Germany), and with FITC-conjugated or PE-conjugated monoclonal antibodies against human CD34 (Serotec; Supplementary Figure I).11
Human Early and Late EPC Cultivation
Peripheral blood samples (20 mL) were obtained from healthy, young volunteers, and total mononuclear cells were isolated by density gradient centrifugation with Histopaque-1077 (Sigma).15
Human EPC Colony-Forming Assay
Isolated mononuclear cells were resuspended in growth medium (EndoCult liquid medium; StemCell Technologies), and 5×106 mononuclear cells were preplated in a fibronectin-coated 6-well plate in duplicate (please see http://atvb.ahajournals.org).16
EPC Number and Proliferation Assay
The number of early EPC and the proliferation of late EPC was determined by direct staining of 6 random high-power microscopic fields (×100) and by 3-(4,5-dimethylthiazol-2-yl)-2,5,diphenyltetrazolium bromide assay, respectively (supplemental material).
EPC Senescence Assay
Cellular aging was determined as previously described (supplemental material).15
EPC Fibronectin Adhesion Assay
EPC fibronectin adhesion test was assessed as previously described (supplemental material).16
EPC Migration Assay
The migratory function of EPC was evaluated by a modified Boyden chamber assay (Transwell; Coster; please see supplemental material).17
EPC Tube Formation Assay
EPC tube formation assay was performed with an in vitro angiogenesis assay kit (Chemicon; supplemental material).17
Western Blotting Analysis and Measurement of Nitrite Levels
Protein extracts were prepared as previously described (supplemental material).18
All data were expressed as the mean±SEM for continuous variables and as the number (percent) for categorical variables. Intergroup comparisons were performed by 1-way ANOVA and a Bonferroni test. P<0.05 was considered statistically significant. The SPSS 9.0 (version 12; SPSS) software package was used for all statistical analyses.
Baseline Characteristics of Study Subjects and Effects of RW on Endothelial Function in Human Study
Eighty healthy subjects participated in the study (mean age, 34±1 years) and were randomized into 4 groups that consumed pure water, RW, beer, or vodka daily for 3 weeks. The baseline characteristics of 80 participants are presented in Supplemental Table I. No significant differences of baseline characteristics were noted among the 4 groups. After pure water, RW, beer, or vodka consumption for 3 weeks, there were no significant changes in body weight, blood pressure, and serum levels of total cholesterol, high-density lipoproteins, triglycerides, and fasting glucose levels. Endothelial function evaluated by endothelium-dependent flow-mediated vasodilation was assessed before and after the study. The baseline diameters of the brachial artery did not differ from each other. However, 3 weeks of RW consumption was associated with a significant enhancement of flow-mediated vasodilation (from 7.4%±0.6% to 10.0%±0.7%; P=0.008); however, it remained unchanged in the pure water, beer, and vodka groups (Figure 1A).
We further determined the plasma levels of NO and asymmetrical dimethylarginine at baseline and after water, RW, beer, or vodka ingestion. As shown in Figure 1B and 1C, RW intake significantly augmented NO concentration (37±4 vs 56±7 mmol/L; P=0.008) and downregulated plasma asymmetrical dimethylarginine levels (0.42±0.02 vs 0.38±0.01 μmol/L; P=0.009). In contrast, subjects in the pure water, beer, or vodka groups did not show significant changes in plasma NO or asymmetrical dimethylarginine concentrations.
The effects of RW or beer consumption on systemic inflammatory markers were determined. No significant differences were observed in plasma levels of high-sensitivity C-reactive protein or TNF-α after 3 weeks of pure water, RW, beer, or vodka intake (Figure 2A, B). We also assessed the effects of RW, beer, or vodka consumption on plasma adiponectin levels and oxidized low-density lipoproteins status, which are biomarkers of lipolysis and oxidative stress. However, there were no significant changes in adiponectin and oxidized low-density lipoproteins responses after RW, beer, or vodka consumption for 3 weeks (Figure 2C, D).
RW Ingestion Increases EPC Colony-Forming Units and Circulating EPC Levels In Vivo
As shown in Figure 3A, there was no significant change in the water, beer, or vodka groups; however, there was a significant increase in the number of EPC colony-forming units noted in the RW group (from 41±9 to 48±8 colony-forming units/well; P=0.001). Flow cytometry was used to determine the numbers of circulating EPC, defined as KDR+CD133+, CD34+CD133+, and CD34+KDR+ double-positive cells. There were no significant differences at baseline in the circulating EPC numbers among the 4 groups. After intake of RW for 3 weeks, the numbers of circulating EPC significantly increased (all P<0.05), but the numbers of circulating EPC remained unchanged in the pure water, beer, and vodka groups (Figure 3B, D).
RW Enhances EPC Number and Reverses TNF-α–Suppressed EPC Proliferation and Colony-Forming Capacity In Vitro
In an in vitro study, we assessed the direct effects of RW on EPC number, proliferation, and colony-forming capacity. After seeding mononuclear cells on 6-well plates, early EPC were incubated with RW (1%), beer (2%), ethanol (0.15%), or resveratrol (50 μmol/L) for 4 days. Incubation of mononuclear cells with RW significantly increased the number of differentiated early EPC, characterized by DiI-acLDL and fluorescein isothiocyanate lectin staining compared with that in the control group (control vs RW, 51±3 vs 71±3/high-power field; P=0.008; Figure 4A), but no significant difference was found between the control and beer or vodka groups. Administration of resveratrol was noted to increase early EPC numbers (control vs resveratrol, 51±3 vs 67±5/high-power field; P=0.003).
The effect of RW on late EPC proliferation was analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5,diphenyltetrazolium bromide assay. As shown in Figure 4B, incubation with TNF-α (T20; 20 ng/mL) in culture medium markedly reduced late EPC proliferation (control vs T20, 1.00±0.03 vs 0.85±0.04; P=0.038). However, administration of RW and resveratrol significantly reversed the reduction in EPC proliferation in the presence of TNF-α (T20 vs T20+RW and T20+ resveratrol, 0.85±0.04 vs 1.08±0.04 and 1.04±0.05; both P<0.05), but this effect was not observed with beer or ethanol. Additionally, the number of EPC colony-forming unit was markedly suppressed by TNF-α, and this suppression was reversed by treatment with RW (T20 vs T20+RW, 26±2 vs 39±2 colony-forming units/well; P=0.002) but not with beer or ethanol (Figure 4C).
RW Upregulates Phosphorylation of Endothelial NO Synthase and Akt and Promotes Bioavailable NO in EPC
We evaluated the effects of RW on endothelial NO synthase (eNOS) and Akt activities by Western blotting in cultured late EPC. After 4 days of incubation, the eNOS phosphorylation at Ser1177 and Akt phosphorylation at Ser473 shown by immunoblotting were significantly upregulated by treatment with RW in late EPC (Figure 5A). This augmentation of eNOS and Akt phosphorylation was associated with an increase in EPC-derived NO production (nitrite levels; T20+RW, 1% vs T20; increased 89.6%; Figure 5B). Coincubation with NO donor sodium nitroprusside significantly ameliorated the inhibition of TNF-α on EPC proliferation (T20+sodium nitroprusside vs T20, increased 57.6%; P=0.001). In contrast, coincubation with eNOS inhibitor l-NAME (100 μmol/L) and PI3K inhibitor (LY294002; 5 μmol/L) markedly attenuated the effect of RW on EPC proliferation (all P<0.001; Figure 5C). These data indicated that RW may augment EPC function by modulating PI3K/Akt-mediated, and NO-mediated mechanisms.
Effects of RW on EPC Senescence, Adhesion, Migration, and Tube Formation In Vitro
Compared with the control group, incubation of late EPC with TNF-α significantly increased the percentage of senescence-associated β-galactosidase–positive EPC (control vs T20; P=0.001; Figure 6A). Administration of RW and resveratrol for 4 days significantly attenuated the percentage of senescence-associated β-galactosidase–positive EPC in the presence of TNF-α (T20 vs T20+RW and T20+resveratrol, both P<0.001); however, beer or ethanol treatment had no significant effect. Administration of l-NAME abolished the antisenescence effect of RW.
As shown in Figure 6B, TNF-α markedly suppressed late EPC adhesion, and administration of RW and resveratrol significantly augmented TNF-α–suppressed EPC adhesion (T20 vs T20+RW and T20+resveratrol, 13±2 vs 39±2 and 35±3 cells/high-power field; both P<0.001), and this effect was inhibited by treatment with l-NAME. However, there was no significant effect of beer or ethanol on recovery of EPC adhesion in the presence of TNF-α (P=0.526 and 0.197, respectively).
A modified Boyden chamber assay with vascular endothelial growth factor as a chemoattractic factor was used to evaluate EPC migration. As shown in Figure 3C, incubation with RW and resveratrol for 4 days significantly enhanced TNF-α–suppressed late EPC migration (T20 vs T20+RW and T20+resveratrol, 37±2 vs 67±3 and 62±2 cells/high-power filed; both P<0.001), but this benefit was not observed after incubation with beer or ethanol. In addition, administration of eNOS inhibitor significantly diminished the effect of RW on EPC migration.
An in vitro angiogenesis assay was performed with late EPC to investigate the effect of RW on EPC neovascularization. After 4 days of culturing, the functional capacity for tube formation of late EPC on ECMatrix gel was significantly reduced in the presence of TNF-α compared with the control group, whereas incubation with RW and resveratrol promoted tube formation in EPC (both P<0.001; Figure 3D) but incubation with beer or ethanol did not. Administration of l-NAME attenuated the effect of RW on EPC tube formation.
The major new findings of this study are that RW consumption increases circulating EPC levels, and this effect seems to be independent of improvement in lipid profiles, inflammation, adiponectin, and oxidized low-density lipoproteins levels. The beneficial effects exerted by RW appear to be related to upregulation of NO bioavailability. In vitro studies support these clinical observations. Incubation of EPC with RW and RW content, resveratrol, can modify the function of EPC, including attenuation of senescence and promotion of EPC adhesion, migration, and tube formation. These data suggest that intake of RW may alter the biology of EPC, and these alterations may contribute to its unique cardiovascular protective effect.
Light to moderate ethanol intake from any type of beverage has been shown to improve lipoprotein metabolism and lower cardiovascular mortality.19,20 However, RW, with its abundant content of phenolic acids, polyphenols, and flavonoids, appears to confer additional healthy benefits.3,4 The protective effect of RW is supported by epidemiological data that show a significantly reduced incidence of coronary artery disease in certain areas of France, despite a high-fat diet, little exercise, and widespread cigarette smoking. This phenomenon has been termed the “French paradox.”21 Evidence for a more pronounced cardioprotective effect of RW as compared with other alcoholic beverages first emerged from the Copenhagen City Heart Study, in which 13 285 men and women were observed for 12 years.22 The results from this study suggested that patients who drank wine had half the risk of death from coronary heart disease or stroke compared with those who never drank wine. Those who drank beer and spirits did not experience this advantage. The additional benefit of RW is supported further by an analysis of 13 studies involving 209 418 participants. This analysis showed a 32% risk reduction of atherosclerotic disease with RW intake, which was >22% risk reduction for beer consumption.23 However, other studies and reviews have not observed the dominant effect of wine. Explanations for these contradictory results may include differences in the risk factors patterns among beer, spirit, and wine drinkers, the pattern of alcohol consumption, the presence of other confounding lifestyle factors, or, alternatively, differences in the type of wine consumed.
A number of mechanisms have been proposed for the beneficial effect of RW, including favorable changes in lipid metabolism, antioxidant effects, changes in hemostasis and platelet aggregation, arterial vasodilation mediated by NO release, insulin sensitization, and lower levels of inflammatory markers.24 However, mechanistic studies suggested that many of these beneficial effects of RW are compatible with the action of endothelium-derived NO,5 implying that NO may be a critical mediator of the cardiovascular protection provided by RW.
A large body of evidence indicates that the integrity and functional activity of the endothelial monolayer play an important role in atherogenesis.25 In humans, extensive endothelial cell damage by cardiovascular risk factors can result in endothelial cell apoptosis with subsequent loss of integrity of the endothelium. The extent of endothelial injury may represent a balance between the magnitude of injury and the capacity for repair and predicts cardiovascular event rates.26 The traditional view suggests that endothelial cell repair is exclusively mediated by the adjacent endothelial cells. However, a series of basic and clinical studies prompted by the discovery of bone marrow-derived EPC have provided new insights into these processes and indicate that circulating EPC play a pivotal role in endothelial cell regeneration.27 Reduced levels of circulating EPC independently predict atherosclerotic disease progression and future cardiovascular events,12 thus supporting an important role for endogenous vascular repair by EPC to modulate the clinical course of coronary artery disease. Therefore, enhancement of circulating EPC level and its functional capacity by lifestyle modification or pharmacological strategies may be of clinical importance and therapeutic potential.
In animal studies, moderate intake of RW promoted EPC numbers in wild-type mice subjected to physical exercise.28 Furthermore, RW improved ischemia-induced neovascularization in hypercholesterolemic apolipoprotein E-deficient mice after hindlimb ischemia by increasing the number and functional activity of EPC.29 Balestrieri et al30 reported that low doses of RW in vitro upregulated the number of circulating EPC during treatment with TNF-α. However, the relationship between RW consumption and circulating EPC remains unclear in humans. In our prospective, randomized study, it was interesting to find that even light consumption of RW for 3 weeks significantly increased circulating EPC levels, implying an additional beneficial effect of RW on EPC and endothelial repair capability. We further confirmed these findings in in vitro study and demonstrated that RW increased EPC numbers and improved TNF-α–suppressed EPC proliferation and colony-formation capacity. RW and resveratrol were also shown to attenuate EPC senescence and restored EPC adhesion, migration, and tube formation, which were suppressed by TNF-α, suggesting resveratrol might be at least partly responsible for the beneficial cardiovascular effects of RW. Besides, the suppressive effect of TNF-α could be ameliorated by coincubation with the NO donor, sodium nitroprusside. In addition, administration of an NOS inhibitor or PI3K inhibitor abolished the beneficial effect of RW on EPC proliferation. These data indicate that RW may promote EPC function by modulating PI3K/Akt-related and NO-related mechanisms.
There is increasing evidence for an interaction of RW and RW polyphenolic compounds with the endothelial NO system. Wallerath et al5 demonstrated that RW upregulated eNOS expression and activity in human endothelial cells by both transcriptional and posttranscriptional (mRNA-stabilizing) mechanisms. Ndiaye et al31 reported that RW polyphenolic compounds induced the redox-sensitive activation of the PI3-kinase/Akt pathway in endothelial cells, which, in turn, caused phosphorylation of eNOS and resulted in an increased formation of NO. Recent evidence suggested that mobilization and differentiation of EPC are modified by NO, and eNOS expressed by bone marrow is essential for the mobilization of stem and progenitor cells.32 In addition, endogenous NOS inhibitors, such as asymmetrical dimethylarginine, were shown to suppress EPC differentiation and function and contributed to impaired endothelial function.33 Additionally, low-density lipoprotein oxidation is a key event in atherogenesis, and previous studies indicated that consumption of RW or its polyphenols was associated with reduced low-density lipoprotein oxidation, low-density lipoprotein aggregation, and decreased foam cell formation.34 However, in this study, there was no significant change in oxidized low-density lipoproteins levels in response to the 3-week intake of RW. The current results may be explained by the short intervention period and the small amount of RW consumed.
This study clearly demonstrated that a low-dose intake of RW can significantly enhance circulating levels of EPC in healthy young subjects by increasing NO bioavailability. These findings may explain the underlying mechanism responsible for the beneficial effects of RW ingestion on the cardiovascular system.
Sources of Funding
This study was partly supported by research grants from the NSC 96-2320-B-039-042 and NSC 97-2314-B-075-039 from the National Science Council, Taiwan; VGH-96DHA0100478, VGH-97DHA0100127, and VGH-ER-2-97DHA0100664 from Taipei Veterans General Hospital, Taipei, Taiwan; CMU95-288, CMU96-188, and CMU96-052 from China Medical University; CI 96-16 and CI-97-14 from the Yen Tjing Ling Medical Foundation, Taipei, Taiwan; and a grant from the Ministry of Education, Aim for the Top University Plan.
P.-H.H. and Y.-H.C. contributed equally to this article.
Received on: March 12, 2009; final version accepted on: December 21, 2009.
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