| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
* CNRS UMR 6214, Angers, France; INSERM UMR 771, Angers, France; Université d’Angers, UFR de Médecine, France;
INSERM, EMI 0335, Angers, France; and
UMR 476 INSERM/1260 INRA, Marseille, France
1Correspondence: Department of Neurovascular Integrated Biology, UMR CNRS6214-INSERM771, Faculté de Médecine, 49045 Angers, France. E-mail: daniel.henrion{at}univ-angers.fr
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
|---|
Key Words: VEGF • remodeling • nitric oxide • blood flow
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
|---|
, 2)
, and ischemic disease. Angiogenesis is a very complex process involving a variety of biologically active substances (3)
.
Epidemiological studies report an inverse association between polyphenol consumption such as fruits and vegetables, tea, and red wine and mortality from cardiovascular diseases and cancers. Polyphenols, particularly red wine polyphenols (RWPC), exert numerous effects including antioxidant and free radical properties, and anti-aggregatory platelet and anti-thrombotic activities. With regard to blood vessels, RWPC are powerful vasodilators and contribute to preserving the integrity of the endothelium, inhibition of vascular cell proliferation and migration, including endothelial and smooth muscle cells, and angiogenesis (4
, 5)
.
Concerning angiogenesis, RWPC exert paradoxical properties. On the one hand, RWPC protect against deleterious effect of cardiac and cerebral ischemia whose correction requires proangiogenic properties to produce new blood vessels to rescue the infracted area, both in human and different experimental models (6
7
8)
. In addition to the antioxidant properties of RWPC, the mechanisms involved mainly the activation of endothelial NO release through an increase in calcium level and activation of the PI3-kinase/Akt pathway by a redox-sensitive pathway in endothelial cells (9
10
11)
. RWPC may also regulate NO activity at the level of endothelial NO synthase (eNOS) protein expression in endothelial cells (12)
, blood vessels, and cardiac tissue (13)
. On the other hand, numerous studies report that RWPC inhibit angiogenesis by acting on different vascular cells. Indeed, RWPC or its content, delphinidin, inhibit endothelial cell proliferation through the involvement of cyclin D1- and A-dependent pathways (14
, 15)
. Moreover, RWPC decrease vascular smooth muscle migration and proliferation through down-regulation of cyclin A expression and inhibition of p38 MAPK and PI3-kinase pathways (16)
. RWPC was recently shown to inhibit expression of the two major proangiogenic factors, vascular endothelial growth factor (VEGF) (17)
and metalloproteinase-2 (MMP-2) (18)
, in smooth muscle cells. All of these effects confer both in vitro and in vivo anti-angiogenic properties of RWPC and its polyphenols content (14
, 17)
.
The mechanisms by which RWPC exert these paradoxical effects on angiogenesis are not understood. In the present study we tested the hypothesis that RWPC, used chronically, might exert a dual effect depending on the dose used. Formation of new blood vessels in chick chorioallantoic membrane (CAM) has been used as a model to test the anti-angiogenic properties of RWPC. However, it is important to consider a valuable in vivo model that takes into account ischemia, the driving force for angiogenesis, activation of NO and hypoxia-inducible factor 1
(HIF1), and the production of VEGF. Therefore, we used a model of femoral artery ligature leading to a 50% reduction in blood vessel density in the ischemic leg musculature, which allowed the characterization of pro- and anti-angiogenic effects of RWPC.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
|---|
. RWPC were dissolved in a solution of 5% glucose (Glc). Consequently, the control group received only 5% glucose (n=12) in the same conditions.
Protocol 2: Involvement of VEGF pathway
To assess the involvement of VEGF, four additional groups were performed: a control group that received 5% glucose by daily gavage (n=5), a control group treated with VEGF neutralizing antibody (2.5 µg, i.p. twice a wk, R&D Systems, Minneapolis, MN, USA; n=5), a group treated with a low dose of RWPC (n=5), and one with or without VEGF neutralizing antibody (19)
.
Protocol 3: Effects of delphinidin in angiogenesis
Delphinidin, a compound of RWPC, was also tested to identify, in vivo, its involvement in the angiogenic process. Consequently, two groups, treated with a high dose (0.6 mg · kg–1 · day–1, n=6) and a low dose (0.06 mg · kg–1 · day–1, n=6) of delphinidin were performed by daily gavage for 15 days. The control group received only 5% glucose (n=5) in the same conditions.
Animals were housed in a regulated environment with a constant ambient temperature of 24°C. They had free access to standard laboratory food and water. As described earlier (19)
, after 7 days of treatment the femoral artery was occluded (3-0 silk suture) under anesthesia (Nesdonal, 50 mg/kg, i.p.). The ligature was performed on the left femoral artery 0.5 cm proximal to the bifurcation to the saphenous and popliteal arteries. After 7 (protocols 2, 3) or 15 (protocol 1) days of ligature, blood flow was measured as described below, followed by an angiographic measurement. The rat was then sacrificed and tissues were sampled for biochemical and histological analysis. The procedure followed in the care and euthanasia of the study animals was in accordance with the European community standards on the care and use of laboratory animals (authorization #00577).
Laser-Doppler whole body imaging
To provide a functional evidence of ischemia, after 15 days of ligature laser Doppler perfusion was performed in anesthetized rats. Animals were settled in an incubator (MMS, Chelles, France) that allowed maintenance of a stable cutaneous temperature (35.0±0.5°C) throughout the experiment. Perfusion was then measured in the foot using a laser Doppler flow probe (PF 408; Perimed). Blood flow was recorded for 5 min. At least two flow measurements were performed per leg. Blood flow perfusion was expressed as a ratio of left (ischemic) to right (nonischemic) leg.
To evaluate the neovascularization (protocol 2), laser Doppler perfusion imaging experiments were performed on day 7 as described (19)
. Briefly, rats were placed on a heating plate at 37°C to minimize temperature variation. To account for variables, including ambient light, temperature, and experimental procedures, blood flow was calculated in the foot and expressed as a ratio of ischemic to nonischemic leg.
Angiography
Arterial density was evaluated by high-definition angiography 1 or 2 wk after ligature, as described (19)
. Briefly, rats were anesthetized (sodium pentobarbital, 50 mg/kg, i.p.) and a contrast medium (barium sulfate, 6g/ml) was injected through a catheter introduced into the abdominal aorta. Two images were acquired per animal using a digital X-ray transducer (Faxitron X-Ray Corporation, Wheeling, IL, USA). Vascular density was expressed as a percentage of pixels per image occupied by vessels in the quantification area. The quantification zone was delineated by the location of the ligature on the femoral artery, the knee, the edge of the femur, and the external limit of the leg. The angiographic score was calculated as the ratio of left (ischemic) to right (nonischemic) leg (L/R ratio). In each experiment, the skeletal muscle from both the ischemic and nonischemic leg were removed and frozen before Western blot and histological analysis, as described before (19)
.
Arteriolar and capillary density
Angiographic analysis was completed by assessing capillary and arteriolar densities, as described (20)
. Briefly, ischemic and nonischemic muscles were dissected and progressively frozen in isopentane solution cooled in liquid nitrogen. Sections (7 µm) were first incubated for 30 min in PBS containing 5% BSA at room temperature, then for 1 h with either mouse monoclonal antibody directed against human smooth muscle actin 1 (dilution 1:50) to identify arterioles or rabbit polyclonal antibody directed against total PECAM (dilution 1:50) to identify capillaries. Capillary-to-myocyte and arteriole-to-myocyte ratios were calculated, and the results were expressed according to ischemic-to-nonischemic ratios. Capillary and arteriolar densities were calculated in three randomly chosen fields of a definite area for each animal.
Western blot in isolated muscles
In each experiment, skeletal muscle from both ischemic and nonischemic leg was removed and frozen. Samples were collected and homogenized (Polytron Pro 250, Bioblock Scientific, Illkirch, France). Proteins were separated by SDS-PAGE (Mini gel protean II system, Bio-Rad (Hercules, CA, USA), 100V, using 300 ml 25 mM Tris, 192 mM glycine, 0.1% SDS) using a stacking gel, followed by a running gel. After migration, proteins were transferred (100 V, 2 h, 4°C using 800 ml 25 mM Tris, 192 mM glycine, 10% methanol) to PVDF blotting membranes (Immobilon-P, Millipore, Billerica, MA, USA). Membranes were then washed in TBS-T buffer (composition: 10 mM Tris/base pH 7.5, 0.1 M NaCl, 1 mM EDTA, 0.1% Tween 20) and blocked for 1 h at room temperature (5% BSA in TBS-T). Membranes were incubated overnight 4°C with the primary antibody (1:500), washed again (3 times for 10 min), then incubated with HRP-conjugated secondary antibody (Amersham; Arlington Heights, IL, USA; 1 h, 30 min RT, 1:2000). Membranes were washed and visualized using the ECL-Plus Chemiluminescence kit (Amersham). Immunodetection was carried out using antibodies directed against Akt, P-Akt, eNOS, P-eNOS, VEGF, caveolin-1, HSP 90, and RhoA (Santa Cruz Biotechnology, Santa Cruz, CA, USA).
Gelatin zymography
Tissue samples were thawed and homogenized in 300 µl of buffer (200 mmol/L sucrose and 20 mmol/L HEPES, pH 7.4) containing protease inhibitors. Protein content was then determined by the method of Bradford (21)
. Samples were mixed in an SDS-PAGE loading buffer (lacking reducing agents) applied to SDS/9% polyacrylamide gels containing 1 mg/ml gelatin (Bio-Rad) and separated by electrophoresis. Subsequently, SDS was removed from the gels by two 15 min washes with 2.5% Triton X-100, then the gels were incubated overnight at 37°C in zymography buffer (50 mmol/L Tris [pH 7.5] and 10 mmol/L CaCl2) and stained with Coomassie Brilliant Blue (Serva, Heidelberg, Germany). Gelatinolytic activity was visualized as clear areas of lysis in the gel. Densitometric analysis was performed using NIH Image software.
Data analysis
Results are expressed as means ± SE. Significance of the differences between groups was determined by analysis of variance (ANOVA), followed by Bonferroni’s test and paired t test. P values of < 0.05 were considered significant.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
|---|
Quantification of neovascularization in the hind limb
Protocol 1: Whole body laser Doppler blood flow
Fifteen days after ligature, foot blood flow as measured using laser Doppler perfusion imaging was significantly lower in the left leg than in the right leg, with an L/R ratio of 76.7 ± 7.4%. A high dose of RWPC decreased by 0.40-fold the L/R ratio (30.9±6.6, P<0.01) compared with control, whereas a low dose increased by 1.48-fold the L/R ratio (114±12.5, P<0.05) compared with control. The intermediate dose of RWPC did not affect the angiogenic process (Fig. 1
A).
|
Angiography
Fifteen days after ligature, the microvascular network density was lower in the left leg than in the right leg, with an L/R ratio of 67.5 ± 4.5 (P<0.05) in the control group (Fig. 1B
). Treatment with a high dose of RWPC significantly decreased the L/R ratio to 38.4±2.5 (P<0.05) compared with control. On the other hand, a low dose of RWPC increased the L/R ratio up to 119.5 ± 15.8 (P<0.001) compared with the control group, whereas 2 mg/kg of RWPC did not affect the vascular density (Fig. 1B
).
Arteriolar and capillary densities
The angiogenic process was not affected by the intermediate dose of ProvinolsTM. Consequently, we concentrated our work on the two others doses (0.2 and 20 mg · kg–1 · day–1). Data from the angiographic analysis were confirmed by arteriolar density measurement. Fifteen days after ligature, the L/R ratio was 85 ± 6%, with an arteriolar density in the left leg increased by 0.94-fold for the control group. A concurrent treatment with a high dose of RWPC decreased by 0.62-fold the L/R ratio (P<0.05) compared with controls, indicating an anti-angiogenic effect (Fig. 2
A). A low dose of RWPC increased by 1.59-fold the L/R ratio (P<0.01) compared with control, indicating a proangiogenic effect (Fig. 2A
).
|
In the control group, capillary density was not affected by the ligature. A high dose of RWPC induced a decrease of L/R ratio by 0.77-fold (P<0.05) compared with the control group (Fig. 2B
). No difference was observed with RWPC low-dose treatment compared with controls.
Western blot analysis
Angiogenesis in the control group (Fig. 3 )
In the control group, the expression level of eNOS, phosphorylated-eNOS, and HSP 90 in the ischemic (L) hind limb was significantly increased compared with nonischemic (R) leg (n=6, P<0.05), as described in the literature. No change with caveolin-1 expression was observed. The ischemia induced a rise of Akt pathway, with an increase of Akt (P<0.05), P-Akt (P<0.05), and PI3K (P<0.05) levels in the ligatured hind limb. p38 and P-p38 were not significantly modified in the left leg (P<0.05). Finally, VEGF and NF-
B expressions were increased in the ischemic leg compared with the nonischemic leg (P<0.05).
|
Effect of RWPC in the aorta (Fig. 4 )
Compared with control, a high dose of RWPC increased expression of eNOS, P-eNOS, and HSP90. This dose also induced an increase of Akt and PI3K expression without affecting P-Akt. Finally, a high dose of RWPC did not affect p38, P-p38, or VEGF expression.
|
Compared with control, a low dose of RWPC increased significantly the expression of NO, Akt/PI3K pathways but decreased that of HSP90. A low dose of RWPC reduced p38 slightly but markedly increased P-p38 expression. Finally, a low dose of RWPC did not affect VEGF expression.
A comparison between the effects of high and low doses of RWPC indicated a greater activation of NO pathway as shown by the difference in the ratio of P-eNOS/eNOS, HSP90 expression, and showed a lower activation of Akt/P-Akt and P-p38 for the former compared with the latter. The two doses of RWPC increased the PI3K level equally and neither affected VEGF expression.
Effect of RWPC in the nonischemic hind limb (Fig. 5 )
A high dose of RWPC reduced eNOS but markedly increased P-eNOS expression, and thus enhanced the ratio P-eNOS/eNOS. HSP90 and caveolin-1 expression was also increased. A high dose of RWPC did not affect Akt, P-Akt levels, but significantly increased that of PI3K. A high dose of RWPC enhanced the p38 MAPK pathway through an increase in P-p38. Finally, it did not change VEGF expression and reduced that of NF-B.
|
A low dose of RWPC increased eNOS but not P-eNOS expression without a significant change in the P-eNOS/eNOS ratio. This dose did not affect HSP90 or caveolin-1 expression. A low dose of RWPC significantly increased Akt expression but did not change that of P-Akt and PI3K. This dose increased the expression of both p38 and P-p38. Finally, a low dose did not affect VEGF expression and significantly enhanced NF-B.
A comparison between the effects of high and very low doses of RWPC showed a greater activation of NO pathway, HSP90, caveolin-1, and PI3K expression for the former compared with the latter. No difference was observed between the two doses of RWPC with regard to P-Akt, p38, or the increase of P-p38. A high dose of RWPC did not affect the Akt level whereas a low dose of RWPC increased its expression. RWPC induced a differential effect on NF-B in which a high dose reduced but a low dose enhanced its expression. The two doses of RWPC did not affect VEGF expression.
Effect of RWPC on angiogenesis (Fig. 6 )
A high dose of RWPC significantly reduced the expression of proteins of the NO and Akt/PI3K pathways as well as HSP90 and caveolin-1 expression. A high dose of RWPC increased p38 without affecting P-p38 expression. This dose reduced both VEGF and NF-B expression.
|
On the other hand, a low dose of RWPC did not modify eNOS but increased P-eNOS expression without any significant change in the ratio P-eNOS/eNOS. This dose did not affect HSP90 and reduced caveolin-1 expression. A low dose of RWPC did not change the expression of Akt but significantly increased that of P-Akt and PI3K. This dose of RWPC increased expression of both p38 and P-p38. Finally, a low dose of RWPC significantly enhanced VEGF expression without affecting that of NF-B.
A comparison between the effects of high and low doses of RWPC showed a lower activation of NO and Akt/PI3K pathways, HSP90 and P-p38 expression for the former compared with the latter. The two doses of RWPC decreased caveolin-1 expression. RWPC induced a differential effect on VEGF in which a high dose reduced and a low dose enhanced its expression. Finally, a high dose reduced NF-B expression but a low dose did not affect its level.
MMPs and angiogenesis (Fig. 7 )
In the control group, MMP activity was significantly increased in the ischemic hind limb compared with the nonischemic hind limb (x1.31), with an L/R ratio of 1.34 ± 0.11 (P<0.01).
|
After 15 days of ligature, a high dose of RWPC reduced vascular density and blood flow (Fig. 1A
, B); this anti-angiogenic effect was associated with a reduced L/R ratio of MMP activity in the ischemic hind limb (Fig. 7)
. However, MMP activity did not differ from a low dose of RWPC compared with control (L/R ratio: 1.25±0.11).
In this study we have demonstrated that a low dose of RWPC induced a proangiogenic effect by up-regulation of VEGF/eNOS pathway. Consequently, we have used a neutralizing VEGF antibody to prove the involvement of VEGF.
Protocol 2: Involvement of VEGF pathway (Fig. 8 )
The changes in vascularization of foot blood flow were measured after 7 days of ligature. In the control group, vascularization of the ischemic foot reached 44.3 ± 3.1% compared with the nonischemic foot blood flow. The injection of neutralizing VEGF antibody twice/wk significantly decreased the L/R ratio by 0.76-fold (P<0.05). Treatment with 0.2 mg/kg of RWPC increased significantly the L/R ratio by up to 54.8 ± 2.8% compared with controls. The neutralizing VEGF antibody treatment decreased by 0.77-fold the L/R ratio (P<0.05) (Fig. 8A
).
|
The quantification of vascular density showed that the neutralizing VEGF antibody decreased significantly the L/R ratio by 0.69- and 0.56-fold in the control and RWPC-treated groups, respectively (P<0.05) (Fig. 8B
).
Protocol 3: Effects of delphinidin in angiogenesis (Fig. 9 )
The effects of delphinidin in the changes in foot vascularization were measured by laser Doppler flowmetry and microangiography. Results showed that the L/R ratio of foot blood flow and vascular density were significantly decreased after a high dose of delphinidin (x0.76- and x0.67-fold, respectively, P<0.05) compared with the control group, but a low dose did not change vascular density (Fig. 9)
.
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
|---|
B expression. The data also highlighted the mechanism by which a low dose of RWPC promotes angiogenesis and this includes activation of NO, Akt/PI3K, p38 MAPK, and VEGF expression without altering either MMP activity or NF-B expression. Thus, NO/VEGF, MMPs, and NF-B are crucial to determine the effect of RWPC on angiogenesis.
The present study provides evidence that in vivo administration of RWPC induced differential effects on angiogenesis depending on the dose used. In general, in vivo effects of RWPC have been conducted at doses ranging between 20 and 40 mg/kg for 1 to 4 wk corresponding to a high dose of RWPC used in the present study. A previous study showed that a high dose of RWPC induces cardiovascular effects, including improvement of endothelial function (22)
and preventing the increase in blood pressure in NO-deficient hypertensive rats (23)
. Also, a high dose of RWPC induces hypotension, decreases cardiac reactivity, infarct size (6)
, and stroke (8)
in rats. Correction of cardiac and cerebral ischemia required proangiogenic properties to produce new blood vessels and rescue the infracted area. In contrast to previous studies, the proangiogenic properties occurred at a dose (0.2 mg/kg) of RWPC that is 100-fold lower. The molecular identity of the compounds responsible for the in vivo effects described in the present study has not been assessed. Nevertheless, it was found that low and high doses of RWPC are adequate to produce a sufficient circulating concentration of compounds able to modulate angiogenesis. A balance between circulating pro- and anti-angiogenic compounds might occur in vivo, and this equilibrium is shifted toward proangiogenic substances at a low dose of RWPC or in tissues with low rate of polyphenols distribution at a high dose of RWPC. After treatment of rats with a high dose of delphinidin, one the major compounds found in RWPC, angiogenesis was significantly inhibited. Consequently, the anti-angiogenic effect of polyphenols was induced, at least in part, by delphinidin. Nevertheless, a low dose of delphinidin had no effect on angiogenesis, suggesting that another compound or the combination of delphinidin and other related compound might be involved in the proangiogenic effect of ProvinolsTM.
We used an in vivo model, which is appropriate to study ischemic angiogenesis, a classical feature of peripheral, cardiac and cerebral ischemic diseases (24)
. This model also allowed the study of both pro- and anti-angiogenic effects of RWPC under the same conditions. In control rats, we found that angiogenesis after ligature was associated with increased eNOS, Akt, VEGF and HSP 90 expression, in agreement with previous studies conducted in the same model (25
, 26)
.
The two doses of RWPC affected neither angiogenesis nor blood flow in the nonischemic area. It is possible that ischemic conditions are needed for RWPC in vivo to modulate angiogenesis, and such conditions are not fulfilled in the nonischemic area. Nevertheless, in the nonischemic area a high dose of RWPC activates multiple pathways reported in previous studies including increase of the Akt/PI3K and NO pathway. A low dose of RWPC produced a lower activation of the NO pathway than a high dose of RWPC. Indeed, RWPC has been shown to activate the PI3-kinase/Akt pathway leading to rapid and sustained activation and enhanced expression of eNOS in endothelial cells (27)
and in different blood vessels (27
, 28)
. In the nonischemic leg, VEGF expression was not altered by the two doses of RWPC. The increase of VEGF gene expression occurs via the NADPH oxidase-dependent formation of reactive oxygen species with subsequent activation of redox-sensitive kinase such as p38 MAPK, resulting in turn in the activation of the transcription factor HIF1 (29
, 30)
. HIF1 might not be activated under nonischemic conditions, although the two doses of RWPC enhanced p38 MAPK activation. In the nonischemic leg, MMP activity was either reduced or unchanged by high and low doses of RWPC, respectively. In accordance with our studies, RWPC has been reported to prevent thrombin-induced activation of MMP-2 in vascular smooth muscles (18)
. Finally, in the nonischemic leg we found that a high dose of RWPC reduced whereas a low dose increased NF-B expression. This differential effect of RWPC has never been reported, but activation of NF-B can lead to the production of secreted factors that enhance growth, survival, and vascularization; this in accordance with the proangiogenic property of RWPC reported in the present study (see below).
A major finding was the effect of RWPC under ischemic conditions. In the ischemic leg, we found that a high dose of RWPC reduced angiogenesis in association with decreased blood flow and a lower arteriolar and capillary density via an inhibition of the NO and Akt/PI3K pathways. However, the mechanism by which a high dose of RWPC reduced the NO pathway under ischemic conditions is not known; this dose of RWPC increases NO expression and activity. We previously reported that delphinidin, a compound that possesses pharmacological properties similar to those of RWPC, inhibits endothelial cell proliferation; this effect is associated with an increase of caveolin-1 expression, which might decrease eNOS activation and therefore exert a negative regulatory effect on proliferation on either endothelial cells (15)
or transformed cells such as NIH-3T3 cells (31)
. RWPCs contained a large number of compounds, including phenolic acids, flavonoids, anthocyanins, and tannings. Since ProvinolsTM is a mixture of different polyphenolic compounds, it is not certain which polyphenolic components are responsible for the pro- or anti-angiogenic process. To further address this question, we performed additional experiments with delphinidin. We showed that a high dose of delphinidin decreased revascularization after 15 days of ligature. Consequently, we demonstrated that delphinidin is probably the key compound (or one of them) implicated in the anti-angiogenic effect of RWPC. Our previous study showed that the antiproliferative effect of delphinidin occurs independent of NO pathway. With regard to Akt/PI3K pathways, polyphenols from tea have been reported to decrease the expression of PI3K and Akt phosphorylation in human prostate cancer cells (32)
. We showed here that inhibition of Akt/PI3K also took place in ischemic legs after a high dose of RWPC. A high dose of RWPC inhibited either the expression or the activity of two major proangiogenic factors, VEGF and MMPs, in association with reduced P-p38 expression. These results are in accordance with those reported in cultured smooth muscle cells in which redox-sensitive inhibition of the p38 MAPK pathway activation leads to inhibition of PDGF-induced VEGF expression (17)
and redox-insensitive mechanisms lead to inhibition of thrombin-induced MMP-2 formation (18)
. Our results also strengthened those reported by Barthomeuf et al. (33)
showing that red grape skin polyphenols inhibit angiogenesis in a Matrigel model and reverse the chemotactic effect of VEGF on bovine aortic endothelial cells. This inhibition is associated with down-regulation of ERK1/2 and p38 phosphorylation. Finally, a high dose of RWPC inhibited NF-B expression in the ischemic legs. Polyphenols from different sources such as tea or hop plant have been reported to possess anti-angiogenic properties: they inhibit growth of a vascular tumor in vivo and repress the NF-B and Akt pathways in endothelial cells (34)
. To the best of our knowledge, our study extended the participation of NF-B and Akt pathways in the anti-angiogenic effect of RWPC.
Altogether, our finding provides a rational explanation for tumor growth inhibition and the anti-atherosclerotic effect of RWPC used at a high dose, although no study had yet clearly demonstrated their anti-angiogenic effect in ischemic conditions, especially in vivo. The anti-angiogenic effect of RWPC is being investigated using in vitro models (i.e., endothelial cell) or tumor cell growth in culture (35)
or in vivo studies predominantly performed in Matrigel and CAM models (33)
.
Finally, and most interesting, we show for the first time that a low dose of RWPC promoted angiogenesis in ischemic conditions in association with increased blood flow and arteriolar density. A low dose of RWPC did not affect capillary density, suggesting that RWPC affect arteriogenesis rather than angiogenesis (Fig. 2B
) (36)
. In our study, the capillary density was evaluated in the bottom of the thigh after 15 days of ligature, which could explain that the capillary density was not affected by the treatment. Furthermore, Deindl et al. (37)
have demonstrated that VEGF was not implicated in arteriogenesis. In our study, the low dose of RWPC increased VEGF expression. In addition, a neutralizing VEGF antibody inhibited neovascularization induced by a low dose of RWPC (Fig. 8)
. Consequently, our results demonstrated the involvement of the VEGF pathway in the proangiogenic effect of polyphenols. Indeed, these results underline that the low dose of RWPC possesses proangiogenic rather than proarteriogenic properties. The mechanism involved activation of NO, Akt/PI3K, p38 MAPK, and VEGF expression without alteration in either MMP activity or NF-B expression. Thus, a low circulating level of RWPC able to improve postischemic neovascularization was present in vivo. The nature of these compounds has not yet been assessed. Anthocyanins such as delphindin and oligomeric-condensed tanning exhibit a pharmacological profile comparable to original RWPC in terms of endothelial NO release (38)
; alternatively, they could activate eNOS, Akt/PI3K, and VEGF. Consequently, we have performed additional experiments in order to test the effect of delphinidin in our study (Fig. 9)
. We found that angiogenesis was not affected by a low dose of delphinidin whereas a high dose decreased the revascularization. Consequently, we demonstrated that delphinidin is probably one of the compounds implicated in the anti-angiogenic effect of RWPC. These data also favor in the hypothesis that the differential effect obtained between a low and a high dose of RWPC might be related to its composition.
RWPC stimulate endothelial NO production through an increase in intracellular calcium levels and through activation of the PI3-kinase/Akt pathway in endothelial cells (9
10
11)
. RWPC may also regulate NO activity at the level of eNOS protein expression in endothelial cells (12)
, blood vessels, and cardiac tissues (13)
. In addition to the NO pathway, we showed the capacity of a low dose of RWPC to increase VEGF expression. Altogether, a low dose of RWPC and, consecutively, moderate consumption of red wine could have a beneficial effect in ischemic diseases requiring angiogenesis and/or an increase in blood flow.
|
CONCLUSIONS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
|---|
|
ACKNOWLEDGMENTS |
|---|
Received for publication November 30, 2006. Accepted for publication May 24, 2007.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
|---|
This article has been cited by other articles:
|
|
 |
|
  A. Walter, N. Etienne-Selloum, D. Brasse, R. Schleiffer, V. Bekaert, P. M. Vanhoutte, A. Beretz, and V. B. Schini-Kerth Red Wine Polyphenols Prevent Acceleration of Neovascularization by Angiotensin II in the Ischemic Rat Hindlimb J. Pharmacol. Exp. Ther., May 1, 2009; 329(2): 699 - 707. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. J. Belin de Chantemele, E. Vessieres, A.-L. Guihot, B. Toutain, M. Maquignau, L. Loufrani, and D. Henrion Type 2 diabetes severely impairs structural and functional adaptation of rat resistance arteries to chronic changes in blood flow Cardiovasc Res, March 1, 2009; 81(4): 788 - 796. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E.J. Belin de Chantemele, K. Retailleau, F. Pinaud, E. Vessieres, A. Bocquet, A.L. Guihot, B. Lemaire, V. Domenga, C. Baufreton, L. Loufrani, et al. Notch3 Is a Major Regulator of Vascular Tone in Cerebral and Tail Resistance Arteries Arterioscler. Thromb. Vasc. Biol., December 1, 2008; 28(12): 2216 - 2224. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. M. Klinge, N. S. Wickramasinghe, M. M. Ivanova, and S. M. Dougherty Resveratrol stimulates nitric oxide production by increasing estrogen receptor {alpha}-Src-caveolin-1 interaction and phosphorylation in human umbilical vein endothelial cells FASEB J, July 1, 2008; 22(7): 2185 - 2197. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. L. Balestrieri, C. Schiano, F. Felice, A. Casamassimi, A. Balestrieri, L. Milone, L. Servillo, and C. Napoli Effect of Low Doses of Red Wine and Pure Resveratrol on Circulating Endothelial Progenitor Cells J. Biochem., February 1, 2008; 143(2): 179 - 186. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
