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Am J Physiol Heart Circ Physiol 288: H2918-H2924, 2005. First published January 21, 2005; doi:10.1152/ajpheart.01194.2004
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Mechanism of potentiation by polyphenols of contraction in human vein-engineered media

M. Diebolt,1,2 L. Germain,2 F. A. Auger,2 and R. Andriantsitohaina1

1Pharmacologie et Physico-Chimie des Interactions Cellulaires et Moléculaires, Unité Mixte de Recherche Centre National de la Recherche Scientifique 7034, Faculté de Pharmacie, Illkirch, France; and 2Laboratoire d'Organogénèse Expérimentale, Hôpital du Saint-Sacrement, Quebec, Quebec, Canada

Submitted 30 November 2004 ; accepted in final form 3 January 2005


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The potential of natural dietary polyphenols in the treatment of vascular diseases originating from veins has been suggested in the literature. However, the mechanisms involved to explain the effects of polyphenols are not yet elucidated. Therefore, the aim of this study was to investigate the mechanisms by which polyphenols from red wine (Provinols) modulated contraction in human veins. We took advantage of a human model previously reported as a new tool for pharmacological research, using tissue-engineered techniques allowing the production of vascular media based exclusively on human smooth muscle cells. Thus human tissue-engineered vascular media (TEVM) were produced with cells originating from umbilical cord vein. TEVM were treated with either vehicle or Provinols. Results showed that treatment of TEVM with Provinols significantly potentiated the contractile responses induced by histamine and bradykinin. The potentiating effect of Provinols was not associated with an enhancement of histamine-induced increase in cytosolic calcium; rather, it implied the presence of a Ca2+-independent signaling pathway. Pharmacological studies indicated that action of Provinols took place at the level of phospholipase A2-Rho-kinase pathway and was associated with an enhancement of myosin light chain kinase activity. These results, obtained using the human TEVM, bring new insights to explain the regulation of venous contraction by polyphenols.

tissue engineering; veins; smooth muscle

NATURAL DIETARY POLYPHENOLS are present in a wide variety of plants and are reported to protect against cardiovascular diseases (23). Indeed, polyphenols possess a multitude of biological activities, including antioxidant, antithrombotic, and antihypertensive properties (20). Furthermore, polyphenols are used in the clinic to treat vascular diseases originating from veins because of their phlebotropic and venoprotective properties (9, 25). Further studies have reported that polyphenolic extracts (i.e., diosmine and hesperidin, which belong to the flavonoids family) improve venous tone in patients (12) and reduce venous capacitance and distensibility (18). However, few studies have addressed the intracellular signaling pathways by which polyphenols regulate contraction in vivo. Our laboratory group (6) recently reported that short-term oral administration of polyphenols from red wine enhances endothelium-dependent relaxation and induces gene expression within the arterial wall. The induction of gene expression (i.e., inducible nitric oxide synthase and cyclooxygenase-2) maintains unchanged agonist-induced contractility in the artery, even though a subtle alteration of the thapsigargin-sensitive component of the contraction linked to Ca2+ release from the intracellular store has been reported in the same vessel (7). Ex vivo studies have proposed that flavonoids increase norepinephrine-induced contraction in human isolated saphenous vein (14) and improve Ca2+ sensitivity of the smooth muscle contractile apparatus in rat isolated femoral vein (25). In addition, in vitro studies have shown that flavonoid components, such as rutin or quercetin, increased myosin ATPase activity, which facilitated actomyosin interaction (29). Nevertheless, an understanding of the intracellular pathways involved to explain the effects of polyphenols in veins has not yet been fully elucidated.

Since the last few decades, the new field of tissue engineering has rapidly evolved with the aim of producing tissues and organs from cells, extracellular matrix (ECM), and biomaterials. Several groups are developing new prostheses for eventual clinical applications in the replacement of small-diameter blood vessels, to substitute artificial grafts that present high risk of thrombosis (5, 16, 21, 27). Recently, our group (16) reported a new tissue-engineering approach for the production of completely biological blood vessels from cultured human cells. The media component, reconstructed from human vascular smooth muscle cells (VSMC) embedded in the ECM that they produced and assembled, constitutes a new human model for pharmacological research (17). Hence, this human tissue-engineered vascular media (TEVM) displays many functional characteristics of the normal vessel from which the cells were originally isolated, including contractile/relaxation responses, cyclic nucleotide sensitivity, and some of the Ca2+-handling mechanisms. However, involvement of pathways beyond the increase of intracellular Ca2+ concentration ([Ca2+]i) in regulating contraction in this model has not been elucidated.

In the present study, we took advantage of the TEVM model to determine the effect of treatment with red wine polyphenolic compounds, Provinols, on venous contraction. In addition, we investigated the implication of phosphorylation cascades including myosin light chain kinase (MLCK) and Rho-kinase pathways in the modulation of contraction induced by polyphenols.


   METHODS
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Tissue culture. Human smooth muscle cells (SMC) from umbilical cord vein were isolated using the explant method of Ross as described previously (17, 24). After a 2-wk period of culture, SMC from human umbilical cord veins migrated from explants and proliferated (17). To obtain TEVM, we cultured human SMC in medium supplemented with 50 µg/ml sodium ascorbate (Sigma, Grenoble, France) to stimulate ECM synthesis. After 2–3 wk of culture, cells formed sheets composed of cells and ECM that could be wrapped around a tubular support (outside diameter 4 mm) to produce a cylinder composed of approximately four concentric sheet layers. Tissues were cultured for a period of ~3 wk, and TEVM were then used for experiments. The total number of tissues produced was >50 TEVM. This study was approved by the Committee of Ethics (CPPRB) of the Hospital of Strasbourg-France.

Vascular reactivity. TEVM were cut into 4-mm-long rings. Four rings were obtained from each TEVM. This allowed us to perform comparative experiments using vasoconstrictor agonists in the absence or presence of inhibitors. The rings were then mounted on a standard organ bath filled with a physiological salt solution (PSS) (in mmol/l: 119 NaCl, 4.7 KCl, 1.25 CaCl2, 1.17 MgSO4, 1.18 KH2PO4, 25 NaHCO3, and 11 glucose). TEVM rings were maintained at 37°C and continuously bubbled with a 95% O2-5% CO2 mixture. Resting tension was adjusted to 0.5 g. Tension was measured with an isometric force transducer. After an equilibration period of 90 min, the vessels were maximally contracted with histamine (10–4 mol/l) to test their maximal contractile capacity.

Experimental protocol. Concentration-response curves to histamine and bradykinin (Sigma) were constructed in a cumulative manner in TEVM. In some experiments, TEVM were treated for 24 and 96 h with Provinols (10–2 g/l) (Société Française de Distilleries, Vallont Pont d'Arc, France). The polyphenol content of Provinols was (in mg/g of dry powder) 18 hydroxycinnamic acid, 38 catechins, 14 flavonols, 61 anthocyanins, and 480 proanthocyanidols. Provinols stock solution was dissolved in culture medium containing 1% DMSO. The final concentration of DMSO was <0.01%. This concentration of Provinols has been shown to induce maximal endothelium-dependent relaxation of precontracted rat aorta (1). To study the component of the Ca2+-dependent pathway, we used ML-9 (Calbiochem, Paris, France), an inhibitor of MLCK, at 10 µmol/l. The mechanism involved in Ca2+-independent signaling pathway was studied using selective inhibitors of phospholipase A2 (PLA2), Rho-kinase, and PKC [i.e., quinacrine (10 µmol/l; Sigma), Y-27632 (5.0 x 10–7 mol/l; Calbiochem), and GF109203X (3 µmol/l; Tocris, Illkirch, France), respectively]. These drugs were incubated 30 min before addition of the agonist. All the inhibitors were used at the maximally effective concentration.

Measurement of [Ca2+]i. Changes in [Ca2+]i were determined by measuring the fluorescence of trapped fura-2 with a dual-excitation wavelength fluorometer (Fluorolog II; SPEX, Edison, NJ) using the method previously described (17). TEVM segments were loaded with fura-2 by incubation with PSS containing 5 µmol/l fura-2 AM (Sigma) and 2% Pluronic acid for 2 h. The increase in [Ca2+]i in response to histamine (10–4 mol/l) was represented by measurement of the ratio of fluorescence (measured at 510 nm) obtained at the two excitation wavelengths (340/380 nm) and calculated after subtraction of the autofluorescence at 340 and 380 nm.

Immunoblotting. TEVM were homogenized in lysis buffer as previously described (19). Total protein (30 µg) from the supernatant fractions was loaded onto gels for 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane. After electrophoresis, immunostaining of contractile proteins, {alpha}-actin, MLCK, desmin, RhoA, RhoB, and RhoC isoforms, and Rho-kinase was achieved using specific monoclonal mouse anti-{alpha}-actin, polyclonal rabbit anti-desmin, and monoclonal mouse anti-MLCK antibodies (Sigma), polyclonal rabbit anti-RhoA, -RhoB, and -RhoC antibodies (Upstate Biotechnology, Euromedex), and monoclonal mouse anti-Rho-kinase I antibody (Transduction Laboratories), reacted with peroxidase-conjugated anti-mouse and anti-rabbit antibodies (Sigma). Antibody-labeled bands on the blots were detected using an enhanced chemiluminescence assay (Amersham). The level of proteins was measured using densitometry. Monoclonal mouse anti-{beta}-actin immunostaining was used to control the homogeneity of proteins between samples.

Rho activity assay. TEVM were lysed in buffer (25 mmol/l HEPES, pH 7.5, 150 mmol/l NaCl, 1% Igepal CA-630, 10 mmol/l MgCl2, 1 mmol/l EDTA, 2% glycerol, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 25 mmol/l sodium fluoride, and 1 mmol/l sodium orthovanadate). Cell lysates (500 µl) were incubated in the presence of rhotekin Rho binding domain (30 µg) at 4°C for 45 min. The beads were microcentrifuged for 5 s at 14,000 g, and the supernatant was removed. The beads were washed three times with wash buffer (50 mmol/l Tris, pH 7.5, 1% Triton X-100, 150 mmol/l NaCl, 10 mmol/l MgCl2, 10 µg/ml leupeptin, 10 µg/ml aprotinin, and 0.1 mmol/l PMSF). Bound Rho proteins were detected by Western blotting by using a rabbit polyclonal antibody against RhoA, RhoB, and RhoC (Upstate Biotechnology, Euromedex).

MLCK activity assay. TEVM were lysed in lysis buffer (20 mmol/l Tris, pH 7.5, 1 mmol/l EDTA, 1 mmol/l DTT, 1 mmol/l sodium orthovanadate, 1 µmol/l okadaic acid, 1 mg/l leupeptin, 1 mg/l pepstatin, 1 mg/l aprotinin, and 1 mg/l PMSF). Soluble homogenate was added to assay mix containing [{gamma}-32P]ATP. Peptide substrate solution (20 µmol/l) was added. After 10 min, radioactivity was counted and expressed as counts per minute. The protein concentration of tissue samples was determined using the method of Lauri.

Expression of results and statistical analysis. Contractions are expressed as percentages of the maximal contractile response obtained with 10–4 mol/l histamine. The maximal responses obtained with histamine and bradykinin were 333 ± 45 mg (n = 33) and 400 ± 45 mg (n = 30), respectively. The increase in [Ca2+]i is expressed as a fluorescence ratio. Results of Western blot analysis were evaluated using densitometric analysis and are expressed as percentages of control. MLCK activity is expressed as picomoles of phosphate per milligram of total protein per minute. Results are expressed as means ± SE of n experiments, and Student's unpaired t-test or ANOVA was used for statistical analysis, as indicated. P < 0.05 was considered significant.


   RESULTS
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Western blot analysis showed that {alpha}-actin, MLCK, and desmin were expressed in both control and Provinols-treated TEVM (Fig. 1). Expression of these proteins was not different between control and Provinols-treated TEVM.



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  		Fig. 1. Expression of {alpha}-actin and desmin in control, and 24- and 96-h Provinols-treated tissue-engineered vascular media (TEVM). Values are representative of 4 TEVM experiments.

 
Both histamine and bradykinin produced concentration-dependent contraction in control or Provinols-treated TEVM (Fig. 2, A and B). The concentration-response curve to histamine was not significantly different between control and 24-h Provinols-treated TEVM, but bradykinin-induced contraction was significantly enhanced under the same experimental conditions (P < 0.05). Moreover, 96-h treatment with Provinols significantly increased both histamine- and bradykinin-induced contraction in TEVM (P < 0.05 and P < 0.001, respectively). As expected, histamine used at the maximally active concentration produced an increase of [Ca2+]i in TEVM. However, this increase in [Ca2+]i was not significantly affected by Provinols treatment at both 24 and 96 h (Fig. 2C).



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  		Fig. 2. Concentration-response curves for histamine (A)- and bradykinin-induced contraction (B) in control and 24- and 96-h Provinols (10–2 g/l)-treated TEVM. Values are means ± SE of 5–6 TEVM. *P < 0.05; ***P < 0.001 vs. control (ANOVA). C: histogram showing measurement of Ca2+ ratio in vein in control and Provinols-treated TEVM. Values are means ± SE of 3 TEVM. Pro, Provinols.

 
Involvement of the MLCK, Rho-kinase, and PKC pathways in histamine- and bradykinin- induced contraction in control and 96-h Provinols-treated TEVM was determined (Table 1). At the concentrations used, ML-9, Y 27632, or GF109203X did not affect contraction induced by the two agonists in control TEVM. However, the inhibitors of MLCK and Rho-kinase significantly reduced the maximal contractile response to histamine (P < 0.001 and P < 0.05, respectively) and bradykinin (P < 0.01) when TEVM was treated for 96 h with Provinols. In contrast, inhibition of PKC pathway did not affect potentiation of contraction induced by histamine and bradykinin in 96-h Provinols-treated TEVM. Because the pathways involved in the effect of Provinols treatment on TEVM contraction were not different at either time course, only those measured at 24 h of treatment have been illustrated.


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  		Table 1. Effects of ML-9, Y-27632, and GF109203X on maximal contraction induced by histamine and bradykinin in control and Provinols-treated TEVM

 
Histamine-induced contraction was not significantly affected by the MLCK inhibitor ML-9 in control TEVM (not shown). However, ML-9 significantly reduced histamine-induced contraction in TEVM treated with Provinols (P < 0.001, Fig. 3A). Basal MLCK activity was not significantly different between control and Provinols-treated TEVM (Fig. 3B). In control TEVM, histamine slightly but not significantly increased MLCK activity. Interestingly, Provinols treatment significantly potentiated this increase in MLCK activity (P < 0.05; Fig. 3B). Both control and Provinols-treated TEVM expressed MLCK, without any significant difference between the two groups (Fig. 3C).



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  		Fig. 3. A: concentration-response curves for histamine-induced contraction in control and 24-h Provinols-treated TEVM in the absence and presence of the MLCK inhibitor ML-9 (10 µmol/l). Values are means ± SE of 5–6 TEVM. ***P < 0.001 vs. control (ANOVA). B: histogram showing the basal level or the level after histamine (10–4 mol/l) stimulation of MLCK activity in control and 24-h Provinols-treated TEVM. Values are means ± SE of 3 TEVM. *P < 0.05 vs. control. ##P < 0.01 vs. basal 24-h Provinols treatment (unpaired t-test). C: expression of MLCK in control and 24-h Provinols-treated TEVM. Values are representative of 4 TEVM experiments.

 
The Rho-kinase inhibitor Y-27632 (5.0 x 10–7 mol/l) did not significantly modify histamine-induced contraction in control TEVM (not shown). However, inhibition of Rho-kinase significantly reduced histamine-induced contraction in Provinols-treated TEVM (P < 0.01; Fig. 4A). Rho-kinase I expression is shown in Fig. 4B. No difference in Rho-kinase I expression was observed between the two groups.



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  		Fig. 4. A: concentration-response curves for histamine-induced contraction in control and 24-h Provinols-treated TEVM in the absence and presence of the Rho-kinase inhibitor Y-27632 (0.3 µmol/l). Values are means ± SE of 5–6 TEVM. B: Western blots of Rho-kinase I expression in control and 24-h Provinols-treated TEVM. Values are representative of 4 TEVM experiments.

 
Basal Rho activity was not significantly different between the two groups. Stimulation of TEVM with histamine did not induce any increase in Rho activity in either the control or the Provinols-treated group (Fig. 5A). RhoA, RhoB, and RhoC isoforms were expressed in both the control and Provinols-treated groups. Provinols treatment did not alter expressions of Rho protein (Fig. 5B).



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  		Fig. 5. A: Western blot representing Rho (RhoA, RhoB, and RhoC) activity in control and 24-h Provinols-treated TEVM at basal level or the level after stimulation with histamine (10–4 mol/l). B: expression of Rho (RhoA, RhoB, and RhoC) in control and 24-h Provinols-treated TEVM. Values are representative of 3 TEVM experiments.

 
Inhibition of PLA2 by quinacrine did not change vascular contraction in control TEVM (not shown), but it significantly reduced histamine-induced contraction in 24-h Provinols-treated TEVM (P < 0.05; Fig. 6A). The combination of quinacrine and Y-27632 did not affect histamine-induced contraction in control TEVM either. Both inhibitors significantly reduced the contractile response to histamine in Provinols-treated TEVM, but this inhibitory effect was not different from that observed in the presence of either quinacrine or Y-27632 alone (Fig. 6B).



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  		Fig. 6. A: concentration-response curves for histamine-induced contraction in control and 24-h Provinols-treated TEVM in the absence and presence of the PLA2 inhibitor quinacrine (10 µmol/l). Values are means ± SE of 5–6 TEVM. *P < 0.05 vs. control (ANOVA). B: concentration-response curves for histamine-induced contraction in control and 24-h Provinols-treated TEVM in the absence and presence of the Rho-kinase inhibitor Y-27632 (0.3 µmol/l) plus quinacrine (10 µmol/l). Values are means ± SE of 5–6 TEVM. **P < 0.01 vs. control (ANOVA).

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
This report describes the modulation of contraction produced by a red wine polyphenolic extract, Provinols, evaluated using a human vascular model produced by tissue engineering. Provinols treatment of TEVM potentiated the contractile responses induced by histamine and bradykinin. This effect of Provinols was not associated with an enhancement of the histamine-induced increase in [Ca2+]i; rather, it implied a Ca2+-independent signaling pathway involving the PLA2-Rho-kinase pathway and was associated with an enhancement of MLCK activity. Therefore, these results reveal new insights to explain the regulation of venous contraction by polyphenols.

Several investigators have described new processes to produce small-diameter tissue-engineered blood vessel substitutes over the past decade. Recently, investigators in our laboratory (16) described the reconstitution of a vascular blood vessel, which contained the three histological layers, the endothelium, the media, and the adventitia. The advantage of this tissue-engineering method was that no synthetic material was used that could be associated with inflammatory reaction after grafting. Furthermore, this tissue-engineered human blood vessel is highly resistant, with a burst strength of >2,500 mmHg. The improved resistance is probably due to the impressive organization of the ECM resulting from the self-assembly approach (16) compared with other previously designed biological tissue-engineered blood vessels (27).

Vasomotricity represented a functional requirement for a vascular graft. This contractile property was attributed to the VSMC. It is well known that VSMC, when placed in culture, rapidly convert to a synthetic phenotype. However, when these cells are organized into a three-dimensional construct, they may revert to a more contractile phenotype (17). Oishi et al. (22) showed that the contractile capacity of SMC from guinea pig stomach mixed in a collagen gel in vitro was restored. Indeed, SMC contracted in response to different contractile agents, including KCl, norepinephrine, serotonin, and histamine, with the mechanism of contraction depending on MLCK and Rho-kinase pathways.

Pharmacological studies remain difficult to perform in monolayer cell cultures because there is a phenotype modification. Moreover, data obtained from single-cell contraction studies may not reflect the global effect of a drug in a tissular context. However, cells cultured in defined favorable conditions have been able to produce and organize their own ECM in a structural tissue (17). The formation of a three-dimensional tissuelike structure provided conditions for adequate cell differentiation and offered the possibility to perform pharmacological study of VSMC in an environment close to in vivo conditions. Investigators in our laboratory (17) have shown that TEVM presents the same characteristics as a native human vessel that responds specifically to a membrane receptor agonist, resulting in an increase in [Ca2+]i. However, the intracellular signaling pathway involved in the mechanism of contraction has not yet been fully studied.

Our results show that vasoconstrictor agents induce a concentration-dependent contraction in TEVM, associated with an increase in [Ca2+]i, as previously reported (17). Agonist-induced activation of SMC resulted in an increase in [Ca2+]i, leading to the Ca2+/calmodulin-dependent MLCK activation and phosphorylation of smooth muscle myosin. Furthermore, it has been shown that Rho-kinase I and PKC increase phosphorylation of myosin light chain (8, 15) in response to various agonists via a Ca2+-independent mechanism. In the present study, inhibitors of MLCK, Rho-kinase, and PKC pathways were used at concentrations that did not affect agonist-induced vasoconstriction so that we could better estimate the involvement of such mechanisms in the modulation of vasomotricity by Provinols. It should be noted that histamine was able to slightly increase MLCK activity in TEVM, and an increasing concentration of Rho-kinase inhibitor (1 µmol/l) totally abolished histamine- and bradykinin-induced contraction (not shown). Altogether, these results suggest the involvement of both MLCK and Rho-kinase pathways for agonist-induced contraction in this model. These data, along with findings of previous work performed in the same model, show that in TEVM, the transduction mechanism leading to contraction in response to vasoconstrictor agents involves a Ca2+-dependent pathway and activation of MLCK and a Ca2+ sensitization through at least the Rho-kinase inhibitor-sensitive pathway. These results are in accordance with a previous study, which showed that [Ca2+]i plays a minor role in phenylephrine-induced contraction in human saphenous vein. The sustained contraction was, however, attributed to an enhancement of Rho-kinase and tyrosine kinase pathways (4).

Natural dietary polyphenols have been used in general to treat vascular diseases originating from veins (18), in addition to their beneficial effects against atherosclerotic plaque development and hypertension (2, 26). Only a few studies have reported the underlying mechanism involved in the phlebotropic and venoprotective properties of polyphenolic compounds in human blood vessels. In rat femoral vein, Savineau (25) demonstrated the venotonic action of a flavone derivative, diosmin, probably through its capacity in increasing Ca2+ sensitivity. In addition, flavonoid components such as rutin or quercetin have been shown in vitro to increase myosin ATPase activity, which facilitated actomyosin interaction (29).

In this work, the effect of Provinols was investigated at two different times (i.e., after 24 and 96 h) to determine its short-term effect and its probable action at the gene expression level that might mediate its physiological modulation of vasomotricity. Indeed, it has been shown that red wine polyphenols alter cyclin A gene expression in rat aorta SMC after a 72-h treatment only. The latter action may contribute to the antiproliferative effect of polyphenols through the inhibition of transcription factor expression (13).

Regarding the expression of cytoskeletal proteins, neither 24- nor 96-h Provinols treatment modified {alpha}-actin or desmin expression in TEVM. Interestingly, Provinols treatment potentiated agonist-induced contraction at both periods of time, except for histamine at 24 h. Because this potentiation was not associated with a change in agonist-induced increase in [Ca2+]i, the effect of Provinols most likely involved an increase of either Ca2+ sensitivity or Ca2+-independent mechanisms.

In the present study, we found that the efficacy of the MLCK inhibitor ML-9 in reducing vasoconstriction induced by histamine was greater in TEVM treated with Provinols. Furthermore, this effect was associated with potentiation of the histamine-induced increase in MLCK activity without alteration of MLCK expression. Taken together, these results strongly support the view that Provinols treatment enhanced MLCK activity in TEVM, and this may play a role in the potentiation of agonist-induced contraction in veins. Several hypotheses could be advanced to explain the mechanism by which Provinols mediated its action. First, Provinols might enhance a local increase in [Ca2+]i that could not be detected by the fura-2 probe but that was sufficient enough to activate the MLCK activity (28). Second, Provinols treatment might potentiate the response at the level of calmodulin, as reported previously (11). We could not distinguish among these possibilities.

Finally the role of the PLA2-Rho-kinase pathway and the involvement of PKC were investigated to explain the potentiating effect of Provinols treatment. Interestingly, the efficiency of the PLA2 and Rho-kinase inhibitors quinacrine and Y-27632, respectively, in reducing agonist-induced vasoconstriction was unmasked by Provinols treatment. However, the combination of both quinacrine and Y-27632 did not produce further inhibition compared with the effect of one of these inhibitors was used separately in TEVM treated with Provinols. Thus Provinols-induced potentiation of vasoconstriction took place partly via mechanisms sensitive to PLA2 and Rho-kinase inhibitors. It is unlikely that the pathway implicated in Rho-kinase activation involved RhoA, RhoB, or RhoC protein, because Provinols treatment was not able to enhance Rho activity in a coimmunoprecipitation assay. However, Provinols-induced Ca2+ sensitization through the activation of Rho-kinase might be caused by an alteration of arachidonic acid production via the activation of PLA2. Indeed, it was recently shown (10) that Ca2+-independent PLA2 was required for the maintenance of basal free arachidonic acid levels that were essential for agonist-induced Ca2+ sensitization of contraction in VSMC. The potentiating effect of contraction in vein TEVM implies the activation of PLA2-Rho kinase pathway in addition to the enhancement of MLCK activity. Improvement of Rho-kinase pathway by Provinols may contribute to explain the vasculoprotector effect of this compound given that deleterious function of this transduction signaling has been reported to play a role in the development of varicose veins (3).

In summary, we report that treatment of TEVM by Provinols potentiated the contractile responses induced by vasoconstrictor agonists. The potentiating effect of Provinols was not associated with an enhancement of histamine-induced increase in [Ca2+]i; rather, it implied a Ca2+-independent signaling pathway. Pharmacological studies indicated that Provinols actions take place at the level of PLA2-Rho-kinase pathway and are associated with an enhancement of MLCK activity. These results bring new insights to explain the regulation of venous contraction by polyphenols, with the use of TEVM as a tool for such pharmacological studies.


   GRANTS
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This work was supported by grants from Société Française de Distilleries, Centre National de la Recherche Scientifique (Ingénierie Tissulaire), and Canadian Institutes for Health Research (CIHR). L. Germain was a recipient of Scholarships from CIHR and holder of a Canadian Research Chair on Stem Cells and Tissue Engineering from CIHR. M. Diebolt was supported by Région Alsace and Société Française de Distilleries.


   ACKNOWLEDGMENTS
 
We thank Mireille Gaire for technical assistance with the MLCK activity assay.


   FOOTNOTES
 

Address for reprint requests and other correspondence: R. Andriantsitohaina, Pharmacologie et Physico-Chimie des Interactions Cellulaires et Moléculaires, UMR CNRS 7034, Faculté de Pharmacie, 74 Route du Rhin, 67401 Illkirch, France (E-mail: Ramaroson.Andriantsitohaina{at}pharma.u-strasbg.fr)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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F. A. Auger, P. D'Orleans-Juste, and L. Germain
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