Contribution of muscarinic receptors to in vitro and in vivo effects of Ruscus extract

Microvascular Research 114 (2017) 1–11

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Contribution of muscarinic receptors to in vitro and in vivo effects of Ruscus extract Isabelle Rauly-Lestienne a, Peter Heusler a, Didier Cussac a, Frédérique Lantoine-Adam a, Fatima Zely Garcia de Almeida Cyrino b, Eliete Bouskela b,⁎ a b

Centre de Recherche Pierre Fabre, 17 Av. Jean Moulin, 81100 Castres, France Laboratory for Clinical and Experimental Research on Vascular Biology (BioVasc), Biomedical Center, State University of Rio de Janeiro, Rio de Janeiro, RJ, Brazil

a r t i c l e

i n f o

Article history: Received 9 April 2017 Accepted 17 May 2017 Available online 19 May 2017 Keywords: Muscarinic receptors Hamster cheek pouch microcirculation Inflammation Venotonic drug In vitro and in vivo studies

a b s t r a c t The objectives of this study were to evaluate, in vitro and in vivo, the contribution of muscarinic receptors to the effects of Ruscus extract. Ruscus extract was tested in competition binding experiments at recombinant human muscarinic receptors, heterologous expressed in Chinese Hamster Ovary (CHO) cells and in cellular assays measuring Ca2+ liberation and activator protein-1 (AP-1) reporter gene activation. The impact of muscarinic blockade on prolonged treatment outcome was evaluated using the hamster cheek pouch (HCP) microcirculation examining macromolecular permeability increase induced by histamine or ischemia/reperfusion (I/R), mean arteriolar and venular diameters, functional capillary density and I/R-induced leukocyte rolling and sticking. Ruscus extract exhibited affinities for muscarinic receptor subtypes at a range of 50–100 μg/ml and behaved as partial agonist at human recombinant M1 and M3 receptors for Ca2+ liberation, confirmed in an AP-1 reporter gene assay. In the HCP model, topical application of atropine completely or partially blocked Ruscus extract-induced reductions of histamine- and I/R-induced increases of macromolecular permeability and leukocyte-endothelium interaction. Our results showed that Ruscus extract in vitro binds and activates different subtypes of muscarinic receptors and in vivo its anti-inflammatory effects are, at least partially, mediated via muscarinic receptors. © 2017 Published by Elsevier Inc.

1. Introduction Varicose veins, a manifestation of venous disease, are enlarged and swollen veins caused by increased venous distensibility and malfunctioning valves. Backflow of the blood results in increased pressure within the post-capillary venules favoring exudation of fluid and edema formation. Insufficient drainage of the tissue causes ischemia which can result in infection, thrombosis and damage to the tissue (Vanhoutte, 1991; Chwala et al., 2015). Venous disease is a socioeconomic problem of increasing magnitude, and experimental results with agents used to treat it are sparse, probably due to a lack of good experimental models for the disease. Ruscus aculeatus (butcher's broom) extract is used in association with the flavonoid hesperidin methylchalcone to increase peripheral venous tone. The venotonic properties of Ruscus are well established, but its mechanism of action has not been fully elucidated. In isolated cutaneous ⁎ Corresponding author at: Laboratório de Pesquisas Clínicas e Experimentais em Biologia Vascular (BioVasc), Universidade do Estado do Rio de Janeiro, Pavilhão Reitor Haroldo Lisboa da Cunha, térreo, Rua São Francisco Xavier, 524, 20550-013 Rio de Janeiro, RJ, Brazil. E-mail address: [email protected] (E. Bouskela).

http://dx.doi.org/10.1016/j.mvr.2017.05.005 0026-2862/© 2017 Published by Elsevier Inc.

veins, Ruscus extract causes contractions owing to activation of both post− junctional α− 1 and α2 adrenoceptors and through direct action on venous smooth muscle cells (Marcelon et al., 1983; Marcelon and Vanhoutte, 1988). Using the hamster cheek pouch (HCP) model, we have previously shown that systemic intravenous administration of Ruscus extract induces venular constriction, but does not affect arteriolar diameter or mean arterial pressure. Topical application of Ruscus extract elicited concentration- and temperature-dependent responses in the vessels studied (Bouskela et al., 1993a). It was possible to block the venular constriction using low concentrations of prazosin (α1-adrenoceptor antagonist) or diltiazem (calcium blocker) and a high concentration of rauwolscine (α2-adrenoceptor antagonist) (Bouskela et al., 1994a). In another series of experiments, we showed that intravenous administration of Ruscus extract resulted in a significant inhibition of the macromolecular permeability-increasing effects of bradykinin, leukotriene B4 and histamine (Bouskela et al., 1993b). The inhibitory effect of Ruscus extract on histamine-induced increases in permeability was blocked by prazosin and by diltiazem, but not by rauwolscine. The results, therefore, indicated that variations in the transmembrane flux of calcium impair the histamine-induced formation of microvascular leakage sites (Bouskela et al., 1994b).

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Muscarinic receptors constitute a heterogeneous population, with five subtypes (M1­M5) having been identified using pharmacological and molecular methods (Eglen, 1997; Caulfield and Birdsall, 1998). The subtypes M1, M2 and M3, are known to be capable of mediating a range of responses to muscarinic agonists in the vascular tissues of different mammals (Eglen and Whiting, 1990): M1 receptors induce contractile responses in venous tissue, M2 elicit relaxant responses in venous and arterial tissues by means of a decrease in sympathetic outflow, and M3 receptors can contract coronary arteries and induce endothelium-derived relaxing factor (EDRF) -dependent relaxant responses in most tissues studied to date. Microcirculatory impairment caused by chronic venous hypertension is one of the first signals of chronic venous disease, frequently associated with its severity (Katsenis, 2005). The increased venous pressure is transmitted to the capillary bed and leads to chronic damage and microcirculatory dysfunction. Cutaneous nutritive capillaries become progressively enlarged, tortuous (Fagrell, 1981; Jünger et al., 2000) and with augmented inter-endothelial space (Cheatle et al., 1991) with consequent increase in macromolecular permeability with plasma, red blood cells and fibrinogen leakage, impairing nutrient exchange (Cheatle et al., 1991). Venous stasis combined with hypertension lead to an inflammatory process (Cheatle et al., 1991) and edema formation (SchmidShöenbein et al., 2001). The reduction in the number of capillaries leads to trophic disorders and leg ulceration (Fagrell, 1981). In order to test for a possible contribution of muscarinic receptors to the venotonic, anti-inflammatory and antioxidant properties of Ruscus extract, we investigated its binding and functional properties at recombinant human muscarinic receptor subtypes in vitro, and its impact on the microvasculature in the HCP preparation in vivo. 2. Materials and methods 2.1. In vitro studies 2.1.1. Ruscus extraction The dry extract of Ruscus titrated in sterolic heterosides was obtained from Ruscus aculeatus L. rhizomes and roots by hydroalcoholic extraction (Pierre Fabre Laboratories). 2.1.2. Competition binding studies at muscarinic receptors Binding studies were performed at human (h) hM1 and hM3 muscarinic receptors using radioligands and membrane preparations from cell lines stably expressing respective receptors. Membranes (10–20 μg of protein) were re-suspended in Tris-HCl 50 mM (pH 7.6) buffer, and incubated for 120 min at 25 °C with different concentrations of Ruscus extract and N-methyl-scopolamine radioligand ([3H]NMS) in a final volume of 0.5 ml. Non-specific binding was defined using excessive concentrations of the specific competing ligand, atropine. Incubations were terminated by rapid filtration through 0.1% polyethylenimine-pre-soaked Whatman GF/B filters using a 96-well filtermate harvester (PerkinElmer Life Science, Boston, MA, USA). Radioactivity retained on filters was determined by liquid scintillation counting by means of a Top-Count microplate scintillation counter (PerkinElmer Life Science, Boston, MA, USA). Experiments were performed at least twice, with duplicate determinations in each case. Isotherms were analyzed by non-linear regression using Prism (GraphPad Software, San Diego, CA, USA) to generate IC50 values. Inhibition constants (Ki) were calculated from IC50 values according to the Cheng-Prusoff equation (Cheng and Prusoff, 1973): Ki = IC50/(1 + L/Kd), where L is the concentration of [3H]-NMS and Kd its dissociation constant for the respective receptor subtype. Averaged IC50 and Ki values are expressed as geometric mean (for evaluations performed at least three times). Kd values of [3H]-NMS at hM1 and hM3 were 0.03 and 0.04 nM, respectively (Heusler et al., 2015). 2.1.3. Ca2+ liberation experiments at muscarinic receptors Chinese Hamster Ovary (CHO) K1 cells were transiently transfected with different sub-types of muscarinic acetylcholine (ACh) receptors,

hM1, hM2, hM3, hM4 and hM5 by electroporation (Pauwels et al., 2000). Briefly, CHO-K1 cells in exponential growth phase were transfected using a gene pulser transfection apparatus (BioRad, Hercules, CA, USA). Cells were electroporated at 25 °C in 0.8 ml of Ham's F12 nutrient medium (Life Technologies, Saint Aubin, France) plus 10% fetal calf serum (FCS) and 1% dimethyl sulfoxide (DMSO) with 10 μg pcDNA3-hM1, 10 μg pcDNA3-hM2, 10 μg pcDNA3-hM3, 10 μg pcDNA3-hM4 or 10 μg pcDNA3-hM5, respectively (voltage: 250 V, capacitor: 960 μF). Transiently transfected CHO-K1 cells were plated into 96-well plates with 0.2 ml of Ham's F12 medium and 10% heat-inactivated FCS at 100,000 cells per well. Cells were assayed for intracellular Ca2+ responses 48 h after plating (Cussac et al., 2008): Culture medium was removed by aspiration and replaced with 0.2 ml of complete growth medium containing 2.5 mM probenicid acid and Fluo-4 fluorescent calcium indicator dye. After incubation for 2 h in 5% CO2, the cells were transferred to μCell, a fluorometric imaging plate reader (Hamamatsu, Massy, France). The μCell transferred 40 μl from the ligand microplate (Hank's Balanced Salt Solution [HBSS] or different muscarinic ACh receptor antagonists) to cells and made fluorescent readings every 6 s for 10 min. Next, μCell transferred 40 μl from the second ligand microplate (HBSS or Ruscus aculeatus rhizome extract for dose-response) and made fluorescent readings every 1 s for 3 min. Maximal fluorescent counts were used to determine molecular activity. The Hamamatsu software normalized the fluorescence reading to give initial values for each well at time zero. Results are presented in Arbitrary Fluorescent Units (AFU). All data are expressed as mean ± standard error of the mean (SEM) of three independent determinations, each performed in duplicate. Isotherms were analyzed by nonlinear regression, using GraphPad Prism (GraphPad Software Inc., San Diego, CA, USA) to generate EC50 values of Ruscus aculeatus rhizome extract in the absence and presence of antagonist compounds. 2.1.4. AP-1 reporter gene assay Activator protein-1 (AP-1) reporter gene assay experiments were performed as previously described (17): Briefly, CHO-K1 cells were transiently transfected with Lipofectamine Plus (Life Technologies, Saint Aubin, France); 3 μg of hM1R or hM3R plasmid and 10 μg of AP-1 luciferase reporter plasmid per P50 petri dish. Twenty-four hours after transfection, the cells were transferred to opaque 96-well plates at a density of 20,000 cells per well. The cells were then left to adhere to the plates overnight. For pharmacological evaluations, transfected cells were incubated with various concentrations (each in duplicate or triplicate) of Ruscus extract or carbachol (CCh) in Optimem 1 (Life Technologies, Saint Aubin, France) without phenol red for 8 h. The antagonist was pre-incubated 15 min before the addition of Ruscus extract. This interval was chosen based on preliminary experiments that indicated optimized assay robustness. Luciferase activity was determined using the BriteLite luciferase assay kit (Perkin Elmer, Courtaboeuf, France). Briefly, 50 μl BriteLite assay reagent was pipetted into each well and luminescence was measured using a TopCount Counter (Perkin Elmer, Courtaboeuf, France) after 5 min incubation in the dark. Luminescence values were normalized to baseline values obtained from untreated cells. It should be noted that the baseline luminescence signal determined in hM1Rtransfeted cells (160,467 ± 13,866 arbitrary luminescence units) was consistently higher than in hM1R-transfected cells (54,892 ± 10,985 arbitrary luminescence units). 2.1.5. Preparation of drugs Radioligand [3H]-N-methyl-scopolamine ([3H]-NMS; 85 Ci/mmol, PerkinElmer Life Sciences (Courtaboeuf, France); atropine (Sigma RBI, France) and Ruscus extract (Pierre Fabre Medicament, Gien, France) were dissolved in 10% DMSO in distilled water at 10−3 M. Subsequent dilutions were prepared in binding buffer.

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2.2. In vivo studies Experiments were performed on the cheek pouch of male golden hamsters (Mesocricetus auratus), aged 7 to 10 weeks. The experimental protocol was approved by the Ethical Committee of the State University of Rio de Janeiro (CEUA 058/2012). For oral treatment, Ruscus extract (50, 150 and 450 mg/kg/day) or placebo (filtered water) was given twice a day, at 8:00 am and 5:00 pm, for 2 weeks. 2.2.1. Hamster cheek pouch preparation Hamsters were anesthetized by intraperitoneal injection of 0.1– 0.2 ml of sodium pentobarbital (Pentobarbital sodique, Sanofi, Paris, France, 60 mg/ml). Anesthesia was maintained using α-chloralose (Sigma Chemicals, St. Louis, MO, USA, 100 mg/kg) administered via the femoral vein. The temperature of the hamsters was maintained at 37.5 °C throughout the surgical and experimental procedures using a heating pad controlled by means of a rectal thermistor. A tracheal tube was inserted to facilitate spontaneous breathing and the femoral artery was cannulated for pressure and heart rate measurements (PowerLab, AD Instruments, New South Wales, Australia) to check the viability of the animal. Hamsters were placed on a microscope stage/platform and the cheek pouches were dissected using the technique described by Duling, (1973) with some minor modifications (Svensjö et al., 1978; Bouskela and Grampp, 1992). The cheek pouches were submerged in a superfusion solution of HEPES-supported HCO3-buffered saline (composition in mM: 110.0 NaCl, 4.7 KCl, 2.0 CaCl2, 1.2 MgSO4, 18.0 NaHCO3, 15.39 HEPES and 14.61 HEPES Na+ − salt) which continuously flushed the pool of the microscope stage; the temperature of the solution was maintained at 36.5 °C, and the pH at 7.40 by continuous bubbling with 5% CO2 in 95% N2 (Bouskela and Grampp, 1992). The superfusion rate varied from 4 ml/min (leukocyte-endothelium interaction and diameter measurements) to 6 ml/min (for macromolecular permeability). 2.2.2. Measurements of arteriolar and venular diameters Pouch preparations were placed under an intravital microscope [Leica DMLFS, Wetzlar, Germany) coupled to a TV camera (Optronics, model 60366-1, Goleta, CA, USA), TV monitor and portable computer] where they were allowed to rest for 30 min. If after this time there was (a) an indication of good vascular tone; (b) brisk blood flow in all parts of the vascular bed, including the larger veins and (c) no tendency for leukocytes to adhere to the vessel wall (Bouskela and Grampp, 1992), the experiments were performed by taking DVD recordings of each of one to four selected arterioles and venules per preparation, both under initial control conditions and after each experimental intervention (Bouskela and Grampp, 1992). The microvessels were selected based on the ability to return to them at the same site, repeatedly, during the experiment. The TV monitor display was used to obtain arteriolar and venular internal diameter measurements by an image shearing monitor (IPM model 907, San Diego, CA, USA). However, DVD replay was used for the final determination of vessel diameters as greater attention could be given to measurement than was possible while the experiment was being conducted. Atropine (solution of 1 mg/ml, Sigma Chemicals, St. Louis, MO, USA) was applied topically in the concentration of 10−8, 10−7 and 10−6 M. 2.2.3. Measurement of macromolecular permeability Thirty minutes after completion of the preparative procedure, fluorescein isothiocyanate (FITC)-dextran (molecular weight 150,000 Da; Bioflor HB, Uppsala, Sweden), with a degree of substitution of FITC molecules 2/1000 glucose molecules in the polysaccharide chain, was administered by intravenous injection at a dose of 25 mg/100 g body weight as a 5% solution in 0.9% saline (Svensjö et al., 1978). Observations were made with an intravital microscope (Leica DMLFS, Wetzlar, Germany) equipped with a 100 W Hg DC lamp and specific filters.

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The total area of the pouch to be observed was 1 cm2 in size and roughly circular. The number of leakage sites (=leaks) was determined by manually scanning the entire area at the following selected time intervals: 2, 5, 7, 10, 15, 20 and 30 min after the initiation of each topical application of histamine (Sigma Chemicals, St. Louis, MO, USA, 5 μM). Atropine was also applied topically in the concentrations mentioned before. 2.2.4. Ischemia/reperfusion Local ischemia was induced by applying a cuff made of thin latex tubing around the neck of the prepared HCP with an intratubular pressure of 200–220 mm Hg (resulting in complete arrest of blood flow within seconds), as described previously (de Souza et al., 2015; Boa et al., 2014). The occlusion period was 30 min. Measurements of macromolecular permeability were performed after I/R as described above. For leukocyte-endothelium interaction, prior to ischemia and 30 min after the onset of reperfusion, the leukocytes were stained with an intravenous infusion of rhodamine G (solution of 200 mg/ml, infusion rate 10 μl/min during 10 min, Sigma Chemicals, St. Louis, MO, USA) and the fluorescent leukocytes adhering to the endothelium or rolling closer to the venular wall were quantified using a UV-light microscope (Leica DMLFS, Wetzlar, Germany). Atropine was also applied topically in the concentrations mentioned before. 2.2.5. Statistical analyses Results were presented as means ± standard deviation (SD), unless otherwise noted. Significance levels (i.e., p-values) in the tests for in vivo studies were obtained using analysis of variance models on data in the groups vehicle/placebo, Ruscus extract 50 mg/kg, Ruscus extract 150 mg/kg, and Ruscus extract 450 mg/kg analyzed together. If the global treatment effect was statistically significant, the effect of each Ruscus extract dose was compared to placebo/vehicle using appropriate contrasts. This results to three t-statistic tests followed by Bonferroni correction for multiplicity. The significance level was set to 0.05 for all comparisons. 3. Results 3.1. In vitro studies 3.1.1. Binding of Ruscus extract to recombinant muscarinic receptors The affinity of Ruscus extract for muscarinic receptors was tested by [3H]-NMS binding to membrane preparations of recombinant human muscarinic receptor subtypes heterologously expressed in CHO-K1 cells. In these experiments, Ruscus extract displayed comparable affinity for hM1 and hM3 subtypes (hM5 not performed) with Ki values in the range of 50–100 μg/ml (Table 1). 3.1.2. Functional activity of Ruscus extract at muscarinic receptors in Ca2+ liberation experiments Functional activity of Ruscus extract at recombinant muscarinic receptor subtypes was first evaluated in Ca2+ liberation experiments. In these assays, receptors were transiently expressed in CHO-K1 cells to allow activation of the Ca2+ pathway. Activation of CHO-K1 cells expressing hM1, hM3 and hM5 subtypes (coupling to endogenous CHOK1 Gq proteins) by the agonist CCh induced robust and reproducible Ca2+ signals that peaked at 15,000–20,000 AFU, with a CCh potency of 47.80, 44.30, 38.15 nM for hM1, hM3 and hM5, respectively (Fig. 1). No Ca2+ signal was observed with CCh stimulation of non-transfected maternal CHO-K1 cell line (data not shown). When Ruscus extract was tested in this assay, it behaved as a partial agonist at all three odd-numbered muscarinic receptors subtypes, displaying potencies of 12 (hM1), 31 (hM3) and 68 μg/ml (hM5) (Fig. 1 and Table 1). In each case, the signal mediated by Ruscus extract was completely abolished by blocking of muscarinic receptors with 1 μM atropine (Fig. 1).

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Table 1 Binding affinities and functional activities of Ruscus extract at recombinant human muscarinic receptor subtypes. Ca2+

Binding

Reporter gene AP-1

Type

IC50 (μg/ml)

Ki (μg/ml)

EC50 (μg/ml)

Emax (% CCh)

EC50 (μg/ml)

Emax (% CCh)

hM1 hM3 hM5

837.6 926.9 –

61.9 96.1 –

12.5 ± 1.0 31.3 ± 2.1 67.8 ± 15.7

62 ± 7 62 ± 4 72 ± 11

64.8 ± 15.2 149.5 ± 14.7 –

64 ± 3 73 ± 12 –

Data are expressed as mean ± SEM calculated from duplicate or triplicate in three independent experiments. Abbreviations: AP-1 = activator protein-1, CCh = carbachol, h = human, M = muscarinic receptor, SEM = standard error of the mean.

3.1.3. AP-1 luciferase reporter experiments and atropine To corroborate the finding of an agonist activity of Ruscus extract at hM1 and hM3 receptors, we performed luciferase reporter gene experiments after transient transfection of CHO-K1 cells with receptors and an AP-1 luciferase reporter gene. With both receptor subtypes, CCh induced an augmentation of the luciferase signal by about 200% relative to its basal activity (Fig. 2). In parallel evaluations, Ruscus extract behaved as a partial agonist at hM1 and hM3, with a mean efficacy similar to that obtained in Ca2+ experiments, albeit with somewhat lower potency (Fig. 2 and Table 1). As for the Ca2+ liberation, the stimulation

observed with Ruscus extract was completely abolished by treatment with the muscarinic antagonist atropine (1 μM; Fig. 2). Atropine by itself acted as an inverse agonist in this assay, i.e. it decreased the luciferase signal below basal values (even in presence of Ruscus extract, Fig. 2), as previously documented (Heusler et al., 2015; Casarosa et al., 2010). 3.2. In vivo studies in the hamster cheek pouch model In placebo treated animals, mean arterial pressure varied from 94.0 to 96.0 mm Hg and heart rate from 394 to 481 beats per min (bpm) and in

Fig. 1. Effects of carbachol (CCh) (left) or Ruscus extract (right) on Ca2+ liberation mediated by muscarinic receptors hM1, hM3 and hM5. Ruscus extract was tested by itself or in the presence of atropine (1 μM). All data are expressed as mean ± standard error of the mean (SEM) of three independent determinations, each performed in duplicate.

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Fig. 2. Effects of carbachol (CCh) (left) or Ruscus extract (right) on luciferase activity mediated by muscarinic receptors hM1 and hM3. Ruscus extract was tested by itself or in the presence of atropine (1 μM). All data are expressed as mean ± standard error of the mean (SEM) of three independent determinations, each performed in quadruplicate.

animals treated with Ruscus extract varied from 94.5 to 96.0 mm Hg and from 432 to 509 bpm for 50 mg/kg/day; from 93.0 to 96.0 mm Hg and from 396 to 491 bpm for 150 mg/kg/day and from 93.0 to 95.5 mm Hg and from 412 to 503 bpm for 450 mg/kg/day. No significant differences could be detected between groups regarding mean arterial pressure or heart rate. 3.2.1. Effects on mean arteriolar and venular diameters Ruscus extract elicited statistically significant constriction of venules compared with placebo (p b 0.001 for all Ruscus concentrations tested), but it did not have a significant effect on arteriolar diameter (Fig. 3). Topical application of atropine in placebo-treated animals provokes arteriolar and venular constriction (Figs. 4 and 5). In animals treated with

different concentrations of Ruscus extract, topical application of atropine elicited arteriolar constriction (Fig. 4) but no significant changes on elicited venular constriction could be observed (Fig. 5).

3.2.2. Effects on histamine-induced macromolecular permeability Topical application of atropine did not significantly affect the macromolecular permeability increase induced by histamine (data not shown). Ruscus extract resulted in a statistically significant dose-dependent reduction in the macromolecular permeability increase induced by topical application of histamine compared with placebo (p b 0.001 for 150 and 450 mg/kg/day and p b 0.01 for 50 mg/kg/day) (Fig. 6). This effect was blocked by topical application of atropine (Fig. 7).

Fig. 3. Effects of different oral doses of Ruscus extract (given twice a day for 2 weeks) on mean arteriolar and venular diameters. Observation in the hamster cheek pouch microcirculation (n = 6 hamsters). ⁎p b 0.05; ⁎⁎p b 0.01 and ⁎⁎⁎p b 0.001.

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Fig. 4. Effects of different oral doses of Ruscus extract (given twice a day for 2 weeks) combined with topical application of different concentrations of atropine on mean arteriolar diameter. Observation in the hamster cheek pouch microcirculation (n = 6 hamsters). ⁎p b 0.05; ⁎⁎p b 0.01 and ⁎⁎⁎p b 0.001.

3.2.3. Effects on ischemia/reperfusion-induced macromolecular permeability Topical application of atropine did not significantly affect the macromolecular permeability increase induced by ischemia/reperfusion (I/R) (Fig. 9). Ruscus extract resulted in a statistically significant dose-dependent reduction in the macromolecular permeability increase induced by I/R compared with placebo (p b 0.001 for all Ruscus concentrations tested) (Fig. 8). The effect of the lowest dose of Ruscus extract (50 mg/kg/day) was blocked by the highest concentration of atropine (10−6 M), applied topically (Fig. 9). However, the effects of the highest doses of Ruscus extract were not blocked by atropine. 3.2.4. Effects on leukocyte rolling/sticking induced by ischemia/reperfusion Ruscus extract resulted in a statistically significant dose-dependent decrease in both the number of rolling leukocytes (p b 0.05, p b 0.01

and p b 0.001 for 50 mg/kg/day, 150 mg/kg/day and 450 mg/kg/day, respectively) and the number of leukocytes adhered to the venular walls (p b 0.001 for all Ruscus concentrations tested) compared with placebo after I/R (Fig. 10). This effect was blocked by topical application of atropine (Figs. 11 and 12). 4. Discussion The aim of this study was to investigate the activity of Ruscus extract at muscarinic receptors and the potential contribution of this activity to its venotonic and anti-inflammatory properties. In the first part of the study we evaluated the activity of Ruscus extract at recombinant human muscarinic receptors, both in binding and in functional studies. Binding affinities of Ruscus at hM1 and hM3 were slightly below 100 μg/ ml, representing a concentration range at which Ruscus extract has

Fig. 5. Effects of different oral doses of Ruscus extract (given twice a day for 2 weeks) combined with topical application of different concentrations of atropine on mean venular diameter. Observation in the hamster cheek pouch microcirculation (n = 6 hamsters). ⁎p b 0.05; ⁎⁎p b 0.01 and ⁎⁎⁎p b 0.001.

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Fig. 6. Effects of different oral doses of Ruscus extract (given twice a day for 2 weeks) on macromolecular permeability increase induced by topical application of histamine. Observation in the hamster cheek pouch microcirculation (n = 6 hamsters). ⁎p b 0.05; ⁎⁎p b 0.01 and ⁎⁎⁎p b 0.001.

previously been shown to have pharmacological activity in the microcirculation (Marcelon et al., 1983; Bouskela et al., 1993a; Bouskela et al., 1993b). On the basis of these binding results, it seemed plausible that the activity of Ruscus at muscarinic receptors could contribute to its pharmacological profile. The functional activity of Ruscus extract at recombinant muscarinic receptor subtypes was then evaluated in Ca2+ liberation experiments. Indeed, the M1, M3 and M5 receptor subtypes are able to activate the phospholipase C inositol triphosphate (PLC-IP3) pathway resulting in the release of calcium from intracellular stores (Bschleipfer et al., 2007). In the present study, activation of CHO-K1 cells expressing hM1, hM3 and hM5 subtypes by the agonist CCh induced a robust and reproducible Ca2+ signal. The potencies observed for CCh are in accordance with other published results (Simon et al., 2012), validating our models. The Ca2+ response measured for Ruscus extract was mediated by the different muscarinic receptors expressed, since this response was lacking in non-transfected CHO-K1 cells. Moreover, an increase in Ca2+ induced in the presence of Ruscus extract was completely abolished by the muscarinic receptor antagonist, atropine. The agonist properties of Ruscus extract at muscarinic receptors were further confirmed by an AP-1 reporter gene assay, yielding essentially

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similar results. In both tests, Ruscus extract surprisingly behaved as an efficacious partial agonist, with an efficacy of N 60% when compared to the reference agonist, CCh. It should be noted, however, that this value was obtained using CHO-K1 cells overexpressing recombinant receptors; an experimental system that might cause a certain overestimation of partial agonist efficacy. Experiments in human saphenous veins of patients with chronic venous disease (C2 Ep As Pr, according to Clinical-Etiology-Anatomy-Pathophysiology [CEAP] classification) have previously shown ACh-induced endothelium-dependent relaxation in the order of 13% compared to control subjects (Carrasco et al., 2010). In preliminary experiments (n = 5), we also evaluated the expression of muscarinic receptors on human saphenous vein preparations using qPCR. These experiments indicated that hM3 receptors are more highly expressed than hM1 (at least twofold higher; data not shown). In the second part of the study, we used an in vivo HCP model to further evaluate the potential impact of Ruscus extract's muscarinic properties on its effects on microcirculation. The HCP model was first described for the purposes of studying microcirculation in the late 1940s and early 1950s (Fulton et al., 1946; Fulton et al., 1947; Lutz and Fulton, 1954) and has since been used in a wide range of studies on microcirculation physiology and microvascular responses. The microvascular bed prepared using the HCP has intact nervous and blood supplies, and is highly vascular with arterial and venous vessels that run parallel to each other down to the level of the small arterioles (all classes of microcirculatory vessels can usually be seen in the microscopic field). The preparation is thus suitable for observing the effects of different drugs on the microvasculature, which are either given orally or added to the superfusion solution (topical application). In the present study, systemic administration of Ruscus extract induced venular constriction in the HCP microcirculation, but did not significantly change mean arteriolar diameter (see Fig. 3). To the best of our knowledge this is the only extract that shows a selective action on arterioles and venules of the microcirculation. The lack of arteriolar constriction is beneficial to the capillary circulation, which is able to keep its blood supply constant. The observed venoconstriction could have resulted from (a) activation of both post-junctional α1- and α2-adrenergic receptors, (b) release of endogenous norepinephrine from adrenergic nerve endings, and/or (c) direct action on venous smooth muscle as described previously for isolated cutaneous veins (Marcelon et al., 1983;

Fig. 7. Effects of different oral doses of Ruscus extract (given twice a day for 2 weeks) combined with different concentrations of atropine applied topically on macromolecular permeability increase induced by topical application of histamine. Observation in the hamster cheek pouch microcirculation (n = 6 hamsters). ⁎p b 0.05; ⁎⁎p b 0.01 and ⁎⁎⁎p b 0.001.

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Fig. 8. Effects of different oral doses of Ruscus extract (given twice a day for 2 weeks) on macromolecular permeability increase induced by ischemia/reperfusion. Observation in the hamster cheek pouch microcirculation (n = 6 hamsters). ⁎p b 0.05; ⁎⁎p b 0.01 and ⁎⁎⁎p b 0.001.

Marcelon and Vanhoutte, 1988; Rubanyi et al., 1984; Harker et al., 1988). These results obtained ex vivo were confirmed in vivo (Bouskela et al., 1993a; Bouskela et al., 1994a). The difference between the responses of arterioles and venules after administration of Ruscus extract may be explained by an increased release of EDRF in the arterioles (Miller et al., 1991), which probably overrides its vasoconstrictor properties. This hypothesis received further support with the observation that topical application of atropine to HCP of animals treated with Ruscus extract elicited arteriolar constriction (Fig. 4). We performed detection of macromolecular permeability in this study using FITC-dextran as a tracer for the following reasons: it is available at varying molecular weights, it is detectable under the microscope at lower concentrations than other vital dyes (Svensjö et al., 1978), the permeability-tracing properties of dextrans are known (Arturson and Granath, 1972), binding of the fluorescent chromophore to dextran is stable (de Belder and Granath, 1973), it appears to have the same properties as unlabeled dextrans (Schroder et al., 1976) and plasma proteins do not bind to it. While the constant flow of superfusion solution continuously removes water-soluble compounds such as FITC-dextran from tissues, localizing extravasation from areas of the microcirculation is nonetheless possible and these macromolecular leakage sites were at post-capillary venules (Persson and Svensjö, 1985). We measured macromolecular permeability using the number of such leakage sites per unit area. It should be noted, however, that not all leakage sites are

detectable by the human eye and FITC-dextran may be removed before the concentration is high enough to be detected. Previous macromolecular permeability studies in the HCP model using FITC-dextran have demonstrated that histamine produces an immediate dose-related increase in the number of leakage (=leaks) sites in venules 9–16 μm in diameter (Björk et al., 1984). The number of leaks per square centimeter correlates with the fluorescent light intensity from the same proportion as measured with a photomultiplier on top of the microscope (Ley and Arfors, 1982). The number of leaks per square centimeter also correlates with the amount of FITC-dextran eliminated by the superfusing buffer for 30 min after stimulation with histamine (Björk et al., 1984). Electron micrographs have shown that leaks occur between adjacent endothelial cells due to separation of loose junctions in the post-capillary venules which creates a gap approximately 1 μm in diameter (Hulström and Svensjö, 1979). Histamine stimulates endothelial cells via specific receptors, although the mechanism by which it induces their separation is not completely understood. There is evidence, however, to suggest that the separation mechanism may involve actomyosin-like contractile elements (Haddy et al., 1972) and Ca++ (Mayhan and Joyner, 1984). An increase in macromolecular permeability due to widened gaps between contracted endothelial cells of the post-capillary venules is an important event in inflammatory reactions and edema formation (Persson and Svensjö, 1985). Our results have shown that Ruscus extract protects against both histamine- and I/ R-induced leakage of FITC-dextran in the HCP model, and this is in agreement with previous findings in the literature (Bouskela et al., 1993b; Bouskela et al., 1994b; Rudofsky, 1984; Lozes and Boccalon, 1984). The microcirculatory inflammatory variables analyzed in this study, including leukocyte rolling/adhesion and macromolecular permeability, were assessed in the post-capillary venules. This is because I/R injury reduces endothelium-dependent relaxation and triggers an intense inflammatory response in the post-capillary venules. Moreover, under an acute stimulus such as 30 min of ischemia, leukocytes tend to roll and adhere on venular (not arteriolar) endothelia, and endothelial barrier disruption due to increased microvascular permeability occurs preferentially in the post-capillary venules (Carden and Granger, 2000; de Souza et al., 2015). I/R injury is associated with leukocyte-endothelial cell interactions that include: rolling of leukocytes along the endothelium, firm adhesion of leukocytes to the endothelium (i.e. ‘sticking’), and transmigration of leukocytes through the endothelium (Carden and Granger, 2000; de Souza et al., 2015). In the present study, Ruscus extract resulted in a

Fig. 9. Effects of different oral doses of Ruscus extract (given twice a day for 2 weeks) combined with different concentrations of atropine applied topically on macromolecular permeability increase induced by 30 min ischemia followed by reperfusion. Observation in the hamster cheek pouch microcirculation (n = 6 hamsters). ⁎p b 0.05; ⁎⁎p b 0.01 and ⁎⁎⁎p b 0.001.

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Fig. 10. Effects of different oral doses of Ruscus extract (given twice a day for 2 weeks) on macromolecular permeability increase induced by ischemia/reperfusion. Observation on the hamster cheek pouch microcirculation (n = 6 hamsters). ⁎p b 0.05; ⁎⁎p b 0.01 and ⁎⁎⁎p b 0.001.

dose-dependent reduction in I/R-induced leukocyte rolling and sticking in the HCP microcirculation. In addition, the macromolecular permeability increase induced by I/R was reduced by Ruscus extract. The results also showed that these effects can be blocked by atropine, indicating that muscarinic receptors play a role in their mediation. It is now accepted that patients with varicose veins have microvascular endothelial dysfunction (Klonizakis et al., 2003; Klonizakis et al., 2006; Li et al., 2013; Virgini-Magalhaes et al., 2006) that apparently persists following surgery (Klonizakis et al., 2006). Venous blood stasis observed in patients with chronic venous disease limits the oxygen supply to venular endothelial cells, which form a barrier between blood and vascular tissue. Hypoxia leads to endothelial cell activation and the release of chemotactic and mitogenic factors (Smith, 2006). Our results in the HCP showing that Ruscus extract reduces macromolecular permeability (induced by either histamine or I/R) and I/R-related leukocyteendothelium interaction, strongly suggest that its action can improve the endothelial dysfunction observed in chronic venous disease.

indicated by our results showing that the muscarinic antagonist atropine blocks several of its effects, namely the reduction of macromolecular permeability (induced by either histamine or I/R) and I/R-related leukocyteendothelium interaction. Therefore, our results suggest that the activity of Ruscus at muscarinic receptors may contribute to its vasoprotective and anti-inflammatory therapeutic effects.

5. Conclusion

Acknowledgments

Our results have shown for the first time that Ruscus extract has binding affinity for muscarinic receptors and also behaves as an efficacious partial agonist, particularly of M1 and M3 receptor subtypes. Moreover, the muscarinic properties of Ruscus seem to have an impact in vivo, as

We would like to thank Mr. Claudio Natalino Ribeiro and Mr. Paulo José Ferreira Lopes for animal care, Marie-Christine Ailhaud and Amélie Tourette for in vitro experiments. We also thank James Kistler (Scinopsis, France) for editorial assistance with the manuscript.

Financial support The study was supported by a grant from Pierre Fabre Laboratories (1301158). Contributions FLA and EB designed the study; IRL, PH and DC performed and wrote the in vitro part; FZGA performed the in vivo part and EB wrote and the in vivo part and the final version of the manuscript.

Fig. 11. Effects of different oral doses of Ruscus extract (given twice a day for 2 weeks) combined with different concentrations of atropine applied topically on the number of rolling leukocytes induced by 30 min ischemia followed by reperfusion. Observation in the hamster cheek pouch microcirculation (n = 6 hamsters). ⁎p b 0.05; ⁎⁎p b 0.01 and ⁎⁎⁎p b 0.001.

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Fig. 12. Effects of different oral doses of Ruscus extract (given twice a day for 2 weeks) combined with different concentrations of atropine applied topically on the number of adherent leukocytes induced by 30 min ischemia followed by reperfusion. Observation in the hamster cheek pouch microcirculation (n = 6 hamsters). ⁎p b 0.05; ⁎⁎p b 0.01 and ⁎⁎⁎p b 0.001.

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