Blockade of cannabinoid receptors reduces ... - Springer Link

Inflamm. Res. (2013) 62:811–821 DOI 10.1007/s00011-013-0638-8

Inflammation Research

ORIGINAL RESEARCH PAPER

Blockade of cannabinoid receptors reduces inflammation, leukocyte accumulation and neovascularization in a model of sponge-induced inflammatory angiogenesis Rodrigo Guabiraba • Remo C. Russo • Amanda M. Coelho • Moˆnica A. N. D. Ferreira Gabriel A. O. Lopes • Ariane K. C. Gomes • Silvia P. Andrade • Luciola S. Barcelos • Mauro M. Teixeira

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Received: 9 March 2013 / Accepted: 16 May 2013 / Published online: 31 May 2013 Ó Springer Basel 2013

Abstract Objective Angiogenesis depends on a complex interaction between cellular networks and mediators. The endocannabinoid system and its receptors have been shown to play a role in models of inflammation. Here, we investigated whether blockade of cannabinoid receptors may interfere with inflammatory angiogenesis. Materials and methods Polyester-polyurethane sponges were implanted in C57Bl/6j mice. Animals received doses (3 and 10 mg/kg/daily, s.c.) of the cannabinoid receptor antagonists SR141716A (CB1) or SR144528 (CB2). Implants were

Responsible editor: Michael J. Parnham. R. Guabiraba (&) R. C. Russo A. M. Coelho L. S. Barcelos M. M. Teixeira (&) Departamento de Bioquı´mica e Imunologia-ICB, Universidade Federal de Minas Gerais (UFMG), Av. Antoˆnio Carlos, 6627. Pampulha, 31270-901 Belo Horizonte, MG, Brazil e-mail: [email protected] M. M. Teixeira e-mail: [email protected] R. C. Russo A. M. Coelho G. A. O. Lopes A. K. C. Gomes S. P. Andrade L. S. Barcelos Departamento de Fisiologia e Biofı´sica-ICB, Universidade Federal de Minas Gerais (UFMG), Av. Antoˆnio Carlos, 6627. Pampulha, 31270-901 Belo Horizonte, MG, Brazil M. A. N. D. Ferreira Departamento de Patologia Geral-ICB, Universidade Federal de Minas Gerais (UFMG), Av. Antoˆnio Carlos, 6627. Pampulha, 31270-901 Belo Horizonte, MG, Brazil R. Guabiraba Institute of Infection, Immunity and Inflammation, Glasgow Biomedical Research Centre, University of Glasgow, 120 University Place, Glasgow G12 8TA, UK

collected at days 7 and 14 for cytokines, hemoglobin, myeloperoxidase, and N-acetylglucosaminidase measurements, as indices of inflammation, angiogenesis, neutrophil and macrophage accumulation, respectively. Histological and morphometric analysis were also performed. Results Cannabinoid receptors expression in implants was detected from day 4 after implantation. Treatment with CB1 or CB2 receptor antagonists reduced cellular influx into sponges at days 7 and 14 after implantation, although CB1 receptor antagonist were more effective at blocking leukocyte accumulation. There was a reduction in TNF-a, VEGF, CXCL1/KC, CCL2/JE, and CCL3/MIP-1a levels, with increase in CCL5/RANTES. Both treatments reduced neovascularization. Dual blockade of cannabinoid receptors resulted in maximum inhibition of inflammatory angiogenesis. Conclusions Blockade of cannabinoid receptors reduced leukocyte accumulation, inflammation and neovascularization, suggesting an important role of endocannabinoids in sponge-induced inflammatory angiogenesis both via CB1 and CB2 receptors. Keywords Cannabinoids Angiogenesis Inflammation Leukocytes Chemokines Cytokines

Introduction The development of new vessels requires a close interaction between cells, extracellular matrix components and soluble factors, and is tightly controlled through the balance between angiogenic and angiostatic factors [1, 2]. A wide range of molecules, including adhesion molecules, growth factors and chemokines, control angiogenesis by acting directly on endothelial cells, or indirectly via

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accessory cells such as monocytes and neutrophils, which in turn secrete angiogenic molecules [3–5]. Angiogenesis occurs in adult tissues in physiologic and pathologic conditions, including inflammation and tumor progression [6, 7]. Both in vitro and in vivo models have been used to characterize the mechanisms involved in angiogenesis and cell trafficking in order to develop drugs and strategies to control or prevent diseases. In one in vivo model, the subcutaneous implantation of a sponge induces a chronic granulomatous response including an intense angiogenesis and accumulation of inflammatory cells [8]. This model has been used to study the biology of several mediators (i.e. cytokines, growth factors, and chemokines) involved in the vascular and inflammatory aspects of granuloma formation in vivo [9–13]. The endocannabinoid system comprises two known cannabinoid receptor subtypes, CB1 and CB2 [14, 15] and a number of endogenous ligands (endocannabinoids) including the compounds anandamide (from the Sanskrit word ananda, which means ‘‘bliss, delight’’) and 2-arachidonoyl glycerol (2-AG) [16–18]. The CB1 receptor is abundantly expressed in the brain [14, 19], and the CB2 receptor is predominantly expressed in several types of inflammatory cells and immune competent cells such as B lymphocytes, natural killer (NK) cells and macrophages/ monocytes [15, 20]. Although anandamide has not been consistently shown to have pro-inflammatory effects, mainly because of its minor role in the periphery, 2-AG is able to induce the migration of HL-60 cells differentiated into macrophage-like cells and human peripheral blood monocytes by acting on the CB2 receptor [21]. Several investigators have also reported that endocannabinoids induce the migration of mouse splenocytes [22]. 2-AG may induce the production of chemokines [23] and rapid actin polymerization [24] in HL-60 cells. Oka and colleagues showed that 2-AG, mainly through the CB2 receptor, stimulates inflammatory reactions and allergic responses by inducing robust adhesion of leukocytes to vascular endothelial cells [25]. In addition, other non-hematopoietic cells, such as endothelial cells, can respond to ligands of CB receptors. Anandamide initiated a transient Ca2 ? elevation through phospholipase C activation that was prevented by the CB2 receptor antagonist SR144528 [26]. Moreover, human microvascular endothelial cells (HMVECs) stimulated with an endocannabinoid-like N-arachidonoyl serine (ARA-S), a CB1 and CB2 agonist, induced angiogenesis and endothelial wound healing in vitro through induction of VEGF [27]. Collectively, these data suggest that agonists of CB1 and CB2 receptors act as powerful pro-angiogenic stimuli for endothelial cells. Cannabinoid receptors represent a new endogenous and complex signaling system that can be targeted pharmacologically for the inhibition of cancer growth and

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neovascularisation, suggesting the use of endocannabinoidbased drugs as anticancer drugs [28]. Regarding some of the most used cannabinoid receptor antagonists, SR141716A (RimonabantÒ) may not only work as an antagonist of cannabinoid-mediated effects but also as an inverse agonist, by blocking CB1 constitutive activity and transduction pathways downstream tyrosine-kinase receptors [29]. Indeed, inhibition of the MAPK and Akt pathways is a possible molecular mechanism for the antiinflammatory and anti-angiogenic effects or SR141716A. Moreover, SR144528 may also act as an inverse agonist in CB2 receptors in the periphery, although the in vivo relevance of these findings is yet to be elucidated [30–32]. It is not well established whether substances capable of targeting the endocannabinoid system can interfere at multiple levels with inflammatory angiogenesis in vivo. Here, we have investigated the effects of antagonists of CB1 and CB2 used alone or in combination on the associated neovascularization and leukocyte accumulation in a model of sponge-induced inflammatory angiogenesis.

Materials and methods Animals Eight to ten-week-old male C57Bl/6 J mice (WT), weighing 18–22 g were obtained from Centro de Bioterismo (CEBIO) of the Universidade Federal de Minas Gerais (UFMG). After sponge implantation, animals were maintained in individual cages with food/water ad libitum and in a controlled environment (temperature and humidity) in the Laboratory of Angiogenesis at the Departamento de Fisiologia e Biofisica (UFMG, Brazil). Ethics Statement This study was carried out in strict accordance with the Brazilian Government’s ethical and animal experiments regulations (COBEA). The experimental protocol was approved by the Committee on the Ethics of Animal Experiments of the Universidade Federal de Minas Gerais (CETEA/UFMG, protocol 147/06). All surgery was performed under controlled procedure, and all efforts were made to minimize suffering. Preparation of cannulated sponge discs and implantation Polyether-polyurethane sponge discs 5 mm thick and 8 mm diameter (Vitafoam, Manchester, UK) were used as the matrix for fibrovascular tissue growth. Then, 12-mm polyvinyl tubing (PE 20, Biovida, Brazil) was secured with silk sutures (Ethicon, UK) to the center of each disc in such

Cannabinoid receptors in inflammatory angiogenesis

a way that the tube was perpendicular to the disc face. The open-end cannula was sealed with removable plugs. The cannulated sponge discs were soaked overnight in 70 % v/v ethanol and sterilized by boiling in distilled water for 15 min before the implantation surgery. The animals were anesthetized (Tribromoethanol, 2.5 % v/v, 1 ml/100 g body weight, i.p.; Sigma), the dorsal hair shaved and the skin wiped with 70 % ethanol. The cannulated sponge discs were aseptically implanted into a subcutaneous pouch, which had been made with curved artery forceps through a 1-cm-long dorsal mid-line incision. The cannula was exteriorized through a small incision in a subcutaneous neck pouch. Mice were allowed to recover under controlled temperature and easy access to soft food and water until 24 h after the procedure. Experimental protocol for CB1 (SR141716A) and CB2 (SR144528) receptor antagonists treatment Mice received subcutaneous treatment with SR141716A (RimonabantÒ, 3 or 10 mg/Kg/day), a CB1 receptor antagonist, SR144528 (3 or 10 mg/Kg/day), a CB2 receptor antagonist or vehicle (PBS/Cremophor EL/DMSO 18:1:1, 200 ll/animal/day). Both compounds were kindly donated by Sanofi-Aventis, France. The vehicle of choice was utilized in previous studies, where DMSO was the main solvent. Cremophor EL helps to keep highly lipophilic compounds in suspension, without presenting any central or peripherical effects in rodents at the concentration used in the present manuscript [33]. The chosen doses for both antagonists (3 to 10 mg/kg, daily or weekly) and the administration route (s.c.) proved to be effective in reducing leukocyte activation and inflammation in different mouse and rat models of disease in a dose-dependent manner [34– 39]. The antagonists were given 30 min before the sponge implantation (considered day 0) and daily thereafter until the day before the excision of the sponge. For the double blockade of cannabinoid receptors, both antagonists were given together at the same dose and route as specified above. The effects of treatment on angiogenesis, neutrophil and macrophage accumulation and cytokine/chemokines production were assessed on days 7 and 14 after sponge implantation. These time points have been shown to be optimal to determine angiogenesis/cell influx and cytokine/ chemokine levels in the sponges [11–13]. Quantification of angiogenesis by hemoglobin measurement Animals were anaesthetized and killed by cervical dislocation and sponge implants were carefully excised, released from the cannula, and weighed. Each implant was homogenized (Tekmar TR-10, Ohio, USA) in 2.0 ml of

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Drabkin’s Reagent (Labtest, Sa˜o Paulo, Brazil) and centrifuged at 10,000g for 15 min. The supernatants were filtered through a 0.22-lm filter (Millipore). Hemoglobin in the samples was quantified colorimetrically at 540 nm in a spectrophotometer (E max-Molecular Devices). The concentration of hemoglobin was calculated from a known amount of hemoglobin assayed in parallel. The results were expressed as lg Hb/ml/mg of wet tissue. Previous studies have shown that hemoglobin detection correlated well with other methods for the detection and quantification of angiogenesis in tissue [40, 41]. Morphometric analysis and blood vessel quantification To examine the degree of neovascularization in the implants of control (vehicle) and SR141716A (RimonabantÒ) or SR144528 (10 mg/kg/day) treated mice, a total of six sponge discs (three for each group) was harvested and stained with Hematoxylin-Eosin. Microscopic images of cross-sections (5 lm) were analyzed with a reticulum inserted in one eyepiece of a binocular microscope (Olympus CHK, Japan) at 4009 magnification (0.053 mm2 per view field). The images were digitized through a micro camera Optronics DEI-470 (Optronics, CA, USA) and processed through the software Image-Pro Plus (v.4.5.1.22; Media Cybernetics). A countable microvessel was defined as a structure with a lumen that contained red blood cells or not as described by [42]. The results were expressed as mean ± SEM of the total number of vessels/view field.

Quantification of neutrophil or macrophage tissue accumulation Pellets from centrifugation of sponge homogenates (see hemoglobin measurement method) were divided into two portions and suspended with different buffers specific for measurement of myeloperoxidase (MPO) or N-acetylglucosaminidase (NAG) activities used as neutrophil and macrophage accumulation indexes, respectively, as described previously [13].

RNA isolation and quantitative PCR RNA was prepared from frozen sponge samples removed at days 4, 7, and 14 after implantation using Trizol separation and RNeasy mini-kits purification following manufacturers’ instructions (Qiagen, Hilden, Germany). Real-time RT-PCR was performed on an ABI PRISM 7900 sequence-detection system (Applied Biosystems, Warrington, UK) by using SYBR Green PCR Master Mix (Applied Biosystems) after a reverse transcription reaction of 1 lg of RNA by using

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M-MLV reverse transcriptase (Promega, Madison, WI, USA). The relative level of gene expression was determined by the comparative threshold cycle method as described by the manufacturer. Levels of cb1 or cb2 gene expression were normalized to those of the housekeeping gene (ribosomal 18s) using the 2-DDCt method and expressed in the graphs as ‘‘relative expression’’. The following primer pairs were used: mouse cb1: 50 -GTACCATCACCACAGACCTCC TC-30 (forward), 50 -GGATTCAGAATCATGAAGCACT CCA-30 (reverse) and mouse cb2: 50 -CCGGAAAAGA GGATGGCAATGAAT-30 (forward) and 50 -CTGCTGAGC GCCCTGGAGAAC-30 (reverse). ELISA for cytokines/chemokines The supernatants from centrifugation of sponge homogenates (see hemoglobin measurement method) were used to examine the levels of VEGF, TNF- a, CXCL1/KC, CCL2/ JE, CCL3/MIP-1a and CCL5/RANTES produced in sponge implants by enzyme-linked immunosorbent assay (ELISA). The assays were performed using Kits from R&D systems and according to the manufacturer’s instructions. The threshold of sensitivity for each cytokine/chemokine was 7.5 pg/ml.

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Results Early expression of CB1 and CB2 receptors in sponge implants during inflammatory angiogenesis is associated to leukocyte infiltration We observed that from 4 to 14 days after implantation, there was detectable expression of both CB1 and CB2 receptors in sponge implants of mice (Fig. 1a, b). The relative expression of CB1 in the implants was higher than that of CB2 receptors in the given time points (at least 1 log difference). The expression of CB1 and CB2 receptors was already maximal at day 4 after implantation, remained elevated at day 7 and decreased at day 14. Expression of CB2 expression decreased more drastically than that of CB1 receptors (Fig. 1b). Neutrophil accumulation, as assessed by measuring MPO, increased from days 4 to 7 and decreased thereafter in the implants (Fig. 1c). Macrophage accumulation (Fig. 1d) and hemoglobin content (Fig. 1e) increased linearly and peaked at day 14 after implantation. Together, these data suggested that CB receptors are induced during sponge-induced implantation and peak expression preceded the influx of leukocytes. CB1 or CB2 receptor blockade reduced the development of sponge-induced inflammatory angiogenesis

Statistical analysis Results are presented as the mean ± SEM. Comparisons between two groups were carried out using Student’s t test for unpaired data. For three or more groups, comparisons were carried out using one-way analysis of variance (ANOVA) followed by Student–Newman–Keuls post hoc analysis. A P value \0.05 was considered significant.

Fig. 1 Kinetics of expression of cb1 and cb2 genes, leukocyte accumulation and vascularization in sponge implants. Polyetherpolyurethane sponge discs were aseptically implanted into a subcutaneous pouch in WT C57Bl/6 mice and surgically removed at days 4, 7 and 14 after implantation. a Relative expression of CB1 receptor and b CB2 receptor was evaluated using specific primers targeting the cb1 and cb2 genes, respectively, and normalized to those of the

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To examine the role of endogenous cannabinoids in inflammatory angiogenesis, leukocyte accumulation was evaluated in sponges implanted in SR141716A-treated (CB1 receptor antagonist), SR144528-treated (CB2 receptor antagonist) or vehicle-treated wild-type mice (WT) at days 7 and 14 post implantation. There was a dose-dependent reduction in neutrophil accumulation in SR141716A-treated

housekeeping gene (ribosomal 18 s). c Neutrophil and d macrophage accumulation in implants were measured by myeloperoxidase (MPO) and N-acetylglucosaminidase (NAG) activities, respectively. e Angiogenesis in implants was measured by hemoglobin. Results represent the mean ± SEM of 5–8 animals for each group and are representative of at least three experiments


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Fig. 2 Leukocyte accumulation in the sponge implants of SR141716A (CB1)- or SR144528 (CB2)-treated mice. SR141716A (CB1 antagonist) or SR144528 (CB2 antagonist), 3–10 mg/kg/day, and vehicle were given subcutaneously as described in ‘‘Materials and methods’’. a Neutrophil and b macrophage influx were evaluated at days 7 and 14 after sponge implantation by measuring MPO and NAG activities, respectively, in the implants. ELISA was performed in the

implants for c CXCL1/KC and d VEGF concentrations. Results represent the mean ± SEM of 5–8 animals for each group and are representative of at least three experiments. Statistical analysis was assessed using one-way analysis of variance (ANOVA) followed by Student–Newman–Keuls post hoc analysis. *P \ 0.05, **P [ 0.01, ***P \ 0.001 when compared to vehicle, #P \ 0.05 when comparing treated groups

mice in days 7 and 14, while SR144528 treatment was only able to reduce this parameter at day 7 (Fig. 2a). Macrophage accumulation (NAG activity) into sponges was reduced only by SR141716A treatment (Fig 2b) in a dose-dependent manner. Thus, our data suggest that inflammatory cell accumulation in the implant is controlled by endogenous cannabinoids by acting especially on the CB1 receptor.

day 7, with a strong effect on pro-angiogenic cytokines at day 14, including CXCL1/KC (Fig. 2c), TNF-a and CCL2/JE (Fig. 3a, b). Treatment with SR144528, a CB2 receptor antagonist, also reduced the production of cytokines and chemokines at day 7, but had a less pronounced inhibitory effect at day 14. SR144528 treatment significantly inhibited the production of VEGF at day 14 in a dose-dependent manner (Fig. 2d). Interestingly, levels of CCL5/RANTES were significantly increased in the sponges of SR141716A- or SR144528-treated mice at day 14 (Fig. 3d). Overall, effects of the CB1 receptor antagonist were more pronounced and sustained. The antagonist of CB2 receptor had significant effects at day 7 which were lost thereafter. However, there was more sustained inhibition of the pro-angiogenic molecule VEGF.

CB1 or CB2 receptor blockade reduced cytokine and chemokine production in implants The production of cytokines and chemokines in the sponge matrix can drive both angiogenic and inflammatory processes [11–13, 43]. To gain insight into potential mechanisms of action of the cannabinoid receptor antagonists on spongeinduced inflammatory angiogenesis, we examined whether the antagonists SR141716A and SR144528 altered the levels of these mediators. The levels of the pro-angiogenic molecules CXCL1/KC and VEGF (Fig. 2c, d) and TNF-a, CCL2/ JE, CCL3/MIP-1a and CCL5/RANTES (Fig. 3a–d) were detected in the sponge of vehicle-treated animals at days 7 and 14. Treatment with SR141716A, the CB1 receptor antagonist, reduced the level of all cytokines and chemokines evaluated at

Treatment with cannabinoid receptor antagonists reduced neovascularization To evaluate the effects of SR141716A (CB1) or SR144528 (CB2) on neovascularization, hemoglobin was measured and histological and morphometric analysis of implants was assessed at days 7 and 14 after sponge implantation. Angiogenesis, as assessed by hemoglobin content, was reduced

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Fig. 3 Production of cytokines and chemokines in sponge implants upon treatment with SR141716A (CB1) or SR144528 (CB2). SR141716A (CB1 antagonist) or SR144528 (CB2 antagonist), 10 mg/kg/day, and vehicle were given subcutaneously as described in ‘‘Materials and methods’’. ELISA was carried out in the implants at day 7 and 14 for a TNF-a, b CCL2/JE, c CCL3/MIP-1a and d CCL5/

RANTES concentrations. Results represent the mean ± SEM of 5–8 animals for each group and are representative of at least three experiments. Statistical analysis was assessed using one-way analysis of variance (ANOVA) followed by Student–Newman–Keuls post hoc analysis. *P \ 0.05, **P \ 0.01, ***P \ 0.001 when compared to vehicle, #P \ 0.05, ###P \ 0.001 when comparing treated groups

Fig. 4 Effects of cannabinoid receptor blockade in neovascularization. SR141716A (CB1 antagonist) or SR144528 (CB2 antagonist), 3–10 mg/kg/day, and vehicle were given subcutaneously as described in ‘‘Materials and methods’’. Angiogenesis in implants was measured by a hemoglobin concentration and b number of blood vessels. Morphometric analyses for blood vessels were performed in samples

from 10 mg/kg treated-mice. Results represent the mean ± SEM of 5–8 animals for each group and are representative of at least three experiments. Statistical analysis was assessed using one-way analysis of variance (ANOVA) followed by Student–Newman–Keuls post hoc analysis. *P \ 0.05, **P \ 0.01, ***P \ 0.001 when compared to vehicle

by SR141716A or SR144528 treatment at both 7 and 14 days after sponge implantation (Fig. 4a). Histological evaluation of the implants concurred with the biochemical findings. Mice given the CB1 or CB2 antagonists also exhibited significant reduction of neovascularization, as assessed by the number of blood vessels in the implants (Fig. 4b). Overall, CB1 or CB2 receptor antagonist-treated mice showed decreased number of congested blood vessels, less cellular accumulation (more consistent in mice treated

with CB1 antagonists) and fibrovascular tissue growth and (Fig. 5, black arrows).

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Double blockade of cannabinoid receptors reduced inflammatory angiogenesis The inhibitory effects of CB1 and CB2 were similar in magnitude but appeared to differ in mechanisms, as seen by the differential effects on leukocyte accumulation and


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Fig. 5 Histological evaluation of sponge implants upon treatment with SR141716A (CB1) or SR144528 (CB2). SR141716A (CB1 antagonist) or SR144528 (CB2 antagonist), 10 mg/kg/day, and vehicle were given subcutaneously as described in ‘‘Materials and

methods’’. Sponge implants stained with hematoxylin and eosin (H&E) were evaluated at a day 7 and b day 14. Black arrows: areas of congested blood vessels, cellular accumulation and fibrovascular tissue growth. SM: sponge matrix 4009 magnification

levels of inflammatory and angiogenic mediators. We then tested the effects of combined daily treatment with SR141716A (CB1) and SR144528 (CB2). There was significant reduction of neovascularization at days 7 and 14 in mice treated with the CB1 ? CB2 antagonists as compared with vehicle-treated mice (Fig. 6a). Overall, inhibition of neovascularization was slightly greater than that of animals treated with either drug alone at day 14 (% inhibition at day 7: 45 % for CB1; 51 % for CB2 and 45 % CB1 ? CB2.; % inhibition at day 14: 43 % CB1; 38 % CB2 and 51 % for CB1 ? CB2). Double blockade of CB1 and CB2 receptors reduced VEGF production (Fig. 6b), neutrophil accumulation (Fig. 6c) and CXCL1/KC production (Fig. 6d). Interestingly, double blockade of CB1 and CB2 receptors also led to an increase in CCL5/RANTES production (Fig. 6e). These results were similar to the maximal effect observed for each parameter when the antagonists were given alone to mice.

conditions [36, 38, 44–47]. However, the role of the endocannabinoid signaling in inflammatory angiogenesis remains unclear. In the present work, we showed that: (1) CB1 and CB2 receptors are expressed during inflammatory angiogenesis in sponge implants; (2) treatment with CB1 or CB2 receptor antagonists led to significant inhibition of neovascularization that was of similar intensity for both antagonists when given alone; (3) this is associated with a reduction of inflammatory cell recruitment by CB1 receptor antagonists; (4) inhibition of cytokine and chemokine levels by CB1 antagonist is in general more sustained than that of CB2 antagonists, but CB2 antagonists have a more prolonged inhibitory effect on VEGF levels; and (5) double blockade of both cannabinoid receptors led to slightly enhanced inhibition of angiogenesis at day 14 after implantation. Expression of CB1 receptor has been shown to be significantly increased during monocyte/macrophage differentiation in experimental atherosclerosis [48]. CB1 receptor blockade by SR141716A increased intracellular cAMP levels and reduced number of circulating neutrophils and monocytes, as well as MCP-1 (a mouse analog of CCL2/JE), IL-8 (a mouse analog of CXCL1/KC) and TNFa in these models [39, 48, 49]. In the present model, only the CB1 antagonist SR141715A was able to reduce

Discussion Endogenous and exogenous cannabinoid receptor agonists are involved in leukocyte activation and cytokine production in different in vitro and in vivo inflammatory

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Fig. 6 Effects of CB1 and CB2 receptor blockade in inflammatory angiogenesis. SR141716A (CB1 antagonist) and SR144528 (CB2 antagonist), 10 mg/kg/day, were given together subcutaneously as described in ‘‘Materials and methods’’. Angiogenesis in implants was measured by a hemoglobin concentration and b VEGF concentration by ELISA. c Neutrophil influx was evaluated by measuring MPO

activity, d CXCL1/KC and e CCL5/RANTES production was measured by ELISA. Results represent the mean ± SEM of 5–8 animals for each group and are representative of at least two experiments. Statistical analysis was assessed using one-way analysis of variance (ANOVA) followed by Student–Newman–Keuls post hoc analysis. *P \ 0.05, **P \ 0.01 when compared to vehicle

macrophage accumulation into the implants. Dual blockade of cannabinoid receptors also led to the same observation, suggesting that this effect is mainly CB1 receptor-dependent. Decreased monocyte recruitment appears to be related to a reduction in the levels of pro-angiogenic chemokines, CCL2/JE and CCL3/MIP-1a, which are known to be involved in the recruitment of these leukocytes [11]. Activation of CXCR1/CXCR2 can modulate angiogenesis by directly activating endothelial cells or via stimulation of pro-angiogenic factors by PMNs and other leukocytes [12, 43, 50–52]. Moreover, VEGF is known to be expressed and released by PMNs in vitro [53]. We observed a reduction of neutrophil accumulation, CXCL1/ KC (a CXCR1/CXCR2 ligand) and VEGF production in sponge implants upon CB1 and CB1?CB2 antagonist treatment, but not in mice treated with CB2 receptor antagonist alone. In a model of indomethacin-induced ulcers in rats, SR 141716A prevented gastric damage and the rise in TNF-a levels and myeloperoxidase activity in the gut [39]. Smith and colleagues [36] showed that SR141716A blocked cytokine modulation when coadministered alone to mice in a model of endotoxemia. Moreover, the inhibition of TNF-a production by SR 141716A through the cannabinoid CB1 receptor blockade is of particular interest, since this cytokine plays an important role in inflammatory angiogenesis through the activation of leukocytes and endothelial cells [7, 13, 54–56].

CB1 blockade is also known to contribute to the inhibition of bFGF- and VEGF-induced endothelial cell proliferation [57]. Therefore, reduced pro-inflammatory cytokine production (i.e. TNF-a, CXCL1/KC) may directly or indirectly contribute to the accumulation and activation of neutrophils within the implants, which together with the local production of VEGF may lead to endothelial cells replication and activation. Kishimoto and colleagues [21] showed that 2-arachidonoylglycerol (2AG) induced the recruitment of macrophages/monocytes through a CB2 receptor-dependent mechanism. Although CB2 receptor blockade did not reduce macrophage accumulation into the sponges, it may contribute to reduce cell activation and cytokine/chemokines acting in autocrine/paracrine loop. It has been shown that SR144528 modulated cytokines when injected i.c.v in Corynebacterium parvum-primed endotoxemic mice [44]. Thus, while anti-inflammatory activity through the blockade of CB2 was consistently observed, it remains unclear where and how endogenous CB2 receptor activation may drive inflammation and leukocyte activation in vivo [44, 45]. In our experimental model, the expression of CB2 receptor peaked at day 7, when neutrophil recruitment is maximal, and was strongly reduced at day 14, when vascularization and macrophage accumulation had their peak. CB2 receptor expression was detected in human neutrophils, even if receptor expression pattern in these cells may

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vary among in vitro systems [58–60]. Although indirect, our data suggest that CB2 receptor is involved in acute production of pro-inflammatory cytokines associated to neutrophil activation/accumulation (i.e. TNF-a and CXCL1/KC) and, consequently, favors chronification and neovascularization (i.e. through VEGF production). VEGF release has been shown to be secondary to the influx of polymorphonuclear cells in various models of angiogenesis [61, 62]. We have shown here that CB1?CB2 receptor blockade reduced VEGF and CXCL1/KC production associated with reduced neutrophil influx and hemoglobin content in sponge implant. The inhibition was similar to the maximum effect observed for CB1 or CB2 antagonists alone. Therefore, we suggest that the combined treatment with both cannabinoid receptor antagonists led to a statistically significant reduction in neovascularization and inflammation, highlighting the mutual participation of cannabinoid receptors in inflammatory angiogenesis. However, the detailed molecular and cellular mechanisms of this important mutual regulation by CB1 and CB2 receptors must be further explored. As reported in the literature, endocannabinoids show a biphasic effect dependent on their concentrations in different animal models, which may help explain their different effects over CB1 or CB2 receptor activation and their association with inflammation and angiogenesis [57, 63]. The sponge implantation technique is useful for the study of inflammatory angiogenesis and provides an environment of defined dimensions and reasonably controlled conditions [10–13, 43]. De Filippis and colleagues [64] showed that cannabinoid receptor activation prevented k-carrageenan-induced granuloma formation and angiogenesis through the inhibition of NF-jB activation. Administration of a CB1/CB2 mixed agonist WIN 55,212-2 decreased inflammation and VEGF expression in the granulomatous tissue. A similar approach was used by Solinas and colleagues [65] using matrigel-soaked sponges, but with a focus on neovascularization. The carrageenansoaked sponge model is based on a powerful stimulus which unspecifically triggers the inflammatory cascade very quickly. Importantly, the authors did not discuss any aspect of leukocyte biology in their findings, contrary to our experimental approach. In addition, WIN 55,212-2, a high endocytotic agonist, may induce cannabinoid receptor desensitization involving both caveolae/lipid-rafts- and clathrin-coated-pits-dependent pathway in a concentrationdependent manner [66]. This may occur with different cannabinoid agonists, including cannabidiol (CBD), but the correlation between receptor downregulation and desensitization is not well explained in vivo [67, 68]. These experimental and pharmacological aspects may help explain why De Filippis and colleagues, and Solinas and

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colleagues, found an inhibition of neovascularization or inflammation using CB1 and/or CB2 agonists in a closely related experimental model. CB2 receptor blockade led to a significant increase in the levels of CCL5/RANTES in the implants at day 14, despite a reduction in the level of other chemokines (Fig. 2). Barcelos and colleagues [11] reported that exogenous CCL5/ RANTES reduced angiogenesis in WT mice possibly through CCR5 receptor activation. Treatment of WT mice with the CCR1/CCR5 antagonist, Met-RANTES, prevented neutrophil and macrophage accumulation, but enhanced sponge vascularization [69]. The unexpected anti-angiogenic effect of CCL5 have analogies with chemokine-like proteins encoded by Kaposi’s sarcoma-associated herpes virus (KSHV) [70], that share sequence similarity with human CCL3 and CCL5 and stimulate tumor angiogenesis [71, 72]. In addition, these proteins are anti-inflammatory [73]. Importantly, there is a similar increase in CCL5/ RANTES production upon treatment with CB1?CB2 antagonists (Fig. 6e). It is still difficult to state that this phenomenon is solely due to a CB2-mediated effect in the present experimental setup. Further studies will help define whether this increase would be mostly mediated by CB1 or CB2 receptors. Raborn and colleagues [74] showed that cannabinoids act through the CB2 receptor to transdeactivate migratory responsiveness to CCL5/RANTES in vitro. Moreover, CCL5/RANTES may be induced by IFN-c, that is known to regulate anigogenesis [7]. These data may bring important insights into the biology of chemokines and cannabinoid receptors during angiogenesis. In conclusion, we showed here that cannabinoid receptors presented discrete roles in our experimental model of inflammatory angiogenesis: while CB1 receptor appeared to be strongly involved in leukocyte accumulation and cytokine production, CB2 receptor is mainly linked to a pro-angiogenic response, especially through early neutrophil activation and VEGF production. Double blockade of cannabinoid receptors favored slightly better inhibition of inflammatory angiogenesis. These data reinforce the role of the cannabinoid system in the context of inflammatory angiogenesis. Acknowledgments This study was supported by research grants of Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq, Brazil) and Fundacao do Amparo a Pesquisas do Estado de Minas Gerais (FAPEMIG, Brazil). We are grateful to Prof. Frederico M. Soriani (UFMG) for his helpful technical assistance.

References 1. Bussolino F, Mantovani A, Persico G. Molecular mechanisms of blood vessel formation. Trends Biochem Sci. 1997;22:251–6. 2. Klagsbrun M, Moses MA. Molecular angiogenesis. Chem Biol. 1999;6:R217–24.

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820 3. Brooks PC, Clark RA, Cheresh DA. Requirement of vascular integrin alpha v beta 3 for angiogenesis. Science. 1994;264:569–71. 4. Odedra R, Weiss JB. Low molecular weight angiogenesis factors. Pharmacol Ther. 1991;49:111–24. 5. Wang H, Keiser JA. Vascular endothelial growth factor upregulates the expression of matrix metalloproteinases in vascular smooth muscle cells: role of flt-1. Circ Res. 1998;83:832–40. 6. Carmeliet P, Jain RK. Angiogenesis in cancer and other diseases. Nature. 2000;407:249–57. 7. Naldini A, Carraro F. Role of inflammatory mediators in angiogenesis. Curr Drug Targets Inflamm Allergy. 2005;4:3–8. 8. Lage AP, Andrade SP. Assessment of angiogenesis and tumor growth in conscious mice by a fluorimetric method. Microvasc Res. 2000;59:278–85. 9. Ford HR, Hoffman RA, Wing EJ, Magee DM, McIntyre L, Simmons RL. Characterization of wound cytokines in the sponge matrix model. Arch Surg. 1989;124:1422–8. 10. Andrade SP, Bakhle YS, Hart I, Piper PJ. Effects of tumour cells on angiogenesis and vasoconstrictor responses in sponge implants in mice. Br J Cancer. 1992;66:821–6. 11. Barcelos LS, Coelho AM, Russo RC, Guabiraba R, Souza AL, Bruno-Lima G Jr. et al. Role of the chemokines CCL3/MIP1alpha and CCL5/RANTES in sponge-induced inflammatory angiogenesis in mice. Microvasc Res. 2009;78(2):148–54. 12. Barcelos LS, Talvani A, Teixeira AS, Cassali GD, Andrade SP, Teixeira MM. Production and in vivo effects of chemokines CXCL1-3/KC and CCL2/JE in a model of inflammatory angiogenesis in mice. Inflamm Res. 2004;53:576–84. 13. Barcelos LS, Talvani A, Teixeira AS, Vieira LQ, Cassali GD, Andrade SP, et al. Impaired inflammatory angiogenesis, but not leukocyte influx, in mice lacking TNFR1. J Leukoc Biol. 2005;78:352–8. 14. Matsuda LA, Lolait SJ, Brownstein MJ, Young AC, Bonner TI. Structure of a cannabinoid receptor and functional expression of the cloned cDNA. Nature. 1990;346:561–4. 15. Munro S, Thomas KL, Abu-Shaar M. Molecular characterization of a peripheral receptor for cannabinoids. Nature. 1993;365:61–5. 16. Mechoulam R, Fride E, Di Marzo V. Endocannabinoids. Eur J Pharmacol. 1998;359:1–18. 17. Guindon J, Hohmann AG. The endocannabinoid system and cancer: therapeutic implication. Br J Pharmacol. 2011;163:1447–63. 18. Smith TH, Sim-Selley LJ, Selley DE. Cannabinoid CB1 receptorinteracting proteins: novel targets for central nervous system drug discovery? Br J Pharmacol. 2010;160:454–66. 19. Cristino L, de Petrocellis L, Pryce G, Baker D, Guglielmotti V, Di Marzo V. Immunohistochemical localization of cannabinoid type 1 and vanilloid transient receptor potential vanilloid type 1 receptors in the mouse brain. Neuroscience. 2006;139:1405–15. 20. Galiegue S, Mary S, Marchand J, Dussossoy D, Carriere D, Carayon P, et al. Expression of central and peripheral cannabinoid receptors in human immune tissues and leukocyte subpopulations. Eur J Biochem. 1995;232:54–61. 21. Kishimoto S, Gokoh M, Oka S, Muramatsu M, Kajiwara T, Waku K, et al. 2-arachidonoylglycerol induces the migration of HL-60 cells differentiated into macrophage-like cells and human peripheral blood monocytes through the cannabinoid CB2 receptordependent mechanism. J Biol Chem. 2003;278:24469–75. 22. Jorda MA, Verbakel SE, Valk PJ, Vankan-Berkhoudt YV, Maccarrone M, Finazzi-Agro A, et al. Hematopoietic cells expressing the peripheral cannabinoid receptor migrate in response to the endocannabinoid 2-arachidonoylglycerol. Blood. 2002;99:2786–93. 23. Kishimoto S, Kobayashi Y, Oka S, Gokoh M, Waku K, Sugiura T. 2-Arachidonoylglycerol, an endogenous cannabinoid receptor ligand, induces accelerated production of chemokines in HL-60 cells. J Biochem. 2004;135:517–24.

123

R. Guabiraba et al. 24. Gokoh M, Kishimoto S, Oka S, Mori M, Waku K, Ishima Y, et al. 2-arachidonoylglycerol, an endogenous cannabinoid receptor ligand, induces rapid actin polymerization in HL-60 cells differentiated into macrophage-like cells. Biochem J. 2005;386:583–9. 25. Oka S, Wakui J, Ikeda S, Yanagimoto S, Kishimoto S, Gokoh M, et al. Involvement of the cannabinoid CB2 receptor and its endogenous ligand 2-arachidonoylglycerol in oxazolone-induced contact dermatitis in mice. J Immunol. 2006;177:8796–805. 26. Zoratti C, Kipmen-Korgun D, Osibow K, Malli R, Graier WF. Anandamide initiates Ca(2 ?) signaling via CB2 receptor linked to phospholipase C in calf pulmonary endothelial cells. Br J Pharmacol. 2003;140:1351–62. 27. Zhang X, Maor Y, Wang JF, Kunos G, Groopman JE. Endocannabinoid-like N-arachidonoyl serine is a novel pro-angiogenic mediator. Br J Pharmacol. 2010;160:1583–94. 28. Bifulco M, Di Marzo V. Targeting the endocannabinoid system in cancer therapy: a call for further research. Nat Med. 2002;8:547–50. 29. Bouaboula M, Perrachon S, Milligan L, Canat X, Rinaldi-Carmona M, Portier M, et al. A selective inverse agonist for central cannabinoid receptor inhibits mitogen-activated protein kinase activation stimulated by insulin or insulin-like growth factor 1. Evidence for a new model of receptor/ligand interactions. J Biol Chem. 1997;272:22330–9. 30. Shire D, Calandra B, Bouaboula M, Barth F, Rinaldi-Carmona M, Casellas P, et al. Cannabinoid receptor interactions with the antagonists SR 141716A and SR 144528. Life Sci. 1999;65:627–35. 31. Bouaboula M, Dussossoy D, Casellas P. Regulation of peripheral cannabinoid receptor CB2 phosphorylation by the inverse agonist SR 144528. Implications for receptor biological responses. J Biol Chem. 1999;274:20397–405. 32. Rhee MH, Kim SK. SR144528 as inverse agonist of CB2 cannabinoid receptor. J Vet Sci. 2002;3:179–84. 33. Gelderblom H, Verweij J, Nooter K, Sparreboom A, Cremophor EL, The drawbacks and advantages of vehicle selection for drug formulation. Eur J Cancer. 2001;37:1590–8. 34. Costa B, Trovato AE, Colleoni M, Giagnoni G, Zarini E, Croci T. Effect of the cannabinoid CB1 receptor antagonist, SR141716, on nociceptive response and nerve demyelination in rodents with chronic constriction injury of the sciatic nerve. Pain. 2005;116:52–61. 35. Lim SY, Davidson SM, Yellon DM, Smith CCT. The cannabinoid CB1 receptor antagonist, rimonabant, protects against acute myocardial infarction. Basic Res Cardiol. 2009;104:781–92. 36. Smith SR, Terminelli C, Denhardt G. Effects of cannabinoid receptor agonist and antagonist ligands on production of inflammatory cytokines and anti-inflammatory interleukin-10 in endotoxemic mice. J pharmacol Exp Ther. 2000;293:136–50. 37. Sugamura K, Sugiyama S, Fujiwara Y, Matsubara J, Akiyama E, Maeda H, et al. Cannabinoid 1 receptor blockade reduces atherosclerosis with enhances reverse cholesterol transport. J Atheroscler Thromb. 2010;17:141–7. 38. Conti S, Costa B, Colleoni M, Parolaro D, Giagnoni G. Antiinflammatory action of endocannabinoid palmitoylethanolamide and the synthetic cannabinoid nabilone in a model of acute inflammation in the rat. Br J Pharmacol. 2002;135:181–7. 39. Croci T, Landi M, Galzin A-M, Marini P. Role of cannabinoid CB1 receptors and tumor necrosis factor-alpha in the gut and systemic anti-inflammatory activity of SR 141716 (rimonabant) in rodents. Br J Pharmacol. 2003;140:115–22. 40. Hu DE, Hiley CR, Smither RL, Gresham GA, Fan TP. Correlation of 133Xe clearance, blood flow and histology in the rat sponge model for angiogenesis. Further studies with angiogenic modifiers. Lab Invest. 1995;72:601–10. 41. Machado RD, Santos RA, Andrade SP. Opposing actions of angiotensins on angiogenesis. Life Sci. 2000;66:67–76. 42. Ferreira MA, Barcelos LS, Campos PP, Vasconcelos AC, Teixeira MM, Andrade SP. Sponge-induced angiogenesis and


43.

44.

45.

46.

47.

48.

49.

50.

51.

52.

53.

54.

55.

56.

57. 58.

inflammation in PAF receptor-deficient mice (PAFR-KO). Br J Pharmacol. 2004;141:1185–92. Bertini R, Barcelos LS, Beccari AR, Cavalieri B, Moriconi A, Bizzarri C, et al. Receptor binding mode and pharmacological characterization of a potent and selective dual CXCR1/CXCR2 noncompetitive allosteric inhibitor. Br J Pharmacol. 2012;165:436–54. Smith SR, Terminelli C, Denhardt G. Modulation of cytokine responses in Corynebacterium parvum-primed endotoxemic mice by centrally administered cannabinoid ligands. Eur J Pharmacol. 2001;425:73–83. Smith SR, Denhardt G, Terminelli C. The anti-inflammatory activities of cannabinoid receptor ligands in mouse peritonitis models. Eur J Pharmacol. 2001;432:107–19. Sacerdote P, Massi P, Panerai AE, Parolaro D. In vivo and in vitro treatment with the synthetic cannabinoid CP55, 940 decreases the in vitro migration of macrophages in the rat: involvement of both CB1 and CB2 receptors. J Neuroimmunol. 2000;109:155–63. Massi P, Fuzio D, Vigano D, Sacerdote P, Parolaro D. Relative involvement of cannabinoid CB(1) and CB(2) receptors in the Delta(9)-tetrahydrocannabinol-induced inhibition of natural killer activity. Eur J Pharmacol. 2000;387:343–7. Sugamura K, Sugiyama S, Nozaki T, Matsuzawa Y, Izumiya Y, Miyata K, et al. Activated endocannabinoid system in coronary artery disease and antiinflammatory effects of cannabinoid 1 receptor blockade on macrophages. Circulation. 2009;119:28–36. Schafer A, Pfrang J, Neumuller J, Fiedler S, Ertl G, Bauersachs J. The cannabinoid receptor-1 antagonist rimonabant inhibits platelet activation and reduces pro-inflammatory chemokines and leukocytes in Zucker rats. Br J Pharmacol. 2008;154:1047–54. Strieter RM, Burdick MD, Gomperts BN, Belperio JA, Keane MP. CXC chemokines in angiogenesis. Cytokine Growth Factor Rev. 2005;16:593–609. Russo RC, Guabiraba R, Garcia CC, Barcelos LS, Roffe E, Souza AL, et al. Role of the chemokine receptor CXCR2 in bleomycininduced pulmonary inflammation and fibrosis. Am J Respir Cell Mol Biol. 2009;40:410–21. Li A, Varney ML, Valasek J, Godfrey M, Dave BJ, Singh RK. Autocrine role of interleukin-8 in induction of endothelial cell proliferation, survival, migration and MMP-2 production and angiogenesis. Angiogenesis. 2005;8:63–71. Scapini P, Calzetti F, Cassatella MA. On the detection of neutrophil-derived vascular endothelial growth factor (VEGF). J Immunol Methods. 1999;232:121–9. Robertson M, Liversidge J, Forrester JV, Dick AD. Neutralizing tumor necrosis factor-alpha activity suppresses activation of infiltrating macrophages in experimental autoimmune uveoretinitis. Invest Ophthalmol Vis Sci. 2003;44:3034–41. Belo AV, Leles F, Barcelos LS, Ferreira MA, Bakhle YS, Teixeira MM, et al. Murine chemokine CXCL2/KC is a surrogate marker for angiogenic activity in the inflammatory granulation tissue. Microcirculation. 2005;12:597–606. Vallien G, Langley R, Jennings S, Specian R, Granger DN. Expression of endothelial cell adhesion molecules in neovascularized tissue. Microcirculation. 2000;7:249–58. Pisanti S, Bifulco M. Endocannabinoid system modulation in cancer biology and therapy. Pharmacol Res. 2009;60:107–16. Oka S, Ikeda S, Kishimoto S, Gokoh M, Yanagimoto S, Waku K, et al. 2-arachidonoylglycerol, an endogenous cannabinoid receptor ligand, induces the migration of EoL-1 human eosinophilic leukemia cells and human peripheral blood eosinophils. J Leukoc Biol. 2004;76:1002–9.

821 59. Deusch E, Kraft B, Nahlik G, Weigl L, Hohenegger M, Kress HG. No evidence for direct modulatory effects of delta 9-tetrahydrocannabinol on human polymorphonuclear leukocytes. J Neuroimmunol. 2003;141:99–103. 60. Kurihara R, Tohyama Y, Matsusaka S, Naruse H, Kinoshita E, Tsujioka T, et al. Effects of peripheral cannabinoid receptor ligands on motility and polarization in neutrophil-like HL60 cells and human neutrophils. J Biol Chem. 2006;281:12908–18. 61. McCourt M, Wang JH, Sookhai S, Redmond HP. Proinflammatory mediators stimulate neutrophil-directed angiogenesis. Arch Surg. 1999;134:1325–31. 62. Kasama T, Shiozawa F, Kobayashi K, Yajima N, Hanyuda M, Takeuchi HT, et al. Vascular endothelial growth factor expression by activated synovial leukocytes in rheumatoid arthritis: critical involvement of the interaction with synovial fibroblasts. Arthritis Rheum. 2001;44:2512–24. 63. Malinowska B, Baranowska-Kuczko M, Schlicker E. Triphasic blood pressure responses to cannabinoids: do we understand the mechanism? Br J Pharmacol. 2012;165:2073–88. 64. De Filippis D, Russo A, De Stefano D, Maiuri MC, Esposito G, Cinelli MP, et al. Local administration of WIN 55,212–2 reduces chronic granuloma-associated angiogenesis in rat by inhibiting NF-kappaB activation. J Mol Med (Berl). 2007;85:635–45. 65. Solinas M, Massi P, Cantelmo AR, Cattaneo MG, Cammarota R, Bartolini D, et al. Cannabidiol inhibits angiogenesis by multiple mechanisms. Br J Pharmacol. 2012;167:1218–31. 66. Wu DF, Yang LQ, Goschke A, Stumm R, Brandenburg LO, Liang YJ, et al. Role of receptor internalization in the agonistinduced desensitization of cannabinoid type 1 receptors. J Neurochem. 2008;104:1132–43. 67. Romero J, Berrendero F, Garcia-Gil L, Ramos JA, FernandezRuiz JJ. Cannabinoid receptor and WIN-55,212–2-stimulated [35S]GTP gamma S binding and cannabinoid receptor mRNA levels in the basal ganglia and the cerebellum of adult male rats chronically exposed to delta 9-tetrahydrocannabinol. J Mol Neurosci. 1998;11:109–19. 68. Romero J, Berrendero F, Garcia-Gil L, de la Cruz P, Ramos JA, Fernandez-Ruiz JJ. Loss of cannabinoid receptor binding and messenger RNA levels and cannabinoid agonist-stimulated [35S]guanylyl-50 O-(thio)-triphosphate binding in the basal ganglia of aged rats. Neuroscience. 1998;84:1075–83. 69. Barcelos LS, Coelho AM, Russo RC, Guabiraba R, Souza AL, Bruno-Lima G Jr, et al. Role of the chemokines CCL3/MIP-1 alpha and CCL5/RANTES in sponge-induced inflammatory angiogenesis in mice. Microvasc Res. 2009;78:148–54. 70. Ahuja SK, Gao JL, Murphy PM. Chemokine receptors and molecular mimicry. Immunol Today. 1994;15:281–7. 71. Boshoff C, Endo Y, Collins PD, Takeuchi Y, Reeves JD, Schweickart VL, et al. Angiogenic and HIV-inhibitory functions of KSHV-encoded chemokines. Science. 1997;278:290–4. 72. Jensen KK, Lira SA. Chemokines and Kaposi’s sarcoma. Semin Cancer Biol. 2004;14:187–94. 73. Chen S, Bacon KB, Li L, Garcia GE, Xia Y, Lo D, et al. In vivo inhibition of CC and CX3C chemokine-induced leukocyte infiltration and attenuation of glomerulonephritis in Wistar-Kyoto (WKY) rats by vMIP-II. J Exp Med. 1998;188:193–8. 74. Raborn ES, Marciano-Cabral F, Buckley NE, Martin BR, Cabral GA. The cannabinoid delta-9-tetrahydrocannabinol mediates inhibition of macrophage chemotaxis to RANTES/CCL5: linkage to the CB2 receptor. J Neuroimmune Pharmacol. 2008;3:117–29.

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