Biochemical Pharmacology 91 (2014) 202–216

Contents lists available at ScienceDirect

Biochemical Pharmacology journal homepage: www.elsevier.com/locate/biochempharm

Cannabinoids inhibit angiogenic capacities of endothelial cells via release of tissue inhibitor of matrix metalloproteinases-1 from lung cancer cells§ Robert Ramer a,1, Sascha Fischer a,1, Maria Haustein a, Katrin Manda b, Burkhard Hinz a,* a b

Institute of Toxicology and Pharmacology, University of Rostock, Schillingallee 70, D-18057 Rostock, Germany Department of Radiotherapy and Radiation Oncology, University of Rostock, Su¨dring 75, D-18059 Rostock, Germany

A R T I C L E I N F O

A B S T R A C T

Article history: Received 27 February 2014 Accepted 17 June 2014 Available online 26 June 2014

Cannabinoids inhibit tumor neovascularization as part of their tumorregressive action. However, the underlying mechanism is still under debate. In the present study the impact of cannabinoids on potential tumor-to-endothelial cell communication conferring anti-angiogenesis was studied. Cellular behavior of human umbilical vein endothelial cells (HUVEC) associated with angiogenesis was evaluated by Boyden chamber, two-dimensional tube formation and fibrin bead assay, with the latter assessing threedimensional sprout formation. Viability was quantified by the WST-1 test. Conditioned media (CM) from A549 lung cancer cells treated with cannabidiol, D9-tetrahydrocannabinol, R(+)-methanandamide or the CB2 agonist JWH-133 elicited decreased migration as well as tube and sprout formation of HUVEC as compared to CM of vehicle-treated cancer cells. Inhibition of sprout formation was further confirmed for cannabinoid-treated A549 cells co-cultured with HUVEC. Using antagonists to cannabinoid-activated receptors the antimigratory action was shown to be mediated via cannabinoid receptors or transient receptor potential vanilloid 1. SiRNA approaches revealed a cannabinoid-induced expression of tissue inhibitor of matrix metalloproteinases-1 (TIMP-1) as well as its upstream trigger, the intercellular adhesion molecule-1, to be causally linked to the observed decrease of HUVEC migration. Comparable anti-angiogenic effects were not detected following direct exposure of HUVEC to cannabinoids, but occurred after addition of recombinant TIMP-1 to HUVEC. Finally, antimigratory effects were confirmed for CM of two other cannabinoid-treated lung cancer cell lines (H460 and H358). Collectively, our data suggest a pivotal role of the anti-angiogenic factor TIMP-1 in intercellular tumor-endothelial cell communication resulting in anti-angiogenic features of endothelial cells. ß 2014 Elsevier Inc. All rights reserved.

Keywords: Cannabinoids Tumor angiogenesis Tissue inhihitor of matrix metalloproteinases-1 Lung cancer cells

1. Introduction §

This study was supported by the Deutsche Forschungsgemeinschaft (Hi 813/61) and by a grant of the FORUN program, Medical Faculty, University of Rostock. Abbreviations: AM-251, [N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide], selective CB1 receptor antagonist; AM-630, [(6-iodo-2-methyl-1-[2-(4-morpholinyl)ethyl]-1H-indol-3-yl) (4-methoxyphenyl)methanone], selective CB2 receptor antagonist; CB1, cannabinoid receptor 1; CB2, cannabinoid receptor 2; CBD, (-)-cannabidiol; CM, conditioned media; DMEM, Dulbecco’s modified Eagle’s medium; EGM, Endothelial Cell Growth Medium; HUVEC, human umbilical vein endothelial cells; JWH-133, (6aR,10aR)-3(1,1-dimethylbutyl)-6a-,7,10,10a-tetrahydro-6,6,9-trimethyl-6H-dibenzo[b,-d]pyran, selective CB2 agonist; MA, R(+)-methanandamide, (R)-N-(2-hydroxy-1methylethyl)-5Z,8Z,11Z,14Z-eicosatetraenamide; NSCLC, non-small cell lung cancer; siRNA, small interfering RNA; THC, D9-tetrahydrocannabinol; TRPV1, transient receptor potential vanilloid 1; WST-1, (4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H5-tetrazolio]-1.6-benzene disulfonate). * Corresponding author. E-mail addresses: [email protected], [email protected] (B. Hinz). 1 The first two authors contributed equally to this work. http://dx.doi.org/10.1016/j.bcp.2014.06.017 0006-2952/ß 2014 Elsevier Inc. All rights reserved.

Angiogenesis poses a crucial event for solid tumors to grow beyond 1–2 mm3 [1,2]. As response to hypoxic conditions and hyponutrition tumors secrete proangiogenic factors such as vascular endothelial growth factor (VEGF) that target endothelial receptor tyrosine kinases to promote neovascularization [3]. Within these tumor–stroma interactions conferring neovascularization several changes in endothelial cell behavior are required such as extracellular matrix degradation, migration towards and into tumor tissue as well as morphological changes of endothelial cells to form tubes. In recent years drugs targeting tumor neovascularization such as bevacizumab as well as the small molecules sunitinib, sorafenib and pazopanib have become an integral component of anticancer therapies (for review see [4]). Within their broad spectrum of anticarcinogenic effects (for review see [5,6]), cannabinoids have been associated with anti-angiogenic

R. Ramer et al. / Biochemical Pharmacology 91 (2014) 202–216

responses in experimental tumors from glioma [7], skin [8], colon [9] and lung [10,11] cancer cells. This anti-angiogenic action has been proposed to be causally associated with downregulation of various proangiogenic proteins known to promote neovascularization of tumor tissue such as angiopoietin-2, placental growth factor and VEGF [7,8,12,13]. With respect to modulation of anti-angiogenic factors by cannabinoids, thrombospondins (TSP), the first proteins proved to act as endogenous inhibitors of angiogenesis [14], have been investigated. However, TSP-1 and -2 were not found to be modulated by cannabinoids in a skin cancer animal model [8]. Regarding a direct impact on endothelial cells, the cannabinoids WIN-55,212-2 and JWH-133 were demonstrated to inhibit vascular endothelial cell survival and migration as part of their anti-angiogenic action [7]. In addition, the cannabinoid derivative HU-331 that exerts topoisomerase inhibitor properties due to a quinone moiety was shown to cause apoptosis of endothelial cells at nanomolar concentrations [9]. Other cannabinoids such as 2methyl-20 -F-anandamide (Met-F-anandamide) and cannabidiol (CBD) revealed as inhibitors of endothelial cell proliferation and tube formation, while sparing a proapoptotic action on endothelial cells [15,16]. Independent thereof, investigations on anti-angiogenic effects of cannabinoids associated with tumor regression currently lack comprehensive inhibitor-based studies addressing the role of tumor-to-endothelial cell communication. Members of the matrix metalloproteinase (MMP) family, a group of zinc-dependent endopeptidases that become secreted by tumor cells, represent a protein family that has been reported to be involved in tumor neovascularization steps [17–19]. Among these proteins endogenous MMP inhibitors such as the tissue inhibitor of matrix metalloproteinases-1 (TIMP-1) have been shown to suppress neovascularization processes [20–22]. In previous studies our group was able to demonstrate the anti-invasive action of D9-tetrahydrocannabinol (THC), R(+)-methanandamide (MA) and CBD to be causally linked to TIMP-1 induction that was found to be triggered by an activation of CB1 and CB2 receptors or transient receptor potential vanilloid 1 (TRPV1) [23,24]. Recently, the intercellular adhesion molecule-1 (ICAM-1) revealed as a pivotal link between upstream cannabinoid receptor and TRPV1elicited p42/44 MAPK activation and downstream TIMP-1dependent inhibition of invasion in the non-small cell lung cancer (NSCLC) cell lines A549, H460 and H358 [25]. In an attempt to merge the two findings of TIMP-1 as an inhibitor of angiogenesis and cannabinoids as TIMP-1 inducers, the present investigation addressed a probable contribution of cannabinoid-induced TIMP-1 to a tumor-to-endothelial cell communication conferring anti-angiogenesis. Here we provide inhibitor-based proof for cannabinoid-induced TIMP-1 release from lung cancer cells to inhibit the angiogenic behavior of human umbilical vein endothelial cells (HUVEC). These findings indicate a novel mechanism within the diverse antitumorigenic effects of cannabinoids. 2. Materials and methods 2.1. Materials AM-251 and AM-630 were obtained from Enzo Life Sciences GmbH (Lo¨rrach, Germany). Leupeptin was purchased from Biomol (Hamburg, Germany). Aprotinin, capsazepine, p-coumaric acid, ethanol, fibrinogen type I from bovine plasma, luminol, orthovanadate, phenylmethylsulfonyl fluoride (PMSF) and thrombin from bovine plasma were bought from Sigma-Aldrich (Taufkirchen, Germany). (-)-CBD was from Biotrend AG (Cologne, Germany). Dimethyl sulfoxide (DMSO), ethylenediaminetetraacetic acid (EDTA), glycerol, hydrogen peroxide (H2O2), sodium chloride (NaCl), Tris–hydrocloride (Tris–HCl) and Tris ultrapure were from

203

AppliChem (Darmstadt, Germany). Dulbecco’s modified Eagle’s medium (DMEM) with 4 mM L-glutamine and 4.5 mg/ml glucose and Endothelial Cell Growth Medium (EGM-2, i.e., Endothelial Basal Growth Medium [EBM-2; CC-3156EA] supplied with EGM-2 SingleQuot Kit Suppl. & Growth Factors [CC-4133EA]) was from Lonza (Cologne, Germany). Penicillin–streptomycin was bought from Invitrogen (Darmstadt, Germany). 4-(2-hydroxyethyl)-1piperazineethanesulfonic acid (HEPES) was from Ferak (Berlin, Germany). JWH-133 was bought from R&D Systems (WiesbadenNordenstadt, Germany). Fetal calf serum (FCS) and phosphatebuffered saline (PBS) were purchased from PAN Biotech (Aidenbach, Germany). MA was purchased from Tocris Bioscience (Wiesbaden-Nordenstadt, Germany). Recombinant TIMP-1 was from Merck KGaA (Darmstadt, Germany). THC was from Lipomed (Weil am Rhein, Germany). Triton1 X-100 was bought from Roth (Karlsruhe, Germany). 2.2. Cell culture A549 lung carcinoma cells were obtained from Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (Braunschweig, Germany). NCl-H460 and NCl-H358 (assigned as H460 and H358) were obtained from American Type Culture Collection (Wesel, Germany). The human bronchial epithelial cell line BEAS-2B was bought from Sigma-Aldrich (Taufkirchen, Germany). A549, H460, H358 and BEAS-2B cells were maintained in DMEM supplemented with 10% (v/v) heat-inactivated FCS, 100 U/ml penicillin and 100 mg/ml streptomycin. HUVEC were obtained from PromoCell (Heidelberg, Germany) and cultivated using the Endothelial Cell Growth Medium Kit (C-22110) from the same company. For experiments cells were used between passages 2 and 7. 2.3. Treatment protocol for evaluation of indirect cannabinoid effects on HUVEC migration, viability and tube formation For evaluation of the effect of conditioned media (CM) obtained from A549, H460, H358 and BEAS-2B on HUVEC migration, viability and tube formation (assessed only for A549 cells), tumor or BEAS-2B cells seeded at a density of 1  105 cells and grown to confluence were treated with the indicated test substances in a final volume of 300 ml serum-free DMEM for 48 h in 24-well plates. Following incubation of the cell lines with vehicle or test substances, CM were collected, centrifuged at 1300  g for 5 min and intermediately stored on ice. Meanwhile, HUVEC were washed, trypsinized and counted. Afterwards, HUVEC were transferred into tubes yielding a density of 1  105 HUVEC per tube and subsequently centrifuged at 100  g for 5 min. After removal of the supernatants, HUVEC pellets were resuspended in the respective CM. 300 ml of HUVEC suspensions containing the preadjusted number of 1  105 HUVEC were seeded into 48-well plates for quantification of cellular viability, onto the upper sides of Boyden chambers for evaluation of migration, or onto the matrigel layers for assessment of tube formation. Subsequently, HUVEC were incubated for 24 h before measurement of viability, migration or tube formation. A scheme of this treatment protocol is indicated in Fig. 2A. Initial experiments with CM from A549 cells (Fig. 3) and key experiments with CM from H460, H358 (Table 1) and BEAS-2B cells (Fig. 8) included a vehicle control with HUVEC suspended in serum-free DMEM in order to quantify the basal effect of the respective cell lines vs. serum-free unconditioned DMEM. As serum-free medium does not contain any components released by tumor cells, these reference groups were omitted in experiments that focused on the impact of TIMP-1 and its upstream targets in cannabinoid- vs. vehicle-treated A549 cells on HUVEC migration. Treatment variations in siRNA transfection

204

R. Ramer et al. / Biochemical Pharmacology 91 (2014) 202–216

Table 1 Impact of conditioned media (CM) obtained from H460 and H358 cells on HUVEC migration and viability. Migration and viability of HUVEC was measured after suspension in serum-free DMEM containing vehicle control (unconditioned control) or in CM from vehicle- or cannabinoid-treated cells. For generation of CM, H460 and H358 lung cancer cells were incubated for 48 h with vehicle or 3 mM CBD, THC, MA or JWH-133. Serum-free media (set as 100%) or CM from the indicated treatment groups were afterwards used to resuspend HUVEC before being subjected to analysis of migration (Boyden chamber assay) or viability (WST-1 tests). Incubation time of HUVEC was 24 h. Values are means  SEM of n = 8 (except migration analyses of H460 cells [n = 4]) experiments. **P < 0.01; ***P < 0.001 vs. vehicle control added to serum-free DMEM; n.s. (not significant, P > 0.05), ##P < 0.01, ### P < 0.001 vs. CM of the corresponding vehicle-treated cells, ANOVA plus posthoc Bonferroni test. Migration (% control) H460

H358

Unconditioned control Vehicle CBD

100.0  15.6 178.1  16.9 ** 61.3  7.7 ###

100.0  22.8 271.7  28.9 *** 80.4  7.8 ###

Unconditioned control Vehicle THC MA JWH-133

100.0  8.2 175.8  2.2 *** 96.4  10.3 ### 88.1  12.9 ### 107.9  10.4 ###

100.0  4.2 177.1  10.9 *** 95.9  6.9 ### 117.9  10.1 ### 128.0  13.6 ##

Viability (% control) H460 Unconditioned control Vehicle CBD THC MA JWH-133

100.0  3.3 143.9  5.1 141.3  4.7 131.3  5.3 138.5  5.6 130.5  1.7

H358 ***

n.s. n.s. n.s. n.s.

100.0  1.6 139.8  6.1 155.5  9.1 143.9  7.6 150.8  4.6 136.7  4.8

***

n.s. n.s. n.s. n.s.

experiments (Figs. 6 and 7 are indicated in the respective section (see Section 2.9). Test substances were dissolved in ethanol or DMSO and diluted with PBS to yield final concentrations of 0.1% (v/v) ethanol (for all cannabinoids) or 0.2% (v/v) DMSO (for AM-251, AM-630, AM-251 plus AM-630, capsazepine). Recombinant TIMP-1 was dissolved in PBS. As vehicle control PBS containing the respective concentration of ethanol or DMSO was used. In experiments with recombinant TIMP-1 equal volumes of PBS served as vehicle control. 2.4. Treatment protocol for evaluation of sprout formation For analyses of direct effects of cannabinoids, fibrin gels containing HUVEC-coated microcarrier 3 beads (three-dimensional culture) were incubated with vehicle or 3 mM of the indicated cannabinoids in freshly added EGM-2 in a final volume of 1 ml. Treatments were repeated every other day following removal of the media. Accordingly, treatments were performed on day 0, 2 and 4. For conditioning of EGM-2 media, A549 cells were seeded at a density of 2  105 cells per well of a 24-well plate and allowed to adhere for 8 h in EGM-2 medium. A549 cells were washed with PBS and incubated with vehicle or test substances in quadruplicate at a final volume of 300 ml EGM-2 medium. As a cell-free vehicle control, four wells of an empty 24-well plate were loaded with 300 ml EGM-2 media containing vehicle. Following an 48-h incubation period, cell culture media were removed from the 24-well plate, and CM from the respective treatment groups were pooled and centrifuged at 1300  g for 5 min. Finally, 1 ml of the cell culture media was added to the three-dimensional cultures of HUVEC following removal of media from the fibrin gels. This procedure was repeated every 48 h. Accordingly, for evaluation of indirect effects on vessel formation of HUVEC, A549 cells were seeded, washed and incubated with vehicle or cannabinoids in three independent experiments carried out every 48 h. Treatment

of A549 cells was therefore performed 2 days before embedding of the HUVEC-coated cytodex 3 microcarrier beads in the fibrin gels for the first incubation with CMs and subsequently at day 0 and day 2. To this end, counting of sprout formation was carried out at day 2, 4 and 6 after embedding of the HUVEC-coated cytodex 3 microcarrier beads in the fibrin gels. A scheme of this treatment protocol is indicated in Fig. 2B. For establishing co-cultures, A549 cells were equilibrated in EGM-2 medium for 5 h. Subsequently, A549 cells were trypsinized, centrifuged and resuspended in fresh EGM-2 medium. Cells were adjusted to yield a density of 1  105 cells per well in the co-culture system and were placed on top of the fibrin layer. Subsequently, vehicle or test substances were added to the co-culture to yield a final volume of 1 ml EGM-2 medium. Treatments were repeated every other day following removal of the old media. Quantification of sprouts in the co-culture system was performed following a 6day treatment period. A scheme of this treatment protocol is indicated in Fig. 2C. 2.5. Analysis of cellular viability Cellular viability of HUVEC was determined by the colorimetric WST-1 test (Roche Applied Science, Mannheim, Germany). This cell viability test is based on the cleavage of the tetrazolium salt WST-1 (4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1.6benzene disulfonate) by mitochondrial succinate-tetrazoliumreductase in metabolically active cells. For assessment of HUVEC viability after direct exposure to cannabinoids or recombinant TIMP-1, HUVEC seeded at a density of 1  105 cells per well in 48well-plates were allowed to adhere for 6 h in Endothelial Cell Growth Medium (Promocell) (Figs. 1A and B and 5A, white bars) or in EGM-2 medium (Lonza) (Fig. 9C, white bars). Subsequently, cells were washed and incubated with vehicle or test substances in serum-free DMEM for 24 h (Figs. 1A and B and 5A, white bars) or in EGM-2 medium for 48 h (Fig. 9C, white bars). For evaluation of the effects of CMs from tumor or BEAS-2B cells on viability of HUVEC (Figs. 3B and C and 8A, white bars, Table 1), cells were treated according to the aforementioned treatment protocols (see Section 2.3). For assessment of the impact of CM from A549 cells on HUVEC viability indicated in Fig. 9C, gray bars, conditioning of EGM-2 media was performed according to the treatment protocol in Section 2.4. Subsequently, CM were used to resuspend HUVEC cells. 300 ml of HUVEC suspensions containing the preadjusted number of 1  105 HUVEC were seeded onto 48-well plates for quantification of cellular viability following a 48-h incubation period. Viability of A549 cells (Fig. 3A) was assessed according to the treatment protocol described in Section 2.3. 2.6. Migration assay The effect of test substances on the migration of HUVEC was determined using uncoated Boyden chambers according to the manufacturer’s instructions (BD Biosciences, Heidelberg, Germany) and as previously described [26]. In this assay, cellular motility is monitored by transmigration through pores with a diameter of 8 mm towards a chemoattractant. After resuspension of HUVEC pellets in the CM obtained from lung cancer cells and BEAS-2B cells, these HUVEC suspensions were seeded into the upper sides of the Boyden chambers (see Section 2.3). For assessment of direct effects of cannabinoids on HUVEC migration (Fig. 1), cells were suspended in serum-free DMEM containing vehicle or test substances. Afterwards, DMEM containing 10% FCS serving as chemoattractant was loaded into the companion plate and the incubations were continued at 37 8C and 5% CO2 atmosphere for another 24 h. Finally, the non-migrated cells on the upper surface of the inserts were removed with a

R. Ramer et al. / Biochemical Pharmacology 91 (2014) 202–216

cotton swab. For calculation of migration, the viability of the migrated cells that adhered to the lower sides of uncoated chambers was determined by the colorimetric WST-1 test (Roche Applied Science). 2.7. Tube formation assay To visualize and quantify the angiogenic potential of cannabinoids or of CM obtained from A549 cells on HUVEC, tube formation assays were performed on Matrigel-coated 48-well plates as described elsewhere [27] with slight modification. This assay is based on the finding that in vitro organization of endothelial cells into capillary-like networks on matrigel layers mimics cellular behavior of an angiogenic process in vivo [28]. In our hands HUVEC did not form tubes yielding a reliable number of intersections when passaged more than twice. Due to this methodical difficulty, tube formation assays were carried out as key experiments only. For tube formation assays presented here HUVEC from passage 2 were used exclusively. 48-well plates were coated with 50 ml ice-chilled Matrigel per well (BD Biosciences) and allowed to polymerize at 37 8C for 2 h. HUVEC were resuspended in serum-free DMEM containing vehicle or in CM of A549 cells (Fig. 3B–D) or in serum-free DMEM containing vehicle, cannabinoids (Fig. 1A–C) or recombinant TIMP1 (Fig. 5A) and seeded at a density of 1  105 cells per well onto Matrigel-coated 48-well plates for a 24-h incubation period. Tube formation was photographed and quantitatively analyzed in total microscopic fields by counting the numbers of tube-like structures forming closed intersections in an investigator-blinded fashion. 2.8. Western blot analysis For analysis of ICAM-1 and b-actin protein levels A549 cells (Fig. 7) were seeded into six-well plates at a density of 2  105 cells per well and were grown to confluence. Subsequently, A549 cells were washed and incubated with vehicle or test substances in serum-free DMEM. Following a 48-h incubation period, lysates of A549 cells were used for further analyses. Cells were lysed in solubilization buffer [50 mM HEPES pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% (v/v) Triton1 X-100, 10% (v/v) glycerol, 1 mM PMSF, 1 mg/ml leupeptin, 10 mg/ml aprotinin], homogenized by sonication, and centrifuged at 10,000 x g for 5 min. Supernatants were used for Western blot analysis. For analysis of TIMP-1 protein levels in siRNA transfection experiments (Fig. 6), A549 cells were seeded into 24-well plates at a density of 1  105 cells per well. Treatment of cells was performed as mentioned below (see Section 2.9). For analyses of TIMP-1 release presented in Fig. 8B, BEAS-2B were seeded into 24well plates at a density of 1  105 cells per well and were allowed to adhere for 24 h before cells were washed with PBS and treated with test substances in serum-free DMEM. Experiments for analyses of TIMP-1 release from A549 cells incubated in EGM-2 medium (Fig. 9B) were performed according to the treatment protocol indicated in Section 2.4. In Western blot experiments monitoring TIMP-1 release from cell culture media, b-actin analyses were performed from cells lysates of the same experiments in 24-well plates to ensure equal loading. Following a 48-h incubation period, CM were collected, centrifuged at 1300  g for 5 min and used for subsequent Western blot analysis of TIMP-1. Total protein was determined using the bicinchoninic acid assay (Pierce, Rockford, IL, USA). Proteins were separated using 10% sodium dodecyl sulfate-polyacrylamide (Applichem) gels and then transferred to nitrocellulose membranes (Roth) that were blocked with 5% milk powder (BioRad, Munich, Germany). Blots were probed with specific antibodies raised to ICAM-1 (Santa Cruz Biotechnology, Heidelberg, Germany), b-actin

205

(Sigma-Aldrich) or TIMP-1 (Merck KGaA). Subsequently, membranes were probed with horseradish peroxidase-conjugated Fabspecific anti-mouse IgG (New England BioLabs GmbH, Frankfurt, Germany). Antibody binding was visualized by a chemiluminiferous solution (100 mM Tris–HCl pH 8.5, 1.25 mM luminol, 200 mM pcoumaric acid, 0.09% [v/v] H2O2, 0.0072% [v/v] DMSO). 2.9. SiRNA transfections Protocols for siRNA transfections are identical with the conditions published previously except incubation times [25]. Transfections were performed to test the impact of CM from A549 cells on HUVEC migration and to analyze knockdown of TIMP-1 and ICAM-1 on the respective protein level. Briefly, A549 cells were seeded at a density of 1  105 cells per well of a 24-well plate for migration assays (Figs. 6 and 7) and TIMP-1 analyses (Fig. 6) or at a density of 2  105 cells per well of a six-well plate for analyses of ICAM-1 (Fig. 7). Following a 3-h attachment, cells were washed and transfected with TIMP-1 siRNA (0.25 mg/ml; Fig. 6), ICAM-1 si RNA (1.25 mg/ml; Fig. 7) or with equal amounts of non-silencing siRNA for 21 h in DMEM containing 10% FCS. Subsequently, cells were washed and retransfected in 300 ml (24-well plates) or 1 ml (6well plates) serum-free DMEM containing the same amounts of siRNA or non-silencing siRNA for another 48 h in the presence of vehicle, CBD, THC, MA or JWH-133 (all cannabinoids at 3 mM). CM were collected, centrifuged at 1300  g for 5 min and used for either preparation of HUVEC suspensions or Western blot analyses of TIMP-1. HUVEC suspended in CM were subsequently subjected to the upper Boyden chambers (1  105 cells in 300 ml CM per insert) to quantify migration following incubation for another 24 h. Cells were transfected using RNAiFect as transfection reagent (Qiagen, Hilden, Germany). TIMP-1 siRNA (Art.-no. SI00745318) and ICAM-1 siRNA (Art.-no. SI00004347) were bought from Qiagen. A non-silencing negative control siRNA was obtained from Eurogentec (Cologne, Germany). 2.10. Fibrin bead assay Fibrin bead assays were carried out to analyze the threedimensional sprouting of HUVEC from the surface of dextrancoated cytodex 3 microcarriers beads (GE Healthcare Europe GmbH, Freiburg, Germany) embedded in fibrin gels. For this purpose the dry Cytodex 3 microcarriers beads were hydrated and autoclaved. Prior to coating of the beads, HUVEC at passage 2 were allowed to equilibrate in EGM-2 medium for 8 h. The supplements added to EBM-2 to yield EGM-2 are fetal bovine serum (FBS, 2% [v/ v] in the medium), human fibroblast growth factor-B (hFGF-B), human epidermal growth factor (hEGF), human vascular endothelial cell growth factor (hVEGF), long R insulin like growth factor-1 (R3-IGF-1), ascorbic acid, hydrocortisone and heparin. HUVEC were mixed with Cytodex 3 microcarrier beads at a cell densitiy of 400 HUVEC per bead in a solution of 2500 beads per 1 ml EGM-2 medium. Beads and HUVEC were co-incubated at 37 8C and 5% CO2 and gently shaken every 15 min for 4 h to allow HUVEC adherence to the bead surface. Following this procedure, beads with cells were transferred to a 25 cm3 tissue culture flask and incubated for 18 h. Subsequently, beads with cells were washed three times with 1 ml of EGM-2 to remove non-coated HUVEC and resuspended at a density of 1000 cell-coated beads/ml in a solution of fibrinogen type I (2.5 mg/ml) with 0.15 U/ml of aprotinin at a pH of 7.4. 12.5 ml of an aquous solution containing 0.625 units of thrombin was transferred to each well of a 24 wellplate. 0.5 ml of the prepared solution containing HUVEC-coated beads and fibrinogen type I was placed over the thrombin drops, gently mixed and allowed to clot for 5 min at room temperature and at 37 8C and 5% CO2 for 20 min to form a gel structure. One

206

R. Ramer et al. / Biochemical Pharmacology 91 (2014) 202–216

milliliter of EGM-2 medium was added to each well and equilibrated with the bead-containing gels for 1 h at 37 8C and 5% CO2. Afterwards, EGM-2 medium was removed from the well and replaced by 1 ml of fresh medium according to the respective treatment protocols. For quantification of sprouts, samples were microscopically scanned from the upper left to the lower right side of the visual field until 30 (direct effects and effects of CMs) or 60 (co-culture) beads were analyzed. A capillary-like structure was counted as one sprout defined as a vessel of length at least equal to the diameter of a bead as previously described [29]. 2.11. Statistics Comparisons between two groups were performed with Student’s t test. Comparisons among more than two groups were carried out with ANOVA plus post hoc Bonferroni test or post hoc Dunnett test. All statistical analyses were undertaken using GraphPad Prism 5.00 (GraphPad Software, San Diego, CA). 3. Results 3.1. Cannabinoids’ direct effects on angiogenic features of HUVEC To investigate the impact of different cannabinoids on endothelial migration and viability as well as on tube and sprout formation, CBD, THC, MA and JWH-133 were added to HUVEC at a final concentration of 3 mM. Based on recent studies that demonstrated inhibition of angiogenesis in a xenograft model of A549 cells in mice treated with CBD [11] or THC [10] and anti-angiogenic effects of JWH-133 in murine glioblastoma xenograft [7] and skin tumor models [8], a decrease of angiogenic parameters was expected, at least in CBD-, THC- and JWH-133-treated HUVEC. However, CBD was found to increase HUVEC tube formation and migration with the latter effect even yielding statistical significance (Fig. 1A). On the other hand, viability was left virtually unaltered (Fig. 1A). In the presence of the other cannabinoids migration and tube formation was likewise found to be increased, with the latter being significantly altered after MA treatment. The modulation of viability by THC and JWH-133 was not significant (Fig. 1B). In the presence of MA, viability of HUVEC was diminished to a slight but significant degree yielding a 15.9% decrease vs. vehicle. The images shown in Fig. 1C depict representative microscopic views of tube formation by HUVEC corresponding to the data presented in Fig. 1A and B. In a three-dimensional angiogenesis assay, HUVEC coated on a dextran surface of microcarrier 3 beads embedded into fibrin gels were tested for their ability to form sprouts in the presence or absence of cannabinoids. In this three-dimensional cell assay, HUVEC form multicellular vessel layers (Fig. 1D, upper left) with side branches and anostomosis occurring between neighbouring vessels. HUVEC arrange to a tube shaped network 2–3 days after establishing the fibrin gel which has also been observed by other authors using a similar setting [29]. Treatments of three-dimensional cultures were performed immediately after fibrin clotting and equilibrating in EGM-2 medium and subsequently every 48 h in fresh EGM-2 medium. Following a 4-day incubation period with treatments carried out twice, CBD and JWH-133 elicited a significant increase of sprout formation (Fig. 1D). This increase remained significant for CBD-treated sprouts as compared to vehicle following a 6-day incubation period comprising three treatments with vehicle or cannabinoids in fresh EGM-2 (Fig. 1D). 3.2. CM of cannabinoid-treated A549 cells confer inhibition of HUVEC migration, tube and sprout formation To address a probable indirect anti-angiogenic action of cannabinoids via modulation of the tumor cell microenviroment

(i.e., cell culture media of A549 cells in the in vitro setting), another experimental setup was used next. To this end A549 cells were treated with vehicle or cannabinoids for 48 h. Subsequently, CM were collected, centrifuged and used to prepare cell suspensions of HUVEC at a final density of 1  105 cells per sample. HUVEC suspended in CM from A549 cells were subjected to Boyden chambers for analysis of migration, to 48-well plates to quantify viability and on Matrigel-coated 48-well plates for monitoring tube formation following an additional 24-h incubation period (Fig. 2A). To analyse indirect effects on three-dimensional sprout formation of HUVEC in fibrin gels, A549 cells were seeded and treated three times in EGM-2 medium (i.e., every 48 h) in parallel. Following a 48-h incubation period, cell culture media were removed from A549 cells, centrifuged and transferred onto the fibrin gels containing HUVEC-coated cytodex 3 microcarrier beads. A scheme of this experimental setting is given in Fig. 2B. Among the cannabinoids used to assess the impact of CM from A549 on HUVEC behavior, CBD at a final concentration of 3 mM elicited a loss of viability of A549 cells treated in serum-free DMEM (Fig. 3A, right) associated with apoptotic characteristics such as blebbing (Fig. 3A, left) which is in line with a recent report from our group [11]. The other cannabinoids did not significantly decrease viability of A549 cells when tested at a final concentration of 3 mM. As shown in Fig. 3B,C and E (lower side), CM obtained from A549 cells treated with vehicle for 48 h significantly increased migration and viability as well as tube and sprout formation of HUVEC as compared to HUVEC suspended in serum-free DMEM or incubated with non-conditioned EGM-2 medium for fibrin bead assays. Notably, EGM-2 medium serving as unconditioned vehicle control (Fig. 3E) was incubated on the same plate but without A549 cells for 48 h at 37 8C to provide identical conditions compared to CM from A549 cells. EGM-2 medium exposed to these conditions elicited less sprout formation than fresh EGM-2 medium that was used as vehicle control in Fig. 1D. HUVEC suspended in CM of A549 treated with 3 mM CBD for 48 h exerted a significant decrease of migration and tube formation as compared to CM from vehicletreated A549 (Fig. 3B). By contrast, cellular viability was not impaired (Fig. 3B). Similar results were obtained from HUVEC suspended in CM of A549 treated with 3 mM of THC, MA or JWH133 with the migration being significantly inhibited by all cannabinoids. The CM-induced tube formation by HUVEC was likewise significantly decreased when A549 cells were treated with cannabinoids as indicated in the histogram of Fig. 3C and depicted in the representative microscopic images (Fig. 3D), respectively. Viability of HUVEC increased by CM obtained from vehicle-treated A549 cells was slightly but significantly diminished in the presence of MA and JWH-133. In fibrin bead assays, CM obtained from cannabinoid-treated A549 cells elicited a decrease of sprout numbers as compared to HUVEC incubated with CM of vehicletreated A549 cells for all cannabinoids tested (Fig. 3E). For further analysis addressing molecular mechanisms underlying the observed anti-angiogenic potential of cannabinoids, the impact of CM on migration of HUVEC was determined. To avoid redundancies, the serum-free DMEM control was omitted in subsequent experiments presented in Figs. 4, 6 and 7. 3.3. Cannabinoids’ antimigratory effect is mediated via CB receptors and TRPV1 In light of our recent findings indicating CB1 and CB2 receptors as well as TRPV1 to be detectable in the membranes of A549 cells [25], the role of these receptors in antimigratory responses was investigated using antagonists to CB1 (AM-251), CB2 (AM-630) and TRPV1 (capsazepine). Receptor antagonists were used at a concentration of 1 mM, which has been reported to be within the range of concentrations inhibiting CB1-, CB2- and TRPV1-dependent

R. Ramer et al. / Biochemical Pharmacology 91 (2014) 202–216

207

Fig. 1. Impact of cannabinoids on migration and viability as well as tube and sprout formation of HUVEC. (A) and (B), Migration (Boyden chamber assays, black bars), viability (WST-1 test, white bars) and tube formation (tube formation assay, gray bars) of HUVEC following incubation with CBD (A), THC, MA and JWH-133 (B) or vehicle for 24 h. (C), Phase contrast images of tube formation on Matrigel layer following a 24-h incubation with vehicle or 3 mM of the indicated cannabinoids. (D) Sprout formation of HUVEC from cytodex 3 microcarrier beads in a fibrin gel following treatment with vehicle or the indicated cannabinoids every 48 h. The microscopic image at the upper left represents a zoomed 400 magnification captured from the root of a single sprout that appeared after a 3-day incubation in EGM-2 medium. Phase contrast images at the upper right were captured at day 6 following the initial stimulation. Percent control represents comparison with vehicle-treated cells (100%) in the absence of test substance. Values are means  SEM of n = 11 (migration, A), n = 3–4 (migration, B), n = 12 (viability, A), n = 7–8 (viability, B) or n = 6 (tube formation, A,B) experiments. Quantification of fibrin bead assays was performed by analyses of 60 individual beads from two independent experiments. Results are presented as means  SEM of sprout numbers per bead. *P < 0.05, ** P < 0.01 vs. corresponding vehicle control, Student’s t-test (A) or ANOVA plus post-hoc Dunnett test (B,D).

events [25,30,31]. As shown in Fig. 4, the inhibition of HUVEC migration in response to CM from A549 cells challenged with 3 mM of the respective cannabinoid was reversed by a 1-h preincubation of A549 cells with AM-251, AM-630 or the combination of both antagonists. The TRPV1 antagonist capsazepine likewise diminished the antimigratory effect of the TRPV1 receptor agonists CBD and MA to a similar extent. As shown in Fig. 4D, HUVEC incubated with CM from A549 cells treated with the receptor antagonists AM-251 and AM-630 in the absence of cannabinoids elicited a slight increase of

HUVEC migration that may, at least in part, contribute to the observed profound reconstitution of migration when cannabinoids were combined with these compounds. 3.4. Recombinant TIMP-1 inhibits HUVEC migration, tube and sprout formation Based on studies indicating TIMP-1 to exhibit anti-angiogenic effects [20–22] and recent findings from our group that revealed

208

R. Ramer et al. / Biochemical Pharmacology 91 (2014) 202–216

Fig. 2. Treatment protocol for quantifying the effect of the cancer cell microenviroment on endothelial cell behavior. (A) CM were generated by incubation of A549 (or in terms of migration and viability analyses additionally with H460, H358 or BEAS-2B cells) with vehicle or 3 mM of the phytocannabinoids CBD and THC, the hydrolysis-stable endocannabinoid analog MA or the specific CB2 agonist JWH-133 for 48 h. Subsequently, HUVEC were resuspended in serum-free DMEM containing vehicle or in CM from lung cancer cells treated with vehicle or cannabinoids for evaluation of migration, tube formation and viability. For evaluation of migration HUVEC suspensions were subjected to Boyden chambers. As a chemoattractant DMEM containing 10% FCS was applied to the lower Boyden chamber. Viability was measured using the colorimetric WST-1 test. The formation of two-dimensional capillary-like structures (tubulogenesis) was quantified by counting the number of closed intersections formed by HUVEC on a Matrigel layer. The incubation period for the experimental section using HUVEC was 24 h. (B) For quantification of sprout formation a three-dimensional in vitro angiogenesis assay was carried out using cytodex 3 microcarrier bead coated with HUVEC that were embedded in fibrin gels. In these experiments three-dimensional sprout formation was tested by initial addition of CM from A549 cells that was replaced by fresh CM after 2 and 4 days. For this purpose A549 cells were trypsinized, centrifuged, seeded and stimulated every 48 h starting 48 h before embedding of HUVEC-attached beads in fibrin gels to obtain the cell culture media for initial incubation. (C) In a co-culture system A549 cells were placed on top of the fibrin gels immediately after establishing the fibrin gel containing HUVEC-coated beads. Thereafter, co-cultures were stimulated with test substances in EGM-2 medium. At days 2 and 4 media were removed and treatment with test substances was repeated using fresh EGM-2 medium. Terminal counting of sprout numbers was performed at day 6.

cannabinoids as potent inducers of TIMP-1 via a mechanism involving CB1 and CB2 receptors as well as TRPV1 in lung cancer cells [23–25], a potential contribution of cannabinoid-induced TIMP-1 release into CM of A549 cells to the observed antimigratory effect on HUVEC was addressed next. First experiments were carried out using recombinant TIMP-1 to mimic the cannabinoidinduced TIMP-1 release from lung cancer cells into CM. According to Fig. 5 exogenously added recombinant TIMP-1 elicited a concentration-dependent inhibition of HUVEC migration and tube formation (Fig. 5A) as well as three-dimensional sprout formation (Fig. 5B). At the concentrations tested, recombinant TIMP-1 left cellular viability of HUVEC suspended in DMEM virtually unaltered (Fig. 5A).

3.5. Reversal of the antimigratory effect of CM from cannabinoidtreated A549 cells on HUVEC by knockdown of TIMP-1 in A549 cells A causal relationship between the cannabinoid-induced TIMP-1 expression and the antimigratory effect of CM from cannabinoidtreated A549 cells was investigated using TIMP-1 siRNA transfections. In a recent study we were able to demonstrate 0.25 mg/ml TIMP-1 siRNA to be sufficient to significantly knockdown TIMP-1 expression induced by a 72-h incubation of A549, H460 and H358 cells with cannabinoids [25]. Using the same protocol, except an incubation period of 48 h instead of 72 h, the knockdown of cannabinoid-induced TIMP-1 was shown to significantly reverse the antimigratory impact on HUVEC suspended in CM from A549

R. Ramer et al. / Biochemical Pharmacology 91 (2014) 202–216

209

Fig. 3. Impact of CM obtained from A549 cells on migration and viability as well as on tube and sprout formation of HUVEC. (A) Viability of A549 cells following a 48-h incubation period with vehicle or 3 mM CBD, THC, MA and JWH-133 in serum-free DMEM (WST-1 test). (B and C) Migration, viability and tube formation of HUVEC after being resuspended in serum-free DMEM or CM from vehicle- or cannabinoid-treated A549 cells. A549 were incubated for 48 h with vehicle or 3 mM of CBD (B), THC, MA or JWH-133 (C). Serum-free media containing vehicle or CM from A549 cells were afterwards used to resuspend HUVEC before being subjected to analysis of migration (Boyden chamber assay, black bars), viability (WST-1 tests, white bars) or capillary-like structures (tube formation assay, gray bars). Incubation time of HUVEC was 24 h. (D) Phase contrast images of tube formation on Matrigel layer following a 24-h incubation period with the respective treatment groups. Images depict one representative view of HUVEC tested in (B) and (C). (E) Sprout formation of HUVEC coated on cytodex 3 microcarrier beads in fibrin gels and challenged with unconditioned EGM-2 media containing vehicle or with CM from A549 cells that were incubated with vehicle or 3 mM of the indicated cannabinoid for 48 h. Treatments were repeated three times every 48 h and CM were transferred to fibrin layers following removal of the old CM from the fibrin layer, respectively. Phase contrast images depict one representative bead per treatment group captured at day 6. Percent control represents comparison with vehicle-treated cells (100%) in the absence of test substance. Values are means  SEM of n = 3-4 (A), n = 7–8 (migration, B), n = 8 (migration, C), n = 4 (viability, B,C), n = 8 (tube formation, B) or n = 3 (tube formation, C) experiments. Quantification of fibrin bead assays was performed by analyses of 60 individual beads from two independent experiments. Results are presented as means  SEM of the sprout number per bead. *P < 0.05 vs. vehicle (A); **P < 0.01, *** P < 0.001 vs. vehicle control added to serum-free DMEM (B,C); ***P < 0.001 vs. vehicle control added to EGM-2 medium (D); #P < 0.05, ##P < 0.01, ###P < 0.001 vs. CM of vehicletreated A549 cells, ANOVA plus post-hoc Bonferroni test or Dunnett test (A).

R. Ramer et al. / Biochemical Pharmacology 91 (2014) 202–216

B

150 ###

50

30 M A

A + C TH

150

##

D

150 125

ap sa

+

C

-6 M A

e ic l

-6 30 M

C +

51 M -2 A

M

A

+

A

M

-2

M A

+ 51

Ve h

-6 30 M

A

A + A M

M

A

+

A

M

M

M A

ic l Ve h

ap sa

0 -6 30

0 -2 51

25

e

25

30

50

A

50

75

-2 51

75

100

M

100

A

Migration (% Control)

125

Migration (% Control)

+

TH

51

C

+ C TH

+ D B C

C

-6

30 -6 M A

e ic l Ve h

C

A

M

C

+ D B C

B D

51

+

+

A

A

M

M A +

B D C

M

-2

-6 30

D B C

Ve hi

***

+

0 ap sa

0 -6 30

25

51

25

M -2

**

##

75

M -2 51

75

###

100

A

100

50

###

125

Migration (% Control)

###

cl e

Migration (% Control)

125

150

###

###

-2

A

TH C

210

Fig. 4. Involvement of cannabinoid receptors (CB1, CB2) and TRPV1 in cannabinoid-induced alteration of CM from A549 leading to inhibition of HUVEC migration. (A–C) Effect of AM-251 (CB1 antagonist, A–C), AM-630 (CB2 antagonist, A–C) and capsazepine (Capsa, TRPV1 antagonist, A,C) on the antimigratory effect of 3 mM CBD (A) THC (B) or MA (C). Cells were pretreated with the respective receptor antagonist (all tested at a final concentration of 1 mM) for 1 h and incubated with vehicle or cannabinoids for another 48 h before CM were collected. (D) Effect of AM-251, AM-630 and capsazepine on HUVEC migration in the absence of cannabinoids following a 49-h incubation period. HUVEC suspended in CM were subjected to the upper Boyden chambers to quantify migration following incubation for another 24-h period. Percent control represents comparison with vehicle-treated cells (100%) in the absence of test substance. Values are means  SEM of n = 11–12 (A) or n = 7–8 (B–D) experiments. **P < 0.01, ***P < 0.001 vs. corresponding vehicle control; ##P < 0.01, ###P < 0.001 vs. the respective cannabinoid-treated group, ANOVA plus post-hoc Bonferroni test.

cells treated with CBD (Fig. 6A), THC (Fig. 6B), MA (Fig. 6C) and JWH-133 (Fig. 6D), respectively. Western blot analyses demonstrated transfection of A549 with TIMP-1 siRNA to be sufficient to decrease cannabinoid-induced TIMP-1 expression (Fig. 6A–D, Western blot images). 3.6. Inhibition of the antimigratory effect of CM from cannabinoidtreated A549 cells on HUVEC by knockdown of the TIMP-1 upstream trigger ICAM-1 in A549 cells In view of our recent findings that demonstrated a neutralizing antibody against ICAM-1 and knockdown of ICAM-1 to abrogate CBD-, THC- and MA-induced TIMP-1 expression [25], further investigations aimed at a probable involvement of ICAM-1 in the antimigratory effect on HUVEC. In line with the proposed role of ICAM-1 as upstream trigger of cannabinoid-induced TIMP-1 expression, experiments using ICAM-1 siRNA transfections of A549 cells provided similar results as compared to the data obtained with TIMP-1 siRNA. Accordingly, knockdown of cannabinoid-induced ICAM-1 expression proven by Western blot analyses

was associated with significant inhibitions of the antimigratory effects of CM obtained from A549 cells treated with CBD (Fig. 7A), THC (Fig. 7B), MA (Fig. 7C) and JWH-133 (Fig. 7D), respectively. 3.7. Cannabinoids inhibit migration indirectly via change of CM from additional lung cancer cell lines To exclude that the indirect cannabinoid-induced antimigratory response on HUVEC was restricted to A549 cells, additional NSCLC cell lines (H460, H358) were treated with vehicle or cannabinoids for 48 h before CM were collected and used to resuspend HUVEC for subsequent Boyden chamber assays and WST-1 tests, respectively. As shown in Table 1, CM from vehicletreated H460 and H358 cells likewise caused significant increases of migration and viability of HUVEC. CM from cannabinoid-treated H460 and H358 cells conferred reduced HUVEC migration as compared to CM from the respective vehicle-treated cell lines (Table 1, upper side), whereas viability remained again virtually unaltered by cannabinoid treatments (Table 1, lower side).

R. Ramer et al. / Biochemical Pharmacology 91 (2014) 202–216

211

Fig. 5. Impact of recombinant TIMP-1 on migration and viability as well as tube and sprout formation of HUVEC. (A, left) Migration (Boyden chamber assay, black bars), viability (WST-1 test, white bars) and tube formation (tube formation assay, gray bars) of HUVEC were determined following a 24-h incubation with vehicle or the indicated concentrations of recombinant TIMP-1. (A, right) Phase contrast images of HUVEC tube formation on Matrigel. (B, left) Sprout formation of HUVEC-coated cytodex 3 microcarrier beads into fibrin gels following treatment with vehicle or the indicated concentrations of recombinant TIMP-1 every 48 h. (B, right) Phase contrast images of HUVEC sprouts at day 6. Percent control represents comparison with vehicle-treated cells (100%) in the absence of test substance. Values are means  SEM of n = 3 (migration), n = 4 (viability) or n = 3 (tube formation) experiments. Quantification of fibrin bead assays was performed by analyses of 60 individual beads from two independent experiments. Results are presented as means  SEM of the sprout number per bead. *P < 0.05, **P < 0.01, ***P < 0.001 vs. corresponding vehicle control, ANOVA plus post-hoc Dunnett test.

3.8. CM from cannabinoid-treated non-cancer bronchial epithelial cells do not confer inhibition of HUVEC migration To analyze whether comparable effects of CM on HUVEC migration also occur when non-cancer cells are treated with cannabinoids, additional experiments were performed with BEAS2B, a cell line established from bronchial epithelium of noncancerous individuals [32] and characterized as normal lung epithelial cell line [33,34]. According to Fig. 8A, CM from vehicletreated BEAS-2B cells were found to significantly increase HUVEC migration as compared to HUVEC suspended in serum-free DMEM. In contrast to CM obtained from the cannabinoid-treated lung cancer cell lines A549, H460 and H358, a 48-h incubation of BEAS2B cells with 3 mM CBD (Fig. 8A, left) or equimolar concentrations of THC, MA and JWH-133 (Fig. 8A, right) did not alter CM to confer significant changes of HUVEC migration as compared to HUVEC suspended in vehicle-treated BEAS-2B (P > 0.05; ANOVA plus post hoc Bonferroni test). As increased TIMP-1 release into CM of A549 cells was determined as the key regulator of the indirect antimigratory effect of cannabinoids on HUVEC, TIMP-1 regulation was likewise addressed in Western blot analyses of cell culture media obtained from BEAS-2B cells. In agreement with the proposed role of TIMP-1 as cannabinoidinduced inhibitor of HUVEC migration, none of the cannabinoids was found to induce TIMP-1 release from BEAS-2B cells (Fig. 8B).

was addressed in a co-culture system using HUVEC-attached cytodex 3 microcarrier beads embedded in and A549 cells placed on top of fibrin gels in EGM-2 media. According to Fig. 9A, a 6-day incubation of HUVEC-coated beads in fibrin gels with A549 cells stimulated every other day in fresh EGM-2 medium revealed a profound inhibition of sprout formation by all cannabinoids tested. Western blot experiments were performed to clarify whether cannabinoid-induced TIMP-1 release is still detectable when A549 cells are treated with test substances in EGM-2 media. As shown in Fig. 9B, upper panel, A549 cells treated in EGM-2 medium maintained their susceptibility to cannabinoid-induced release of TIMP-1. On the other hand, TIMP-1 release into EGM-2 media was left virtually unaltered when HUVEC were treated with cannabinoids for 48 h (Fig. 9B, lower panel). Viability analyses of HUVEC exposed to EGM-2 medium of A549 cells treated with vehicle or cannabinoids for 48 h revealed none of the cannabinoids to significantly inhibit HUVEC viability (Fig. 9C, gray bars). Similar results were obtained when HUVEC were directly exposed to test substances in EGM-2 medium (Fig. 9C, white bars). Interestingly, CBD even increased viability of HUVEC to a slight but significant extent. In both experimental settings HUVEC were incubated for 48 h to mimic a single treatment cycle of the co-culture system and the conditions for analyses of the effects of CM in the fibrin bead assays presented in Fig. 3E. 4. Discussion

3.9. Cannabinoids inhibit sprout formation in a A549–HUVEC coculture Finally, a probable effect of cannabinoids on tumor-toendothelial cell communication conferring anti-angiogenesis

Investigations of the last decade have provided evidence for inhibition of tumor neovascularization by cannabinoids in various murine xenograft models. However, only a few studies particularly focused on the anti-angiogenic mechanisms of cannabinoids. So

212

R. Ramer et al. / Biochemical Pharmacology 91 (2014) 202–216

Fig. 6. Influence of TIMP-1 siRNA transfection of A549 cells on migration of HUVEC suspended in CM from cannabinoid-treated A549 cells. A549 were transfected with 0.25 mg/ml TIMP-1 siRNA or non-silencing siRNA (nonsi) for 21 h in DMEM containing 10% FCS. Subsequently, cells were transfected in serum-free DMEM with the same amounts of siRNA or non-silencing siRNA for another 48 h in the presence of vehicle or 3 mM CBD (A), THC (B), MA (C) and JWH-133 (D) before CM were collected. HUVEC suspended in CM from A549 were subjected to the upper Boyden chambers to quantify migration following incubation for another 24 h. Monitoring of TIMP-1 knockdown was performed in parallel experiments in 24-well plates under the same conditions by Western blot analyses of the cell culture media obtained from A549 cells following a 48-h incubation period. Percent control represents comparison with vehicle-treated cells (100%) in the absence of test substance. Values are means  SEM of n = 4 (A–C) or n = 3–4 (D) experiments. **P < 0.01, ***P < 0.001 vs. corresponding vehicle control; ###P < 0.001 vs. the respective cannabinoid-treated group without siRNA, ANOVA plus post-hoc Bonferroni test. Western blot images depict one representative experiment. Equal loading of proteins in cell culture media is indicated by analyses of b-actin in the respective cell lysates.

far, results concerning this matter were obtained from experiments addressing direct effects of cannabinoids on endothelial cells or modulation of angiogenic parameters in cancer cells or xenograft tissues. On the other hand, investigations on potential tumor-to-endothelial cell interactions have been largely neglected so far. The results from the present study provide the first-time inhibitor-based proof for cannabinoid-induced TIMP-1 release from lung cancer cells as a key event in cannabinoids’ antiangiogenic action on endothelial cells. There are several lines of evidence supporting this notion. First, all CM obtained from cannabinoid-treated A549 lung cancer cells were found to confer profound inhibition of HUVEC migration as well as tube and sprout formation. Second, post-transcriptional knockdown of ICAM-1, a parameter recently demonstrated to act as an upstream trigger of cannabinoid-induced TIMP-1 release in A549 cells [25], was demonstrated to abrogate the observed inhibition of HUVEC migration by CM from cannabinoid-treated A549 cells. Third, a reversal of the antimigratory action of these CM was achieved by blocking cannabinoid receptors (CB1, CB2) or TRPV1 with specific antagonists which is in agreement with the recently established cannabinoid receptor- and TRPV1-dependent upregulation of ICAM-1 and TIMP-1 expression [25]. Fourth,

knock-down of TIMP-1 expression by siRNA transfection of A549 cells was found to inhibit the antimigratory action of CM from cannabinoid-treated A549 cells. The proposed TIMP-1 induction by cannabinoids as a specific shift of lung cancer cell gene expression conferring antiangiogenesis was further substantiated by experiments using recombinant TIMP-1. Thus, exogenously added TIMP-1 caused a concentration-dependent inhibition of HUVEC migration, tube and sprout formation, while sparing effects on cellular viability. Finally, to exclude the possibility that the observed effects were restricted to one lung cancer cell line, an antimigratory response of HUVEC was confirmed by use of CM from two additional cannabinoidtreated NSCLC cell lines that have recently been demonstrated to be likewise susceptible to cannabinoid-induced ICAM-1-dependent TIMP-1 release [25]. The data presented indicate an indirect effect of cannabinoids on vascular cells that requires an intermediate cell type involved in pathological processes such as cancer and inflammation. This finding is in line with a recent report that demonstrated the endocannabinoid-like substance palmitoyl ethanolamide to alter CM of activated mastocytic cells thereby causing a decrease of HUVEC proliferation [35]. In the cited study the indirect anti-angiogenic

R. Ramer et al. / Biochemical Pharmacology 91 (2014) 202–216

213

Fig. 7. Influence of ICAM-1 siRNA transfection of A549 cells on migration of HUVEC suspended in CM from cannabinoid-treated A549 cells. A549 were transfected with 1.25 mg/ml ICAM-1 siRNA or non-silencing siRNA (nonsi) for 21 h in DMEM containing 10% FCS. Subsequently, cells were transfected in serum-free DMEM with the same amounts of siRNA or non-silencing siRNA for another 48 h in the presence of vehicle or 3 mM CBD (A), THC (B), MA (C) and JWH-133 (D) before CM were collected. HUVEC suspended in CM were subjected to the upper Boyden chambers to quantify migration following incubation for another 24 h. Monitoring of ICAM-1 knockdown was performed in parallel experiments in six-well plates under the same conditions by Western blot analyses of the cell lysates obtained from A549 cells following a 48-h incubation period. Percent control represents comparison with vehicle-treated cells (100%) in the absence of test substance. Values are means  SEM of n = 3 (A,B) or n = 4 (C,D) experiments. ***P < 0.001 vs. corresponding vehicle control; ##P < 0.01, ###P < 0.001 vs. the respective cannabinoid-treated group without siRNA, ANOVA plus post-hoc Bonferroni test. Western blot images depict one representative experiment. Equal loading of proteins in cell lysates is indicated by hybridization of membranes to an antibody raised against bactin.

effect of palmitoyl ethanolamide was considered as a potential tool to treat pathological angiogenesis in context with inflammatory diseases. Noteworthy, results from initial experiments demonstrating pro- rather than anti-angiogenic effects of cannabinoids when added to HUVEC directly (e.g., migration and sprout formation in CBD-, and tube formation in MA-treated A549 cells) are partly in contrast to some reports indicating cannabinoids to directly act anti-angiogenically on endothelial cells. Accordingly, Bla´zquez et al. [7] were able to demonstrate low concentrations of JWH-133 to inhibit HUVEC migration in Boyden chamber experiments when using lysophosphatidic acid as chemoattractant. In the cited study HUVEC were pretreated with JWH-133 for 18 h and subsequently trypsinized and subjected to Boyden chambers for 3 h which differs from the method used here. Another study demonstrated CBD to inhibit HUVEC migration using collagen-coated Boyden chambers with different media and freshly prepared HUVEC [16]. In the latter study CBD was further found to inhibit proliferation of HUVEC with an IC50 of approximately 10 mM and to inhibit migration quantified by wound healing assays at concentrations greater than or equal to 9 mM. Another study demonstrated suppression of bovine aortic endothelial cell proliferation by 2.3 mM CBD and 9.4 mM THC with inhibition values of approximately

40% and 60%, respectively [9]. Notably, the latter investigation found CBD and THC to act biphasic in the rat aortic ring assay with inductions of angiogenesis at very low concentrations (50 nM), no effect at 2.3 mM and an anti-angiogenic action at higher concentrations. Finally, Pisanti et al. [15] found Met-F-anandamide to inhibit proliferation and tube formation of pig aortic endothelial cells in the presence of basic fibroblast growth factor (bFGF), whereas no antiproliferative effect was observed when cells were challenged with up to 10 mM Met-F-anandamide in the absence of bFGF. Thus, the discrepancy between the data presented here and the aforementioned studies may be due to differences in experimental settings and inhibition of angiogenic capacities of endothelial cells exposed to higher cannabinoid concentrations. In agreement with the CBD-mediated increased migration of HUVEC reported here, several other studies were able to demonstrate promigratory effects of cannabinoids on various cell types. As such, promigratory effects were proven for HU-210, anandamide and WIN55,212-2 on human embryonic kidney cells [36], WIN55,212-2 on human corneal epithelial cells. [37], 2arachidonyl glycerol, anandamide and several other fatty acid ethanolamides on microglial cells [38,39] as well as CBD and JWH133 on human mesenchymal stem cells [40]. Furthermore, cannabinoid receptor activation has been shown to trigger

214

R. Ramer et al. / Biochemical Pharmacology 91 (2014) 202–216

Fig. 8. Analysis of cannabinoid-modulated TIMP-1 expression in non-cancer bronchial epithelial BEAS-2B cells and impact of CM obtained from BEAS-2B cells on migration and viability of HUVEC. (A) BEAS-2B cells were incubated for 48 h with vehicle or 3 mM CBD (left panel), THC, MA or JWH-133 (right panel). Serum-free DMEM containing vehicle or CM from BEAS-2B cells were afterwards used to resuspend HUVEC before being subjected to analysis of migration (Boyden chamber assays, black bars) or viability (WST-1 test, white bars). Incubation time of HUVEC was 24 h. (B) Western blot analyses of TIMP-1 release into cell culture media by BEAS-2B cells treated with vehicle or the indicated cannabinoid at a final concentration of 3 mM for 48 h. Values are means  SEM of n = 4 experiments. Values above the blots represent means  SEM of densitometric analyses in comparison with vehicle-treated cells (100%) in the absence of test substance of n = 4 experiments. Equal loading of proteins in cell culture media is indicated by analyses of b-actin from cell lysates. *P < 0.05, **P < 0.01, ***P < 0.001 vs. corresponding vehicle control, ANOVA plus post-hoc Dunnett test.

migration of neuroblastic cells [41]. Concerning the cell types involved in vessel formation the endocannabinoid-like substance N-arachidonoyl serine was demonstrated to elicit migration and tube formation of human dermal microvascular endothelial cells [42]. Taken into account that the cannabinoids tested here elicit antiangiogenic properties by modifying microenviroments of tumor cells and recent data indicating cannabinoids to contribute to maintenance of vascular integrity [43–45], it is tempting to speculate that cannabinoids’ anti-angiogenic effects are sitespecifically restricted to the tumor tissue. In line with this notion, the present study found that CM obtained from cannabinoidtreated BEAS-2B cells, a normal bronchial epithelial cell line [33,34], did not inhibit HUVEC migration as compared to CM obtained from vehicle-treated BEAS-2B. In agreement with the proposed role of TIMP-1 as inhibitor of HUVEC migration, BEAS-2B lacked susceptibility to cannabinoid-induced TIMP-1 release. Noteworthy, the sparing of unwanted side effects on vascularization of healthy tissue or an even protective action on physiologically essential blood vessels may represent an advantage of cannabinoids as compared to several currently used chemotherapeutics with vascular toxicity conferring a cardiovascular risk (for review see [46]). However, to deduce a potential site-specificity of cannabinoids’ anti-angiogenic effects being restricted to tumor microenviroments further in vivo evaluation of vascularization of healthy tissue exposed to cannabinoids is required. Referring to the role of TIMP-1 during tumor neovascularization the results presented here are in agreement with several investigations demonstrating TIMP-1 to inhibit tumor progression via its anti-angiogenic action [21,47]. In line with this notion, ectopical overexpression of TIMP-1 was shown to significantly

suppress tumor neovascularization [48,49]. Regarding the antiangiogenic action on endothelial cells, recombinant TIMP-1 as well as cell culture media of TIMP-1-overexpressing cancer cells have been demonstrated to inhibit migration of endothelial cells [48]. In further agreement with our data indicating no significant effects of CM from three different cannabinoid-treated NSCLC cell lines on viability of HUVEC (except significant but only partial effects of MA- and JWH-133-treated A549), TIMP-1 was previously shown to leave proliferative activity of endothelial cells virtually unaltered [50]. However, the precise intracellular mechanism by which TIMP-1 released from cancer cells causes inhibition of migration and tube formation of HUVEC remains to be elucidated. In this context inhibition of MMP-2 has been described as a crucial step conferring blockade of HUVEC migration [51]. On the other hand, numerous studies reported TIMP-1 to elicit effects independent of modulation of proteolytic action [52–55]. Thus, a shifting paradigm for TIMP-1’s diverse molecular actions has been proposed [55]. Concerning the mechanism underlying TIMP-1 release by cannabinoids in lung cancer cells, a prior increased expression of ICAM-1 was recently demonstrated to confer this response [25]. Although upregulation of ICAM-1 was proven to depend on cannabinoid receptor and TRPV1-elicited p42/44 MAPK activation [25], the downstream target of ICAM-1 in conferring TIMP-1 expression remains to be determined. In light of findings demonstrating an AP-1 binding site within the TIMP-1 promotor region [56], an ICAM-1-triggered activation of AP-1 [57] appears feasible. Collectively, our data suggest cannabinoids to increase the release of TIMP-1 from lung cancer cells via activation of CB1 and CB2 receptors as well as TRPV1 and a subsequent induction of ICAM-1 expression, thereby altering the cancer cell microenvironment and suppressing the angiogenic potential of endothelial cells.

R. Ramer et al. / Biochemical Pharmacology 91 (2014) 202–216

215

Fig. 9. Analysis of cannabinoid-modulated TIMP-1 expression in HUVEC and A549 cells treated in EGM-2 medium and impact of cannabinoids on sprout formation in a coculture of A549 cells and HUVEC. (A) Sprout formation of HUVEC-coated cytodex 3 microcarrier beads in fibrin gels when co-cultured with A549 following treatment with vehicle or 3 mM of the indicated cannabinoids every 48 h. Quantification of fibrin bead assays was performed by analyses of 60 individual beads from one experiment 6 days after embedding of HUVEC-coated beads in a fibrin gel. Results are presented as means  SEM of the sprout number per bead. Phase contrast images depict one representative bead per treatment group. (B) Western blot analyses of TIMP-1 release into cell culture media by A549 cells (upper panel) or HUVEC (lower panel) treated with vehicle or the indicated cannabinoids at a final concentration of 3 mM for 48 h in EGM-2 medium. Values above the blots represent means  SEM of densitometric analyses in comparison with vehicle-treated cells (100%) in the absence of test substance of n = 3 experiments. (C) Viability analyses of HUVEC exposed to vehicle or 3 mM of the indicated cannabinoids in EGM-2 medium for 48 h. Results are indicated as white bars (direct effects). Gray bars (indirect effects) indicate viability of HUVEC exposed to EGM-2 medium obtained from A549 cells that were treated with vehicle or 3 mM of the indicated cannabinoid for 48 h. Incubation time of HUVEC was 48 h. Values are means  SEM of n = 3 (C, direct effects) or n = 4 (C, indirect effects) experiments. **P < 0.01, ***P < 0.001 vs. vehicle control; ANOVA plus post-hoc Dunnett test.

These findings implying an anti-angiogenic action of cannabinoids that requires the presence of tumor cells as trigger of antiangiogenesis represents a specific novel anti-angiogenic mechanism within the diverse antitumorigenic effects of cannabinoids. Cannabinoids may therefore represent an interesting tool in the clinic armamentarium for the treatment of lung cancer.

References [1] Folkman J. Tumor angiogenesis: therapeutic implications. N Engl J Med 1971;285:1182–6. [2] Folkman J. Anti-angiogenesis: new concept for therapy of solid tumors. Ann Surg 1972;175:409–16. [3] Ferrara N, Gerber HP, LeCouter J. The biology of VEGF and its receptors. Nat Med 2003;9:669–76. [4] Iacovelli R, Alesini D, Palazzo A, Trenta P, Santoni M, De Marchis L, et al. Targeted therapies and complete responses in first line treatment of metastatic renal cell carcinoma. a meta-analysis of published trials. Cancer Treat Rev 2014;40:271–5. [5] Freimuth N, Ramer R, Hinz B. Antitumorigenic effects of cannabinoids beyond apoptosis. J Pharmacol Exp Ther 2010;332:336–44. [6] Velasco G, Sa´nchez C, Guzma´n M. Towards the use of cannabinoids as antitumour agents. Nat Rev Cancer 2012;12:436–44. [7] Bla´zquez C, Casanova ML, Planas A, Go´mez Del Pulgar T, Villanueva C, Fer˜ ero MJ, et al. Inhibition of tumor angiogenesis by cannabinoids. na´ndez-Acen FASEB J 2003;17:529–31. ˜ ero [8] Casanova ML, Bla´zquez C, Martı´nez-Palacio J, Villanueva C, Ferna´ndez-Acen MJ, Huffman JW, et al. Inhibition of skin tumor growth and angiogenesis in vivo by activation of cannabinoid receptors. J Clin Invest 2003;111:43–50. [9] Kogan NM, Bla´zquez C, Alvarez L, Gallily R, Schlesinger M, Guzma´n M, et al. A cannabinoid quinone inhibits angiogenesis by targeting vascular endothelial cells. Mol Pharmacol 2006;70:51–9.

[10] Preet A, Ganju RK, Groopman JE. D9-Tetrahydrocannabinol inhibits epithelial growth factor-induced lung cancer cell migration in vitro as well as its growth and metastasis in vivo. Oncogene 2008;27:339–46. [11] Ramer R, Heinemann K, Merkord J, Rohde H, Salamon A, Linnebacher M, et al. COX-2 and PPAR-g confer cannabidiol-induced apoptosis of human lung cancer cells. Mol Cancer Ther 2013;12:69–82. [12] Portella G, Laezza C, Laccetti P, De Petrocellis L, Di Marzo V, Bifulco M. Inhibitory effects of cannabinoid CB1 receptor stimulation on tumor growth and metastatic spreading: actions on signals involved in angiogenesis and metastasis. FASEB J 2003;17:1771–3. [13] Bla´zquez C, Gonza´lez-Feria L, Alvarez L, Haro A, Casanova ML, Guzma´n M. Cannabinoids inhibit the vascular endothelial growth factor pathway in gliomas. Cancer Res 2004;64:5617–23. [14] Good DJ, Polverini PJ, Rastinejad F, Le Beau MM, Lemons RS, Frazier WA, et al. A tumor suppressor-dependent inhibitor of angiogenesis is immunologically and functionally indistinguishable from a fragment of thrombospondin. Proc Natl Acad Sci U S A 1990;87:6624–8. [15] Pisanti S, Borselli C, Oliviero O, Laezza C, Gazzerro P, Bifulco M. Anti-angiogenic activity of the endocannabinoid anandamide: correlation to its tumor-suppressor efficacy. J Cell Physiol 2007;211:495–503. [16] 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. [17] Zetter BR. Angiogenesis and tumor metastasis. Annu Rev Med 1998;49:407–24. [18] Murphy G, Gavrilovic J. Proteolysis and cell migration: creating a path? Curr Opin Cell Biol 1999;11:614–21. [19] Stetler-Stevenson WG. Matrix metalloproteinases in angiogenesis: a moving target for therapeutic intervention. J Clin Invest 1999;103:1237–41. [20] Moses MA, Sudhalter J, Langer R. Identification of an inhibitor of neovascularization from cartilage. Science 1990;248:1408–10. [21] Johnson MD, Kim HR, Chesler L, Tsao-Wu G, Bouck N, Polverini PJ. Inhibition of angiogenesis by tissue inhibitor of metalloproteinase. J Cell Physiol 1994;160:194–202. [22] Seandel M, Noack-Kunnmann K, Zhu D, Aimes RT, Quigley JP. Growth factorinduced angiogenesis in vivo requires specific cleavage of fibrillar type I collagen. Blood 2001;97:2323–32.

216

R. Ramer et al. / Biochemical Pharmacology 91 (2014) 202–216

[23] Ramer R, Hinz B. Inhibition of cancer cell invasion by cannabinoids via increased expression of tissue inhibitor of matrix metalloproteinases-1. J Natl Cancer Inst 2008;100:59–69. [24] Ramer R, Merkord J, Rohde H, Hinz B. Cannabidiol inhibits cancer cell invasion via upregulation of tissue inhibitor of matrix metalloproteinases-1. Biochem Pharmacol 2010;79:955–66. [25] Ramer R, Bublitz K, Freimuth N, Merkord J, Rohde H, Haustein M, et al. Cannabidiol inhibits lung cancer cell invasion and metastasis via intercellular adhesion molecule-1. FASEB J 2012;26:1535–48. [26] Ramer R, Hinz B. Cyclooxygenase-2 and tissue inhibitor of matrix metalloproteinases-1 confer the antimigratory effect of cannabinoids on human trabecular meshwork cells. Biochem Pharmacol 2010;80:846–57. [27] Pan Q, Pan H, Lou H, Xu Y, Tian L. Inhibition of the angiogenesis and growth of Aloin in human colorectal cancer in vitro and in vivo. Cancer Cell Int 2013;13:69. [28] Grant DS, Tashiro K, Segui-Real B, Yamada Y, Martin GR, Kleinman HK. Two different laminin domains mediate the differentiation of human endothelial cells into capillary like structures in vitro. Cell 1989;58:933–43. [29] Nakatsu MN, Sainson RC, Aoto JN, Taylor KL, Aitkenhead M, Pe´rez-del-Pulgar S, et al. Angiogenic sprouting and capillary lumen formation modeled by human umbilical vein endothelial cells (HUVEC) in fibrin gels: the role of fibroblasts and angiopoietin-1. Microvasc Res 2003;66:102–12. [30] Mukherjee S, Adams M, Whiteaker K, Daza A, Kage K, Cassar S, et al. Species comparison and pharmacological characterization of rat and human CB2 cannabinoid receptors. Eur J Pharmacol 2004;505:1–9. [31] Ramer R, Rohde A, Merkord J, Rohde H, Hinz B. Decrease of plasminogen activator inhibitor-1 may contribute to the anti-invasive action of cannabidiol on human lung cancer cells. Pharm Res 2010;27:2162–74. [32] Reddel RR, Ke Y, Gerwin BI, McMenamin MG, Lechner JF, Su RT, et al. Transformation of human bronchial epithelial cells by infection with SV40 or adenovirus-12 SV40 hybrid virus, or transfection via strontium phosphate coprecipitation with a plasmid containing SV40 early region genes. Cancer Res 1988;48:1904–9. [33] Amstad P, Reddel RR, Pfeifer A, Malan-Shibley L, Mark 3rd GE, Harris CC. Neoplastic transformation of a human bronchial epithelial cell line by a recombinant retrovirus encoding viral Harvey ras. Mol Carcinog 1988;1:151–60. [34] Wan YW, Raese RA, Fortney JE, Xiao C, Luo D, Cavendish J, et al. A smokingassociated 7-gene signature for lung cancer diagnosis and prognosis. Int J Oncol 2012;41:1387–96. [35] Cantarella G, Scollo M, Lempereur L, Saccani-Jotti G, Basile F, Bernardini R. Endocannabinoids inhibit release of nerve growth factor by inflammationactivated mast cells. Biochem Pharmacol 2011;82:380–8. [36] Song ZH, Zhong M. CB1 cannabinoid receptor-mediated cell migration. J Pharmacol Exp Ther 2000;294:204–9. [37] Yang H, Wang Z, Capo´-Aponte JE, Zhang F, Pan Z, Reinach PS. Epidermal growth factor receptor transactivation by the cannabinoid receptor (CB1) and transient receptor potential vanilloid 1 (TRPV1) induces differential responses in corneal epithelial cells. Exp Eye Res 2010;91:462–71. [38] Franklin A, Stella N. Arachidonylcyclopropylamide increases microglial cell migration through cannabinoid CB2 and abnormal-cannabidiol-sensitive receptors. Eur J Pharmacol 2003;474:195–8. [39] Walter L, Franklin A, Witting A, Wade C, Xie Y, Kunos G, et al. Nonpsychotropic cannabinoid receptors regulate microglial cell migration. J Neurosci 2003;23:1398–405. [40] Schmuhl E, Ramer R, Salamon A, Peters K, Hinz B. Increase of mesenchymal stem cell migration by cannabidiol via activation of p42/44 MAPK. Biochem Pharmacol 2014;87:489–501.

[41] Oudin MJ, Gajendra S, Williams G, Hobbs C, Lalli G, Doherty P. Endocannabinoids regulate the migration of subventricular zone-derived neuroblasts in the postnatal brain. J Neurosci 2011;31:4000–11. [42] Zhang X, Maor Y, Wang JF, Kunos G, Groopman JE. Endocannabinoid-like Narachidonoyl serine is a novel pro-angiogenic mediator. Br J Pharmacol 2010;160:1583–94. [43] Rajesh M, Mukhopadhyay P, Ba´tkai S, Hasko´ G, Liaudet L, Drel VR, et al. Cannabidiol attenuates high glucose-induced endothelial cell inflammatory response and barrier disruption. Am J Physiol Heart Circ Physiol 2007;293:H610–9. [44] Lu TS, Avraham HK, Seng S, Tachado SD, Koziel H, Makriyannis A, et al. Cannabinoids inhibit HIV-1 Gp120-mediated insults in brain microvascular endothelial cells. J Immunol 2008;181:6406–16. ˜ as L, Martı´nez-Orgado JA, Benito C, Milla´n A, Tolo´n RM, Romero [45] Ruiz-Valdepen J. Cannabidiol reduces lipopolysaccharide-induced vascular changes and inflammation in the mouse brain: an intravital microscopy study. J Neuroinflammation 2011;8:5. [46] Lo´pez-Miranda V, Herrado´n E, Gonza´lez C, Martı´n MI. Vascular toxicity of chemotherapeutic agents. Curr Vasc Pharmacol 2010;8:692–700. [47] Martin DC, Sanchez-Sweatman OH, Ho AT, Inderdeo DS, Tsao MS, Khokha R. Transgenic TIMP-1 inhibits simian virus 40T antigeninduced hepatocarcinogenesis by impairment of hepatocellular proliferation and tumor angiogenesis. Lab Invest 1999;79:225–34. [48] Guedez L, McMarlin AJ, Kingma DW, Bennett TA, Stetler-Stevenson M, StetlerStevenson WG. Tissue inhibitor of metalloproteinase-1 alters the tumorigenicity of Burkitt’s lymphoma via divergent effects on tumor growth and angiogenesis. Am J Pathol 2001;158:1207–15. [49] Ikenaka Y, Yoshiji H, Kuriyama S, Yoshii J, Noguchi R, Tsujinoue H, et al. Tissue inhibitor of metalloproteinases-1 (TIMP-1) inhibits tumor growth and angiogenesis in the TIMP-1 transgenic mouse model. Int J Cancer 2003;105:340–6. [50] Murphy AN, Unsworth EJ, Stetler-Stevenson WG. Tissue inhibitor of metalloproteinases-2 inhibits bFGF-induced human microvascular endothelial cell proliferation. J Cell Physiol 1993;157:351–8. [51] Montiel M, Urso L, de la Blanca EP, Marsigliante S, Jime´nez E. Cisplatin reduces endothelial cell migration via regulation of type 2 matrix metalloproteinase activity. Cell Physiol Biochem 2009;23:441–8. [52] Avalos BR, Kaufman SE, Tomonaga M, Williams RE, Golde DW, Gasson JC. K562 cells produce and respond to human erythroid-potentiating activity. Blood 1988;7:1720–5. [53] Chesler L, Golde DW, Bersch N, Johnson MD. Metalloproteinase inhibition and erythroid potentiation are independent activities of tissue inhibitor of metalloproteinases-1. Blood 1995;86:4506–15. [54] Jung KK, Liu XW, Chirco R, Fridman R, Kim HR. Identification of CD63 as a tissue inhibitor of metalloproteinase-1 interacting cell surface protein. EMBO J 2006;25:3934–42. [55] Chirco R, Liu XW, Jung KK, Kim HR. Novel functions of TIMPs in cell signaling. Cancer Metastasis Rev 2006;25:99–113. [56] Edwards DR, Rocheleau H, Sharma RR, Wills AJ, Cowie A, Hassell JA, et al. Involvement of AP1 and PEA3 binding sites in the regulation of murine tissue inhibitor of metalloproteinases-1 (TIMP-1) transcription. Biochim Biophys Acta 1992;1171:41–55. [57] Koyama Y, Tanaka Y, Saito K, Abe M, Nakatsuka K, Morimoto I, et al. Crosslinking of intercellular adhesion molecule 1 (CD54) induces AP-1 activation and IL-1b transcription. J Immunol 1996;157:5097–103.