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Abstract
In this study, we have assessed the activation of the cannabinoid CB2 receptor (CB2-R) in a model of mouse myocardial ischemia/reperfusion (I/R). The results show that treatment of animals with WIN55212-2, a CB1/CB2-R agonist, given 30 min before induction of I/R, significantly reduced the extent of infarct size (IS) in the area at risk, as measured 2.5 h later, with almost a 51% inhibition observed at the dose tested of 3.5 mg/kg intraperitoneally (i.p.). The protective effect of WIN55212-2 was almost abolished by the selective CB2-R antagonist AM630 (1 mg/kg i.p.) and not affected by the selective CB1-R antagonist AM251 (3 mg/kg i.p.). The CB2-R antagonist administered alone produced a slight but significant (P<0.05) increase in IS compared with vehicle alone. The protection afforded by WIN55212-2 was paralleled by lower values of myeloperoxidase activity and interleukin-1β and of the CXC chemokine ligand 8 into the injured tissue. In conclusion, we demonstrate for the first time that exogenous and endogenous CB2-R activation reduces the leukocyte-dependent myocardial damage associated with an I/R procedure.
INTRODUCTION
Myocardial infarct following experimental ischemia and reperfusion (I/R) has long been shown to be mediated by a number of inflammatory mediators and processes (for review, see refs. [1 , 2]).
The cannabinoids are an important group of substances newly discovered to be anti-inflammatory mediators [3 4 5 6 7]. The cabnnabinoids are the active component of cannabis sativa and act in part as pan-cytokine inhibitors. Several papers have shown that cannabinoids or cannabinoid receptor (CB-R) agonists inhibit cytokine synthesis and their release from target cells in vitro and also reduce cytokine levels in inflammatory exudates [4 , 8 , 9]. Natural and synthetic cannabinoids are effective in modulating pain [5 6 7] and down-regulating the host inflammatory response by inhibiting the generation and release of proinflammatory cytokine and mediators, thus playing a putative role in maintaining homeostasis.
Two CB-Rs have been identified so far, and recent data suggest that the type 2 or CB2-R, is the main peripheral molecular target responsible for the inhibitory properties of the cannabinoids [10 , 11] and more importantly, for the cardioprotection afforded by these compounds [12 13 14 15] in models of inflammation-based tissue damage. However, a clear molecular mechanism has not been identified underlying cardioprotection afforded by CB2-R stimulation.
The aim of this present study is to expand the knowledge on the potential, protective actions of CB2-R activation in models of inflammation-mediated tissue injury. To address this, we have used an integrated approach with pharmacological and biochemical assays being used to challenge the hypothesis that CB2-Rs could be targeted to achieve beneficial effects in a mouse model of heart infarct.
MATERIALS AND METHODS
Male C57Bl.6 mice (20—22 g body weight) were purchased from Tuck (Battlesbridge, Essex, UK) and maintained on a standard chow pellet diet with tap water ad libitum using a 12-h light/dark cycle. Animal experimental work was performed according to the Animal Care and Use Committee of the Medical College of Naples (Italy).
In vivo I/R injury of the mouse heart
Surgical procedure
A model successfully used in the rat [16] was modified to be used in mice. Animals were anesthetized with Inactin [100 mg/kg intraperitoneally (i.p.)+30 mg/kg i.p. supplemental dose if required; RBI, St. Albans, UK] before coronary artery occlusion. A tracheotomy was performed using a polythene cannula to permit artificial ventilation (Harvard 50-1718, Edenbridge, Kent, UK), and the left jugular vein was cannulated to allow administration of further anesthetic. The tidal volume of the respirator was set at 1.0 ml/min, and the rate was set at 110 strokes/min and supplemented with 100% oxygen. The right carotid artery was cannulated for blood pressure measurement. After an equilibration period of 20 min, a left thoracotomy was performed (between the fourth and the fifth ribs, ∼3 mm from the sternum), and the pericardium was removed to expose the heart. The chest walls were retracted by use of a 5-0 or 6-0 silk or monofilament suture, and slight rotation of the mouse to the right allowed for better exposure of the left ventricle (LV). The left auricle was slightly retracted, exposing the entire left main coronary artery system. Ligation of the left anterior descending coronary artery (LADCA) was performed using a 7-0 silk suture (attached to a 10-mm micropoint reverse cutting needle; W593 7/0 BVl, Ethicon, Edinburgh, UK). The mean arterial blood pressure (MABP) was continuously recorded by a MacLab system (Ugo Basile, Comerio VA, Italy). The heart rate (HR) was automatically calculated from the blood pressure. A rectal thermometer was inserted, and the mice were kept at a constant body temperature of 37—38°C by a homeothermic blanket.
After completion of the surgical procedures, mice were allowed to stabilize for 30 min before occlusion of LADCA. Both ends of the ligature around the coronary artery were threaded through a small polythene button placed in contact with the heart. Coronary artery occlusion was achieved by applying tension to it and clamping the ligature against the button with a small, light-weight, rubber-sheathed artery clip, without damaging the artery. After a 25-min period of myocardial ischemia, the clip was removed so that the tension on the ligature was released, and reperfusion occurred for 2 h.
Measurement of area at risk (AR) and infarct size (IS)
At the end of the reperfusion period, LADCA was reoccluded 2 h after the reperfusion period, and Evans blue dye [0.5 ml of a 1% w/v solution in phosphate-buffered saline (PBS); Sigrna-Aldrich, Poole, Dorset, UK] was injected into the carotid artery catheter to delineate the AR. The heart was removed, and the LV was excised and weighed. After this procedure, the heart was sectioned transversely into five sections; one section was made at the site of ligature, and each section was weighed. Sections of the ventricle above the site of the ligature were uniformly completely blue. Sections of the ventricle from the level of the ligature to the apex that were not blue (defining the AR) were then incubated in 1.5% w/v triphenyltetrazolium chloride in PBS (Sigma-Aldrich) staining; viable myocardium stains brick red, and the infarct appears pale white. The area of infarction for each slice was determined by computerized planimetry using an image analysis software program (NIH Image Software, Bethesda, MD). Color enhancement was used to accentuate the differences among areas stained in blue or red or which remain pale. The size of infarction was determined by the following equation: weight of infarction = (Al—WT1) + (A2—WT2) + (A3—WT3) + (A4—WT4), where A is percent area of infarction by planimetry from subscripted numbers 1—4 representing section, and WT is weight of the same numbered sections. Percentage of infarcted LV is obtained from (WT of infarction/WT of LV) — 100.
AR is then obtained as percentage of LV calculated by (WT of LV—WT of LV stained blue)/WT of LV [17]. The weight of LV stained blue was calculated in a similar manner by the sum of products of the percent area of each slice subtracted by the weight of the respective slice.
Drug treatment
WIN55212-2 {(R)-(+)-[2,3-dihydro-5-methyl-3-[(4-morpholinyl)methyl]pyrrolo[1,2,3-de]1,4-benzoxazin-6-yl](1-naphthyl)methanone}was purchased from Research Biochemicals International (Natick, MA); the potent and selective antagonist of the CB1-R, AM251, 1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-L-1-piperidinil-1H-pyrazole-3-carboxamide trifluoroacetate salt, was purchased from Sigma-Aldrich (Milan, Italy), and AM630, CB2-R antagonist/inverse agonist, [6-iodo-2-methyl-1-[2-(4-morpholinyl-)ethyl]-1H-indol-3-yl](4-methoxyphenyl)methanone, was from Tocris Cookson Ltd. (Avonmouth, UK).
WIN55212-2 was prepared in methylcellulose and administered i.p. AM251 and AM630 were prepared in saline containing 5% propylene glycol and 2% Tween 80 and were administered i.p.
Cohorts of mice were allocated randomly to one of the six following groups: vehicle control; WIN55212-2 (nonselective CB1/CB2-R agonists, 3.5 mg/kg i.p.); WIN55212-2 + AM251 (3 mg/kg i.p.); WIN55212-2 + AM630 (1 mg/kg i.p.); two different groups of mice treated with the CB1-R and the CB2-R antagonists alone. All drugs were administered 30 min before the I/R procedure when given alone. In contrast, AM251 and AM630 were also administered 30 min before WIN55212-2 in the antagonist experiments. All doses of drugs were chosen and prepared in accordance with those shown to be active in other models of inflammation [4 , 18 19 20 21].
The number (n=6) of animals used in each group refers to the mice that survived up to the end of the reperfusion period. Overall mortality was 4% throughout the entire study.
Biochemical determinations
Selected experiments were repeated with n = 5 mice per group. Following the I/R injury procedure, the LV was excised without performing any staining procedure, and tissue was frozen at −80°C before the analyses described below. The tail vein also took blood samples (0.25—0.2 ml) into a heparinized Eppendorff vial, and aliquots were used for peripheral blood counts [16].
Myeloperoxidase (MPO) activity
The MPO reaction was performed as described by Mullane et al. [22], using a total volume of 200 μl containing 2 mM 3,3′,5,5′-tetramethyl-benzidine (Sigma-Aldrich, Poole, Dorset, UK and Milan, Italy) in 50 mM acetate buffer (pH 6) as a substrate and hydrogen peroxide (20 μl of a solution 30% v/v). Absorbance was read at 620 nm wavelength and interpolated on a standard curve constructed with 0—2000 mU/l human neutrophil MPO (Calbiochem, Nottingham, UK). Data are reported as units of MPO activity per gram of tissue protein (U/g).
A standard curve was constructed with different numbers of mouse neutrophils that were elicited into the peritoneal cavity at 4 h in response to application of 5 ml thioglycollate [23]. Pellets were subjected to three cycles of freeze-thawing. Samples (100 μl) were obtained after a final centrifugation at 13,000 rpm for 30 min on a benchfuge. The MPO reaction was produced with a polymorphonuclear neutrophil (PMN) number ranging from 0 to 225 × 103, obtaining a linear relationship with the absorbance at 620 nm following the equation: A620 = 4.504 × no. of PMN + 33.74, with a R2 = 0.97983. Data are reported as equivalent numbers of PMN per mg of tissue.
Cytokine quantification by enzyme-linked immunosorbent assay (ELISA)
Tissue interleukin (IL)-1β levels were determined using a commercially available ELISA, specific for the mouse cytokine, purchased from R&D Systems (Abingdon, UK). In brief, plasma and tissue supernatant aliquots (50 μl) were assayed for IL-1β and compared with a standard curve constructed with 0—1 ng/ml of the standard cytokine. The ELISA showed negligible (<1%) cross-reactivity with several murine cytokines and chemokines (data as furnished by the manufacturer). A similar procedure was followed for determination of the CXC chemokine ligand (CXCL)8 by ELISA (R&D Systems) and was used according to the manufacturer's instructions.
Parameters of systemic activation
Hemodynamic parameters
Following measurement of the MABP (mmHg), HR was calculated from the blood pressure trace and expressed in beats/min−1. The pressure rate index (PRI), a relative indicator of myocardial oxygen consumption [24], was calculated as the product of MABP and HR and was expressed in mmHg/min/103.
Leukocyte profile
Total white cell counts were determined using a standard in vivo protocol [16]. Differential counts were obtained from blood smears stained with Turk's solution (crystal violet 0.01% w/v in acetic acid 3% v/v), and the total number of each leukocyte type was then calculated. Data are reported as 106 cells per ml blood.
CD11b levels on circulating neutrophils
Fluorescein-activated cell sorter analysis was performed using a whole blood protocol [16]. Briefly, blood aliquots (200 μl) were incubated with 5 μg/ml of a specific rat anti-mouse CD11b monoclonal antibody (clone 5C6; Serotec, Oxord, UK) for 60 min on ice. Control tubes received the same amounts of rat immunoglobulin G (IgG). After two washes in PBS, cells were then stained with a fluorescein isothiocyanate-conjugate F(ab′)2 fragment of goat anti-rat IgG antibody for 5 min at room temperature. After two further washes in PBS, red blood cell lysis was performed with Immuno-Lyse (Coulter, Luton, UK) for 1 min. For storage purposes, cells were fixed with an equal volume of 2% paraformaldehyde in PBS. Three distinct peripheral leucocyte populations (lymphocytes, monocytes, and polymorphonuclear leucocytes) were discriminated using forward- and side-scatter characteristics. Data were analyzed as mean fluorescence intensity (MFI) units measured in an FL1 (green) channel (wavelength of 530 nm).
Statistical analysis
All values are expressed as mean ± SEM of number (n) of mice for the in vivo experiments. Statistical analysis was assessed by Student's t-test (when only two groups were compared) or one-way ANOVA followed by Dunnett's test (more than two experimental groups). A probability P value less than 0.05 was considered significant to reject the null hypothesis.
RESULTS
In vivo myocardial injury
Occlusion of the LADCA and subsequent reperfusion produced a marked damage in the mouse LV, which was reliably measured at the 2-h time-point (Fig. 1 ). Evans blue staining underlying the AR showed 52% of the LV remained unstained (Fig. 1A) , and ∼46% of this portion of the ventricle was infarcted (Fig. 1B) , giving a ratio of IS/LV between 30 and 35% (Fig. 1C) .
Administration (i.p.) of the CB1/CB2-R agonist WIN55212-2, 30 min before the I/R period, afforded a marked protection against the myocardial injury, measured 2 h later, as estimated by the IS and the ratio of IS/LV (Fig. 1B and 1C) . A protective effect of 51% was obtained (51.1% of inhibition vs. vehicle-treated mice, n=6, P<0.01). In contrast, WIN55212-2 treatment did not alter the AR values (Fig. 1A) . The protective effect of WIN55212-2 on IS was abolished by 30 min pretreatment with the CB2-R antagonist AM630 and was not affected by the CB1-R antagonist AM251 (Fig. 1B) . The CB2 compound (AM630) but not the CB1 antagonist (AM251) slightly, but significantly (P<0.05), increased the IS/AR ratio and the IS/LV value when administered alone, before I/R (Fig. 1B and 1C) .
Occlusion and subsequent reperfusion of LADCA (control vehicle) did not produce a marked modification in MABP, HR, or PRI with regard to the values measured 5 min prior to occlusion (Table 1 ). Mice treated with WIN55212-2 produced a slight, but not significant, decrease of MABP and HR with respect to the group treated with vehicle (Table 1) . No effects on these parameters were seen following treatment with the CB1-R antagonist or the CB2-R antagonist (Table 1) .
PMN-associated myocardial damage
Parallel changes in MPO activity measured in the AR and myocardial injury were observed in the mice subjected to the I/R procedure. Figure 2 shows this increase in MPO activity in the infarcted heart versus the hearts collected from vehicle-control mice. Administration of WIN55212-2, given here at the doses that produced a significant reduction in IS, produced a parallel attenuation in MPO activity, as measured 2 h post-reperfusion (Fig. 2) . This attenuation in MPO activity was absent in the mice treated with the WIN55212-2 + AM630 but was still present in the mice treated with WIN55212-2 + AM251. AM630 given alone to the mice significantly (P<0.05) increased the levels of MPO activity with respect to those expressed in the vehicle-control mice (Fig. 2) . Sham operation did not alter the MPO activity from the baseline level of 115 ± 23 × 103 PMN equivalent per mg of protein (n=5).
In separate experiments, we related the MPO activity with the extension of the injured area into the myocardium. For this, we performed some separate experiments measuring the MPO values and the IS, 30 min post-reperfusion. In these experiments, MPO activity was 463 × 103 PMN equivalent per mg of protein (n=5), and the myocardial parameters were as follows: AR, 50 ± 3.2%; IS/AR, 11.2 ± 2%; IS/LV, 7.6 ± 0.4% (n=5 in all cases, P<0.01 vs. 2-h values shown in Fig. 1 ). Together, these data underlie a causal relationship between PMN influx and heart damage.
Cytokine levels in the infarcted myocardium
I/R of the heart showed levels of the two cytokines, IL-1β and CXCL8, up to values of 137 ± 18 pg/mg tissue and 725 ± 50 pg/mg tissue, respectively. Treatment of mice with the CB1/CB2-R agonist WIN55212-2 reduced CXCL8 by 60% and IL-1β by 49% following I/R (Fig. 3 ). The CB2-R antagonist AM630 reversed this inhibitory effect of WIN55212-2 and was unaffected by the CB1-R antagonist AM251 (Fig. 3) . It is interesting that AM630 caused a slight increase in IL-1β and CXCL8 compared with control (infarcted; Fig. 3 ). Sham-operated mice (n=4) showed values of IL-1β and CXCL8 of 22 ± 5 pg/mg tissue and 156 ± 12 pg/mg tissue, respectively.
Effects of CB-R activation on systemic PMN counts and CD11b expression
I/R of the LADCA was associated with alterations in circulating leukocytes. A neutrophilia was observed at the end of the reperfusion period with respect to the values of PMN counted 5 min before the induction of the I/R procedure (Fig. 4 ). Treatment with WIN5521-2 (3.5 mg/kg) did not modify the number of circulating PMN at any of the time-points tested (Fig. 4) . Similarly, none of the CB-R antagonists tested here, alone or in conjunction with WIN55212-2, affected the PMN counts (data not shown).
In contrast to the observed increase in PMN counts, the I/R induced a significant reduction in the number of circulating lymphocytes while not affecting the number of monocytes when compared with the values obtained 5 min before ischemia (Table 2 ). None of the treatments tested have modified these effects.
I/R-induced neutrophilia was associated with PMN activation, as indicated by the increase in cell-surface CD11b. A marked up-regulation of this β2-integrin was detected at the end of the reperfusion period with an increase of 60 ± 3% with respect to the 5-min preocclusion value (Fig. 5 ). WIN55212-2 (3.5 mg/kg) did not significantly change the CD11b expression with respect to vehicle-treated mice at any time point considered (Fig. 5) . Similarly, AM251 (3 mg/kg) and AM630 (1 mg/kg) did not affect systemic blood counts (data not shown).
DISCUSSION
The present study demonstrates that CB2-R activation protects against myocardial damage associated with I/R. The inhibitory effects produced by activation of this receptor are functionally related to a reduced recruitment of blood-borne PMNs into the damaged tissue, and the end-point is cardioprotection.
Since the seminal studies on cannabinoids, their pharmacological action has been attributed to central and peripheral actions, such as analgesia, anticonvulsion, anti-inflammation, alleviation of intraocular pressure and emesis, and attenuation of cardiovascular pathologies [25]. These effects are mediated by the activation of specific G protein-coupled receptors [26 , 27], characterized and cloned from mammalian tissues CB1 [28] and CB2 [29]. The central and most of the peripheral effects of cannabinoids rely on CB1-R activation. This receptor is found in high abundance in the central nervous system, where it mediates cannabinoid psychoactivity. It is also present in peripheral nerve terminals, as well as in extra-neural sites, such as testis, uterus, vascular endothelium, eye, spleen, and tonsils [26 27 28 29 30]. By contrast, the CB2-R is expressed in cells and organs of the immune system with a rank order of B cells > natural killer > monocytes > polymorphonuclear neutrophil cells > T8 cells [31] and is unrelated to cannabinoid psychoactivity [26 , 27]. In addition, macrophages express high levels of CB2-Rs [32].
Lagneux and Lamontagne [12] and Krylatov et al. [13 , 14] have proposed a pivotal role for the CB2-R, having inhibitory effects on cardiovascular pathologies. In these studies, they have reported the ability of CB2-R ligands to attenuate myocardial arrhythmias and myocardial damage following I/R.
In the present study, the synthetic ligand of the CB2-R WIN55212-2, which displays sevenfold higher affinity for the CB2-R over CB1-R [33], was administered 30 min before induction of the I/R procedure, producing a reduction in the tissue damage measured as IS or IS/LV 2 h later in the mouse heart being observed. The effect of the agonist was pronounced with almost a 52% inhibition of the injured area. The protective effect of WIN55212-2 was almost completely abolished by the highly selective CB2 antagonist AM630 and was unaffected by the CB1 antagonist AM251, thus suggesting that CB2-Rs are the major receptors involved.
The data obtained with AM251 in this present study are of particular importance, as they suggest that AM251 or CB1-R activation is not involved in altering the outcome of myocardial experimental I/R pathology. However, it should be noted that CB1s have been implicated in protection against brain I/R injury [34].
The molecular mechanisms behind CB-2R-mediated cardioprotection are difficult to dissect in the present working system. However, based on the evidence that I/R of the mouse heart was associated with a pronounced increase in the levels of MPO, a marker used to monitor tissue infiltration by PMNs [22], we propose that the anti-inflammatory properties of the CB2 agonist are responsible for its attenuation of I/R-induced heart injury.
The causal link between recruitment of white blood cells to the myocardium and subsequent damage was confirmed by the inhibition produced by the WIN55212-2 on local generation of leukocyte activators, cytokines, and chemokines, which are known to promote the leukocyte-endothelium interaction (for review, see ref. [35]).
We chose to investigate IL-1β as an example of a multipotent cytokine, able to increase the adhesive properties of the endothelial wall [36 , 37], and CXCL8 as an example of a chemokine able to recruit neutrophils in rodent species during experimental inflammation [22 , 38 , 39]. In addition, IL-1β has been implicated in the pathology associated with experimental myocardial infarction [40 , 41]. Occlusion and reopening of the LADCA led to a marked increase in IL-1β and CXCL8. The fact that WIN55212-2 produced a significant inhibition in these mediators together with the fact that AM630 slightly increased them may suggest a reduced PMN accumulation, possibly via reduced macrophage activation. In fact, activated PMNs are one of the sources of IL-1β [42], but macrophages can be a major cell source of these cytokines during an ongoing, inflammatory reaction [43]. It is interesting that several studies about mouse and rat peritoneal macrophages and cell lines in culture showed that various CB-R ligands affect macrophage functions including phagocytosis, cytolisis, and protein expression [44 , 45], and CB2-R activation leads to reduced activation/migration of macrophages in vitro [10].
Therefore, the inhibitory effect on IL-1β and CXCL8 production seen with the CB2-R agonist treatment may explain, at least in part, the reduced degree of leukocyte accumulation. This is likely to be a local effect, as in contrast to the protection afforded in the heart by treatment of rats with the CB2 agonist, no such reduction was seen on the systemic alterations provoked by the I/R procedure. As expected, the reperfusion phase was characterized by systemic leukocytosis [46], and it is noteworthy that changes in neutrophil numbers and in their CD11b expression on the plasma membrane (as a marker of generalized cell activation) occurred in parallel over the time course analyzed. In fact, ischemia itself produced a modest but significant increase in both parameters, and they were augmented in a more marked way at the end of the reperfusion period [16]. Treatment of rats with WIN55212-2 at a dose that was effective in reducing myocardial infarction did not modify systemic alterations linked to this experimental pathology.
In conclusion, we have demonstrated a novel action for the CB2-R. Its activation inhibits the leukocyte-dependent damage of an infarcted myocardium. The molecular mechanism(s) responsible for these actions of CB2-R remains elusive. However, we wish to suggest a decreased macrophage activation and reduced cytokine/chemokine-induced chemotaxis of PMN into the injured tissue. Although further investigations are warranted, we cannot exclude involvement of a direct mechanism involving the CB2-R present on the PMN cell surface [31], possibly affecting the adhesion/migration of PMN in these experimental conditions.
Source, Graphs and Figures: Cannabinoid CB2 receptor activation reduces mouse myocardial ischemia-reperfusion injury: involvement of cytokine/chemokines and PMN
In this study, we have assessed the activation of the cannabinoid CB2 receptor (CB2-R) in a model of mouse myocardial ischemia/reperfusion (I/R). The results show that treatment of animals with WIN55212-2, a CB1/CB2-R agonist, given 30 min before induction of I/R, significantly reduced the extent of infarct size (IS) in the area at risk, as measured 2.5 h later, with almost a 51% inhibition observed at the dose tested of 3.5 mg/kg intraperitoneally (i.p.). The protective effect of WIN55212-2 was almost abolished by the selective CB2-R antagonist AM630 (1 mg/kg i.p.) and not affected by the selective CB1-R antagonist AM251 (3 mg/kg i.p.). The CB2-R antagonist administered alone produced a slight but significant (P<0.05) increase in IS compared with vehicle alone. The protection afforded by WIN55212-2 was paralleled by lower values of myeloperoxidase activity and interleukin-1β and of the CXC chemokine ligand 8 into the injured tissue. In conclusion, we demonstrate for the first time that exogenous and endogenous CB2-R activation reduces the leukocyte-dependent myocardial damage associated with an I/R procedure.
INTRODUCTION
Myocardial infarct following experimental ischemia and reperfusion (I/R) has long been shown to be mediated by a number of inflammatory mediators and processes (for review, see refs. [1 , 2]).
The cannabinoids are an important group of substances newly discovered to be anti-inflammatory mediators [3 4 5 6 7]. The cabnnabinoids are the active component of cannabis sativa and act in part as pan-cytokine inhibitors. Several papers have shown that cannabinoids or cannabinoid receptor (CB-R) agonists inhibit cytokine synthesis and their release from target cells in vitro and also reduce cytokine levels in inflammatory exudates [4 , 8 , 9]. Natural and synthetic cannabinoids are effective in modulating pain [5 6 7] and down-regulating the host inflammatory response by inhibiting the generation and release of proinflammatory cytokine and mediators, thus playing a putative role in maintaining homeostasis.
Two CB-Rs have been identified so far, and recent data suggest that the type 2 or CB2-R, is the main peripheral molecular target responsible for the inhibitory properties of the cannabinoids [10 , 11] and more importantly, for the cardioprotection afforded by these compounds [12 13 14 15] in models of inflammation-based tissue damage. However, a clear molecular mechanism has not been identified underlying cardioprotection afforded by CB2-R stimulation.
The aim of this present study is to expand the knowledge on the potential, protective actions of CB2-R activation in models of inflammation-mediated tissue injury. To address this, we have used an integrated approach with pharmacological and biochemical assays being used to challenge the hypothesis that CB2-Rs could be targeted to achieve beneficial effects in a mouse model of heart infarct.
MATERIALS AND METHODS
Male C57Bl.6 mice (20—22 g body weight) were purchased from Tuck (Battlesbridge, Essex, UK) and maintained on a standard chow pellet diet with tap water ad libitum using a 12-h light/dark cycle. Animal experimental work was performed according to the Animal Care and Use Committee of the Medical College of Naples (Italy).
In vivo I/R injury of the mouse heart
Surgical procedure
A model successfully used in the rat [16] was modified to be used in mice. Animals were anesthetized with Inactin [100 mg/kg intraperitoneally (i.p.)+30 mg/kg i.p. supplemental dose if required; RBI, St. Albans, UK] before coronary artery occlusion. A tracheotomy was performed using a polythene cannula to permit artificial ventilation (Harvard 50-1718, Edenbridge, Kent, UK), and the left jugular vein was cannulated to allow administration of further anesthetic. The tidal volume of the respirator was set at 1.0 ml/min, and the rate was set at 110 strokes/min and supplemented with 100% oxygen. The right carotid artery was cannulated for blood pressure measurement. After an equilibration period of 20 min, a left thoracotomy was performed (between the fourth and the fifth ribs, ∼3 mm from the sternum), and the pericardium was removed to expose the heart. The chest walls were retracted by use of a 5-0 or 6-0 silk or monofilament suture, and slight rotation of the mouse to the right allowed for better exposure of the left ventricle (LV). The left auricle was slightly retracted, exposing the entire left main coronary artery system. Ligation of the left anterior descending coronary artery (LADCA) was performed using a 7-0 silk suture (attached to a 10-mm micropoint reverse cutting needle; W593 7/0 BVl, Ethicon, Edinburgh, UK). The mean arterial blood pressure (MABP) was continuously recorded by a MacLab system (Ugo Basile, Comerio VA, Italy). The heart rate (HR) was automatically calculated from the blood pressure. A rectal thermometer was inserted, and the mice were kept at a constant body temperature of 37—38°C by a homeothermic blanket.
After completion of the surgical procedures, mice were allowed to stabilize for 30 min before occlusion of LADCA. Both ends of the ligature around the coronary artery were threaded through a small polythene button placed in contact with the heart. Coronary artery occlusion was achieved by applying tension to it and clamping the ligature against the button with a small, light-weight, rubber-sheathed artery clip, without damaging the artery. After a 25-min period of myocardial ischemia, the clip was removed so that the tension on the ligature was released, and reperfusion occurred for 2 h.
Measurement of area at risk (AR) and infarct size (IS)
At the end of the reperfusion period, LADCA was reoccluded 2 h after the reperfusion period, and Evans blue dye [0.5 ml of a 1% w/v solution in phosphate-buffered saline (PBS); Sigrna-Aldrich, Poole, Dorset, UK] was injected into the carotid artery catheter to delineate the AR. The heart was removed, and the LV was excised and weighed. After this procedure, the heart was sectioned transversely into five sections; one section was made at the site of ligature, and each section was weighed. Sections of the ventricle above the site of the ligature were uniformly completely blue. Sections of the ventricle from the level of the ligature to the apex that were not blue (defining the AR) were then incubated in 1.5% w/v triphenyltetrazolium chloride in PBS (Sigma-Aldrich) staining; viable myocardium stains brick red, and the infarct appears pale white. The area of infarction for each slice was determined by computerized planimetry using an image analysis software program (NIH Image Software, Bethesda, MD). Color enhancement was used to accentuate the differences among areas stained in blue or red or which remain pale. The size of infarction was determined by the following equation: weight of infarction = (Al—WT1) + (A2—WT2) + (A3—WT3) + (A4—WT4), where A is percent area of infarction by planimetry from subscripted numbers 1—4 representing section, and WT is weight of the same numbered sections. Percentage of infarcted LV is obtained from (WT of infarction/WT of LV) — 100.
AR is then obtained as percentage of LV calculated by (WT of LV—WT of LV stained blue)/WT of LV [17]. The weight of LV stained blue was calculated in a similar manner by the sum of products of the percent area of each slice subtracted by the weight of the respective slice.
Drug treatment
WIN55212-2 {(R)-(+)-[2,3-dihydro-5-methyl-3-[(4-morpholinyl)methyl]pyrrolo[1,2,3-de]1,4-benzoxazin-6-yl](1-naphthyl)methanone}was purchased from Research Biochemicals International (Natick, MA); the potent and selective antagonist of the CB1-R, AM251, 1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-L-1-piperidinil-1H-pyrazole-3-carboxamide trifluoroacetate salt, was purchased from Sigma-Aldrich (Milan, Italy), and AM630, CB2-R antagonist/inverse agonist, [6-iodo-2-methyl-1-[2-(4-morpholinyl-)ethyl]-1H-indol-3-yl](4-methoxyphenyl)methanone, was from Tocris Cookson Ltd. (Avonmouth, UK).
WIN55212-2 was prepared in methylcellulose and administered i.p. AM251 and AM630 were prepared in saline containing 5% propylene glycol and 2% Tween 80 and were administered i.p.
Cohorts of mice were allocated randomly to one of the six following groups: vehicle control; WIN55212-2 (nonselective CB1/CB2-R agonists, 3.5 mg/kg i.p.); WIN55212-2 + AM251 (3 mg/kg i.p.); WIN55212-2 + AM630 (1 mg/kg i.p.); two different groups of mice treated with the CB1-R and the CB2-R antagonists alone. All drugs were administered 30 min before the I/R procedure when given alone. In contrast, AM251 and AM630 were also administered 30 min before WIN55212-2 in the antagonist experiments. All doses of drugs were chosen and prepared in accordance with those shown to be active in other models of inflammation [4 , 18 19 20 21].
The number (n=6) of animals used in each group refers to the mice that survived up to the end of the reperfusion period. Overall mortality was 4% throughout the entire study.
Biochemical determinations
Selected experiments were repeated with n = 5 mice per group. Following the I/R injury procedure, the LV was excised without performing any staining procedure, and tissue was frozen at −80°C before the analyses described below. The tail vein also took blood samples (0.25—0.2 ml) into a heparinized Eppendorff vial, and aliquots were used for peripheral blood counts [16].
Myeloperoxidase (MPO) activity
The MPO reaction was performed as described by Mullane et al. [22], using a total volume of 200 μl containing 2 mM 3,3′,5,5′-tetramethyl-benzidine (Sigma-Aldrich, Poole, Dorset, UK and Milan, Italy) in 50 mM acetate buffer (pH 6) as a substrate and hydrogen peroxide (20 μl of a solution 30% v/v). Absorbance was read at 620 nm wavelength and interpolated on a standard curve constructed with 0—2000 mU/l human neutrophil MPO (Calbiochem, Nottingham, UK). Data are reported as units of MPO activity per gram of tissue protein (U/g).
A standard curve was constructed with different numbers of mouse neutrophils that were elicited into the peritoneal cavity at 4 h in response to application of 5 ml thioglycollate [23]. Pellets were subjected to three cycles of freeze-thawing. Samples (100 μl) were obtained after a final centrifugation at 13,000 rpm for 30 min on a benchfuge. The MPO reaction was produced with a polymorphonuclear neutrophil (PMN) number ranging from 0 to 225 × 103, obtaining a linear relationship with the absorbance at 620 nm following the equation: A620 = 4.504 × no. of PMN + 33.74, with a R2 = 0.97983. Data are reported as equivalent numbers of PMN per mg of tissue.
Cytokine quantification by enzyme-linked immunosorbent assay (ELISA)
Tissue interleukin (IL)-1β levels were determined using a commercially available ELISA, specific for the mouse cytokine, purchased from R&D Systems (Abingdon, UK). In brief, plasma and tissue supernatant aliquots (50 μl) were assayed for IL-1β and compared with a standard curve constructed with 0—1 ng/ml of the standard cytokine. The ELISA showed negligible (<1%) cross-reactivity with several murine cytokines and chemokines (data as furnished by the manufacturer). A similar procedure was followed for determination of the CXC chemokine ligand (CXCL)8 by ELISA (R&D Systems) and was used according to the manufacturer's instructions.
Parameters of systemic activation
Hemodynamic parameters
Following measurement of the MABP (mmHg), HR was calculated from the blood pressure trace and expressed in beats/min−1. The pressure rate index (PRI), a relative indicator of myocardial oxygen consumption [24], was calculated as the product of MABP and HR and was expressed in mmHg/min/103.
Leukocyte profile
Total white cell counts were determined using a standard in vivo protocol [16]. Differential counts were obtained from blood smears stained with Turk's solution (crystal violet 0.01% w/v in acetic acid 3% v/v), and the total number of each leukocyte type was then calculated. Data are reported as 106 cells per ml blood.
CD11b levels on circulating neutrophils
Fluorescein-activated cell sorter analysis was performed using a whole blood protocol [16]. Briefly, blood aliquots (200 μl) were incubated with 5 μg/ml of a specific rat anti-mouse CD11b monoclonal antibody (clone 5C6; Serotec, Oxord, UK) for 60 min on ice. Control tubes received the same amounts of rat immunoglobulin G (IgG). After two washes in PBS, cells were then stained with a fluorescein isothiocyanate-conjugate F(ab′)2 fragment of goat anti-rat IgG antibody for 5 min at room temperature. After two further washes in PBS, red blood cell lysis was performed with Immuno-Lyse (Coulter, Luton, UK) for 1 min. For storage purposes, cells were fixed with an equal volume of 2% paraformaldehyde in PBS. Three distinct peripheral leucocyte populations (lymphocytes, monocytes, and polymorphonuclear leucocytes) were discriminated using forward- and side-scatter characteristics. Data were analyzed as mean fluorescence intensity (MFI) units measured in an FL1 (green) channel (wavelength of 530 nm).
Statistical analysis
All values are expressed as mean ± SEM of number (n) of mice for the in vivo experiments. Statistical analysis was assessed by Student's t-test (when only two groups were compared) or one-way ANOVA followed by Dunnett's test (more than two experimental groups). A probability P value less than 0.05 was considered significant to reject the null hypothesis.
RESULTS
In vivo myocardial injury
Occlusion of the LADCA and subsequent reperfusion produced a marked damage in the mouse LV, which was reliably measured at the 2-h time-point (Fig. 1 ). Evans blue staining underlying the AR showed 52% of the LV remained unstained (Fig. 1A) , and ∼46% of this portion of the ventricle was infarcted (Fig. 1B) , giving a ratio of IS/LV between 30 and 35% (Fig. 1C) .
Administration (i.p.) of the CB1/CB2-R agonist WIN55212-2, 30 min before the I/R period, afforded a marked protection against the myocardial injury, measured 2 h later, as estimated by the IS and the ratio of IS/LV (Fig. 1B and 1C) . A protective effect of 51% was obtained (51.1% of inhibition vs. vehicle-treated mice, n=6, P<0.01). In contrast, WIN55212-2 treatment did not alter the AR values (Fig. 1A) . The protective effect of WIN55212-2 on IS was abolished by 30 min pretreatment with the CB2-R antagonist AM630 and was not affected by the CB1-R antagonist AM251 (Fig. 1B) . The CB2 compound (AM630) but not the CB1 antagonist (AM251) slightly, but significantly (P<0.05), increased the IS/AR ratio and the IS/LV value when administered alone, before I/R (Fig. 1B and 1C) .
Occlusion and subsequent reperfusion of LADCA (control vehicle) did not produce a marked modification in MABP, HR, or PRI with regard to the values measured 5 min prior to occlusion (Table 1 ). Mice treated with WIN55212-2 produced a slight, but not significant, decrease of MABP and HR with respect to the group treated with vehicle (Table 1) . No effects on these parameters were seen following treatment with the CB1-R antagonist or the CB2-R antagonist (Table 1) .
PMN-associated myocardial damage
Parallel changes in MPO activity measured in the AR and myocardial injury were observed in the mice subjected to the I/R procedure. Figure 2 shows this increase in MPO activity in the infarcted heart versus the hearts collected from vehicle-control mice. Administration of WIN55212-2, given here at the doses that produced a significant reduction in IS, produced a parallel attenuation in MPO activity, as measured 2 h post-reperfusion (Fig. 2) . This attenuation in MPO activity was absent in the mice treated with the WIN55212-2 + AM630 but was still present in the mice treated with WIN55212-2 + AM251. AM630 given alone to the mice significantly (P<0.05) increased the levels of MPO activity with respect to those expressed in the vehicle-control mice (Fig. 2) . Sham operation did not alter the MPO activity from the baseline level of 115 ± 23 × 103 PMN equivalent per mg of protein (n=5).
In separate experiments, we related the MPO activity with the extension of the injured area into the myocardium. For this, we performed some separate experiments measuring the MPO values and the IS, 30 min post-reperfusion. In these experiments, MPO activity was 463 × 103 PMN equivalent per mg of protein (n=5), and the myocardial parameters were as follows: AR, 50 ± 3.2%; IS/AR, 11.2 ± 2%; IS/LV, 7.6 ± 0.4% (n=5 in all cases, P<0.01 vs. 2-h values shown in Fig. 1 ). Together, these data underlie a causal relationship between PMN influx and heart damage.
Cytokine levels in the infarcted myocardium
I/R of the heart showed levels of the two cytokines, IL-1β and CXCL8, up to values of 137 ± 18 pg/mg tissue and 725 ± 50 pg/mg tissue, respectively. Treatment of mice with the CB1/CB2-R agonist WIN55212-2 reduced CXCL8 by 60% and IL-1β by 49% following I/R (Fig. 3 ). The CB2-R antagonist AM630 reversed this inhibitory effect of WIN55212-2 and was unaffected by the CB1-R antagonist AM251 (Fig. 3) . It is interesting that AM630 caused a slight increase in IL-1β and CXCL8 compared with control (infarcted; Fig. 3 ). Sham-operated mice (n=4) showed values of IL-1β and CXCL8 of 22 ± 5 pg/mg tissue and 156 ± 12 pg/mg tissue, respectively.
Effects of CB-R activation on systemic PMN counts and CD11b expression
I/R of the LADCA was associated with alterations in circulating leukocytes. A neutrophilia was observed at the end of the reperfusion period with respect to the values of PMN counted 5 min before the induction of the I/R procedure (Fig. 4 ). Treatment with WIN5521-2 (3.5 mg/kg) did not modify the number of circulating PMN at any of the time-points tested (Fig. 4) . Similarly, none of the CB-R antagonists tested here, alone or in conjunction with WIN55212-2, affected the PMN counts (data not shown).
In contrast to the observed increase in PMN counts, the I/R induced a significant reduction in the number of circulating lymphocytes while not affecting the number of monocytes when compared with the values obtained 5 min before ischemia (Table 2 ). None of the treatments tested have modified these effects.
I/R-induced neutrophilia was associated with PMN activation, as indicated by the increase in cell-surface CD11b. A marked up-regulation of this β2-integrin was detected at the end of the reperfusion period with an increase of 60 ± 3% with respect to the 5-min preocclusion value (Fig. 5 ). WIN55212-2 (3.5 mg/kg) did not significantly change the CD11b expression with respect to vehicle-treated mice at any time point considered (Fig. 5) . Similarly, AM251 (3 mg/kg) and AM630 (1 mg/kg) did not affect systemic blood counts (data not shown).
DISCUSSION
The present study demonstrates that CB2-R activation protects against myocardial damage associated with I/R. The inhibitory effects produced by activation of this receptor are functionally related to a reduced recruitment of blood-borne PMNs into the damaged tissue, and the end-point is cardioprotection.
Since the seminal studies on cannabinoids, their pharmacological action has been attributed to central and peripheral actions, such as analgesia, anticonvulsion, anti-inflammation, alleviation of intraocular pressure and emesis, and attenuation of cardiovascular pathologies [25]. These effects are mediated by the activation of specific G protein-coupled receptors [26 , 27], characterized and cloned from mammalian tissues CB1 [28] and CB2 [29]. The central and most of the peripheral effects of cannabinoids rely on CB1-R activation. This receptor is found in high abundance in the central nervous system, where it mediates cannabinoid psychoactivity. It is also present in peripheral nerve terminals, as well as in extra-neural sites, such as testis, uterus, vascular endothelium, eye, spleen, and tonsils [26 27 28 29 30]. By contrast, the CB2-R is expressed in cells and organs of the immune system with a rank order of B cells > natural killer > monocytes > polymorphonuclear neutrophil cells > T8 cells [31] and is unrelated to cannabinoid psychoactivity [26 , 27]. In addition, macrophages express high levels of CB2-Rs [32].
Lagneux and Lamontagne [12] and Krylatov et al. [13 , 14] have proposed a pivotal role for the CB2-R, having inhibitory effects on cardiovascular pathologies. In these studies, they have reported the ability of CB2-R ligands to attenuate myocardial arrhythmias and myocardial damage following I/R.
In the present study, the synthetic ligand of the CB2-R WIN55212-2, which displays sevenfold higher affinity for the CB2-R over CB1-R [33], was administered 30 min before induction of the I/R procedure, producing a reduction in the tissue damage measured as IS or IS/LV 2 h later in the mouse heart being observed. The effect of the agonist was pronounced with almost a 52% inhibition of the injured area. The protective effect of WIN55212-2 was almost completely abolished by the highly selective CB2 antagonist AM630 and was unaffected by the CB1 antagonist AM251, thus suggesting that CB2-Rs are the major receptors involved.
The data obtained with AM251 in this present study are of particular importance, as they suggest that AM251 or CB1-R activation is not involved in altering the outcome of myocardial experimental I/R pathology. However, it should be noted that CB1s have been implicated in protection against brain I/R injury [34].
The molecular mechanisms behind CB-2R-mediated cardioprotection are difficult to dissect in the present working system. However, based on the evidence that I/R of the mouse heart was associated with a pronounced increase in the levels of MPO, a marker used to monitor tissue infiltration by PMNs [22], we propose that the anti-inflammatory properties of the CB2 agonist are responsible for its attenuation of I/R-induced heart injury.
The causal link between recruitment of white blood cells to the myocardium and subsequent damage was confirmed by the inhibition produced by the WIN55212-2 on local generation of leukocyte activators, cytokines, and chemokines, which are known to promote the leukocyte-endothelium interaction (for review, see ref. [35]).
We chose to investigate IL-1β as an example of a multipotent cytokine, able to increase the adhesive properties of the endothelial wall [36 , 37], and CXCL8 as an example of a chemokine able to recruit neutrophils in rodent species during experimental inflammation [22 , 38 , 39]. In addition, IL-1β has been implicated in the pathology associated with experimental myocardial infarction [40 , 41]. Occlusion and reopening of the LADCA led to a marked increase in IL-1β and CXCL8. The fact that WIN55212-2 produced a significant inhibition in these mediators together with the fact that AM630 slightly increased them may suggest a reduced PMN accumulation, possibly via reduced macrophage activation. In fact, activated PMNs are one of the sources of IL-1β [42], but macrophages can be a major cell source of these cytokines during an ongoing, inflammatory reaction [43]. It is interesting that several studies about mouse and rat peritoneal macrophages and cell lines in culture showed that various CB-R ligands affect macrophage functions including phagocytosis, cytolisis, and protein expression [44 , 45], and CB2-R activation leads to reduced activation/migration of macrophages in vitro [10].
Therefore, the inhibitory effect on IL-1β and CXCL8 production seen with the CB2-R agonist treatment may explain, at least in part, the reduced degree of leukocyte accumulation. This is likely to be a local effect, as in contrast to the protection afforded in the heart by treatment of rats with the CB2 agonist, no such reduction was seen on the systemic alterations provoked by the I/R procedure. As expected, the reperfusion phase was characterized by systemic leukocytosis [46], and it is noteworthy that changes in neutrophil numbers and in their CD11b expression on the plasma membrane (as a marker of generalized cell activation) occurred in parallel over the time course analyzed. In fact, ischemia itself produced a modest but significant increase in both parameters, and they were augmented in a more marked way at the end of the reperfusion period [16]. Treatment of rats with WIN55212-2 at a dose that was effective in reducing myocardial infarction did not modify systemic alterations linked to this experimental pathology.
In conclusion, we have demonstrated a novel action for the CB2-R. Its activation inhibits the leukocyte-dependent damage of an infarcted myocardium. The molecular mechanism(s) responsible for these actions of CB2-R remains elusive. However, we wish to suggest a decreased macrophage activation and reduced cytokine/chemokine-induced chemotaxis of PMN into the injured tissue. Although further investigations are warranted, we cannot exclude involvement of a direct mechanism involving the CB2-R present on the PMN cell surface [31], possibly affecting the adhesion/migration of PMN in these experimental conditions.
Source, Graphs and Figures: Cannabinoid CB2 receptor activation reduces mouse myocardial ischemia-reperfusion injury: involvement of cytokine/chemokines and PMN
