The cannabinoid WIN55,212-2 protects against oxidized LDL-induced inflammatory response in murine macrophages.

The endocannabinoid system has recently been attracted interest for its anti-inflammatory and anti-oxidative properties. In this study, we investigated the role of the endocannabinoid system in regulating the oxidized low-density lipoprotein (oxLDL)-induced inflammatory response in macrophages. RAW264.7 mouse macrophages and peritoneal macrophages isolated from Sprague-Dawley (SD) rats were exposed to oxLDL with or without the synthetic cannabinoid WIN55,212-2. To assess the inflammatory response, reactive oxygen species (ROS) and tumor necrosis factor alpha (TNF- α) levels were determined, and activation of the mitogen-activated protein kinase (MAPK) and nuclear factor (NF)-kappa B signaling pathways were assessed. We observed that: i) oxLDL strongly induced ROS generation and TNF- α secretion in murine macrophages; ii) oxLDL-induced TNF- α and ROS levels could be lowered considerably by WIN55,212-2 via inhibition of MAPK (ERK1/2) signaling and NF-kappa B activity; and iii) the effects of WIN55212-2 were attenuated by the selective CB2 receptor antagonist AM630. These results demonstrate the involvement of the endocannabinoid system in regulating the oxLDL-induced inflammatory response in macrophages, and indicate that the CB2 receptor may offer a novel pharmaceutical target for treating atherosclerosis.

and the SYBR ® Premix Ex Taq TM (Perfect Real Time) kit from Takara Inc. (Japan). The mouse and rat TNF-␣ /TNFSF1A kits were purchased from R and D Systems Inc. (USA). The NE-PER Nuclear and Cytoplasmic Extraction Reagents and the LightShift EMSA kit were obtained from Pierce (USA). The ECL Western Blotting Detection Kit was purchased from Amersham Pharmacia Biotech (Germany). The following antibodies were used: rabbit anti-phospho-p44/42 MAPK (ERK1/2) and rabbit anti total-p44/42 MAPK (ERK1/2) (Cell Signaling Technology; MA); rabbit polyclonal anti-GAPDH antibody (Proteinech Group; USA); and donkey anti-rabbit secondary antibody (Santa Cruz Biotechnology, Santa Cruz, CA). The ROS assay kit was purchased from Sigma (St. Louis, MO), and the BCA Protein Assay Kit was from Beyotime Institute of Biotechnology (China).

Isolation and oxidative modifi cation of LDL
LDL was isolated from human endotoxin-free heparin plasma by sequential ultracentrifugation (density range, 1.019-1.063 g × ml -1 ), and dialyzed against PBS at 4°C. LDL protein concentration was determined by a modifi cation of the Lowry method using bovine albumin as the standard. LDL was oxidized with CuSO 4 and stored at 4°C as described previously ( 12 ).

Cell culture
Peritoneal macrophages were isolated from SD rats (250-350g) and cultured with DMEM with 2 mM L-glutamine, 100 units/ml penicillin, 100 g/ml streptomycin, and 10% FBS under standard conditions as previously described ( 13 ). The mouse RAW264.7 macrophage line (ATCC; Manassas, VA) was cultured in the same culture medium. Before stimulation, nonadherent cells were removed by washing twice with DMEM, and then incubated with oxLDL, WIN55,212-2, AM251, AM630, PD98059, or U0126 alone or in combination. The treated cells were subsequently harvested for the following procedures.

Flow cytometry for measurement of ROS
ROS generation in RAW264.7 macrophages and peritoneal macrophages isolated from SD rats was determined with the ROS assay kit and by fl ow cytometry monitoring the oxidation of 2 ′ ,7 ′ -dichlorodihydrofl uorescein (DCFH). After a 30-min pretreatment with WIN55,212-2, PD98059, U0126, AM251, and/or AM630, cells were treated with oxLDL for 1 h. Cells were then loaded with 5 M DCFH-diacetate for 30 min in the dark, and then resuspended in PBS for fl ow cytometry. DCF fl uorescence was read at 488-nm excitation and 525-nm emission.

ELISA analysis of TNF-␣ protein levels
TNF-␣ levels in the cell culture media were determined by mouse and rat TNF-␣ /TNFSF1A kits according to the manufacturer's instructions. Concentrations were expressed as pg/ml. Three separate experiments were conducted with duplicate samples. Sensitivity for the TNF-␣ enzyme immunoassays was 5.1 pg/ml, and intra-assay and inter-assay coeffi cients of variation were less than 10%.
Important cross talk has been revealed between infl ammation, generation of reactive oxygen and nitrogen species, and lipid metabolism in the pathogenesis of atherosclerosis. A recent study by Bátkai ( 11 ) demonstrated that the endocannabinoid neurotransmitter anandamide (AEA) dose-dependently attenuated TNF-␣ -induced intercellular adhesion molecule (ICAM)-1 and vascular cell adhesion molecule (VCAM)-1 expression, nuclear factor (NF)-kappa B activation in human coronary artery endothelial cells (HCAEC), and the adhesion of monocytes to HCAECs in a CB1-and CB2-dependent manner. It was also shown that the inhibition of the endocannabinoid AEA metabolizing enzyme, the fatty acid amide hydrolase (FAAH) by gene knock-out method, decreased age-related cardiac dysfunction, myocardial nitrative stress, infl ammatory gene expression, and apoptosis in mice. In our most recent study, we reported activation of the endocannabinoid system by oxLDL in macrophages; the activated endocannabinoid system promotes cellular cholesterol accumulation in macrophages by upregulating CD36 receptor expression and downregulating ATP-binding cassette transporter (ABCA1) expression ( 4 ). In the present study, we hypothesized that the endocannabinoid system is directly involved in regulating oxLDL-induced infl ammation and oxidative stress in macrophages.

Materials
The protocol involved animals was approved by the Experimental Animal Ethics Committee in Shanghai Jiaotong University School of Medicine. Sprague-Dawley (SD) rats were purchased from the Chinese Academy of Sciences, Shanghai Laboratory Animal Center. All cell culture reagents, including FBS, were purchased from Gibco Life Technologies (USA), and culture materials were purchased from Corning Life Science (USA). MEK1 inhibitor PD98059 and MEK 1/2 signaling pathway inhibitor U0126 were obtained from Merck (USA), and WIN55,212-2 (synthetic nonselective CB1/CB2 agonist), AM251 (selective CB1 antagonist), and AM630 (selective CB2 antagonist) were purchased from Tocris (USA). The Revertaid TM First Strand cDNA Synthesis Kit was from Fermentas International Inc. (Canada), pretreatment, with AM630 demonstrating a stronger effect than AM251 ( Fig. 1C ). Interestingly, increased intracellular ROS by oxLDL treatment (12 g/ml) was also signifi cantly decreased by coincubation with PD98059 or U0126, two inhibitors of the MEK signaling pathway ( Fig.  1B ). A similar phenomenon was also observed in peritoneal macrophages isolated from SD rats ( Fig. 1D-F ), although higher doses of oxLDL (10-50 g/ml) were required to achieve similar intracellular ROS generation in these cells.
WIN55,212-2 decreased oxLDL-induced phosphorylation of ERK1/2. Western blot analysis was performed to determine GGT C-3 ′ . Total RNA (2 g) was fi rst reverse-transcribed to cDNA with the Revertaid TM First Stand cDNA Synthesis Kit. Real-time polymerase chain reaction (PCR) was performed as follows: preliminary denaturation at 95°C for 10 s, followed by 40 cycles of 95°C for 5 s, and 60°C for 30 s. Relative expression was determined with the ⌬ ⌬ Ct method using GAPDH as the internal control.
Western blot analysis RAW264.7 macrophages and peritoneal macrophages isolated from SD rats were lysed in 100 l RIPA buffer containing 50 mM Tris (pH 7.5), 150 mM NaCl, 10 mM EDTA, 1% Tergitoltype NP-40, 0.1% sodium dodecyl sulfate (SDS), 1 mM phenylmethanesulphonylfl uoride (PMSF), 10 g/ml aprotinin, and 2 mM NaF. Insoluble materials were removed by centrifugation at 12,000 rpm at 4°C for 20 min, and then protein concentration was determined using the Enhanced BCA Protein Assay Kit. After boiling for 5 min with 5× Laemmli sample buffer, equal protein amounts (20 g) were resolved by 10% SDS-polyacrylamide gel electrophoresis and electroblotted onto nitrocellulose membranes at 4°C. After blocking for 1 h with 5% nonfat dry milk in TBS containing 0.1% Tween 20, membranes were incubated overnight at 4°C with primary antibodies (dilution, 1:2000 for p-ERK1/2, t-ERK1/2, and GAPDH), and then incubated with secondary antibodies and visualized by chemiluminescence using an ECL Western Blotting Detection Kit. Finally, densitometric analysis of the immunoblots was performed with Quantity One 4. 4.0 software (Bio-Rad, Hercules, CA).

EMSA to detect NF-kappa B activity
Nuclear extract preparation and conditions for the electrophoretic mobility shift assay (EMSA) reactions were described previously ( 14 ). The 22-mer synthetic double-stranded oligonucleotides used as NF-kappa B probes in the gel shift assay were 5 ′ -AGT TGA GGG GAC TTT CCC AGG C-3 ′ and 3 ′ -TCA ACT CCC CTG AAA GGG TCC G -5 ′ .

Statistical methods
One-way ANOVA was followed by the Student-Newman-Keuls posthoc test to compare means between different groups. Continuous data were expressed as mean ± standard error of the mean (SEM). P < 0.05 (two-tailed) was accepted as statistically signifi cant.
Cannabinoids have been used therapeutically for the past several millennia ( 19 ), but it is only in the last few decades that the biological basis for their effects has begun to be studied. Owing to enormous differences in outcomes according to dosage, type of endocannabinoid, and cell type, only some of the mechanisms have been elucidated. Based on the affi rmative evidence, endocannabi-RAW264.7 mouse macrophages and in rat peritoneal macrophages were considerably reduced by the synthetic cannabinoid WIN55,212-2; (ii) WIN55,212-2 reduced TNF-␣ expression and ROS production, mainly via the CB2 receptor, and partially linked with the CB1 receptor; (iii) MAPK and NF-kappa B were inhibited by WIN55,212-2activated CB1/CB2 receptor signaling, suggesting their involvement in the attenuation of TNF-␣ expression and ROS generation.
Atherosclerosis is no longer considered simply a lipid metabolism disorder, but rather a subacute infl ammatory condition of the vascular system. The initiating event in atherosclerosis remains unclear, but it is known that activation of the acute phase response can result in a shift to a more oxidizing/infl ammatory lipid profi le, which may play a crucial role in atherosclerosis initiation and progression. The binding of oxLDL to the LOX-1 receptor leads from the market because of the increased risk of depression by acting on the cerebral CB1 receptor. The CB2 receptor, which, unlike the CB1 receptor, does not induce major side effects, has been shown to be closely linked with immunoregulation and therefore shows potential for treating chronic infl ammatory diseases, such as multiple sclerosis ( 26 ), colonic infl ammation ( 27 ), and atherosclerosis ( 28 ).
The endocannabinoid system has recently attracted wide interest for its anti-infl ammatory and anti-oxidative properties. Accumulating evidence indicates that synthetic or endogenous cannabinoids protect against central nervous system injury by factors including ischemic/reperfusion ( 29,30 ) and hyperglycemia ( 31 ). Although the exact mechanisms of these neuroprotective effects are not completely understood, numerous CB receptor-dependent as noids are ubiquitous lipid signaling molecules that exert a number of central and peripheral actions mediated primarily by the specifi c receptors CB1 and CB2. CB1 receptors are expressed mainly in the brain but also in some peripheral tissues ( 20 ), and CB2 receptors have been detected mainly in immune cells ( 21 ). Studies have reported that increased endocannabinoids and activated CB1 receptors in peripheral tissues are closely linked with multiple cardiometabolic risk factors, including obesity and increased serum lipid production in rodents and humans ( 22,23 ), and as well as cellular cholesterol accumulation in macrophages in vitro ( 4 ). Selective CB1 receptor antagonists, such as rimonabant, have been implicated to reduce body weight and waist circumference, reduce triglyceride, and increase high density lipoprotein cholesterol ( 24,25 ). Controversially, they have been withdrawn dation, and expression of adhesion molecule ICAM-1 in vivo. Accordingly, I/R-induced hepatic damage and infl ammation are increased in CB2 Ϫ / Ϫ mice ( 6 ). In addition, low doses of tetrahydrocannabinol (THC), the major psychoactive cannabinoid compound of marijuana, appear to inhibit the progression of established atherosclerotic lesions. This therapeutic effect is associated with reduced proliferation and interferon (IFN)-␥ secretion by lymphoid cells as well as reduced macrophage infi ltration into atherosclerotic lesions ( 28 ). These antiatherosclerotic effects of THC appear to be concentration-dependent.
Di Filippo et al. ( 7 ) demonstrated that treatment with the synthetic nonselective CB1/CB2 receptor agonist WIN55,212-2 signifi cantly reduced the extent of myocardial infarct in mice suffering from myocardial I/R injury; cardioprotection was accompanied by lower myeloperoxidase well as receptor-independent processes have been suggested, of which the CB2 receptor-mediated anti-infl ammatory and anti-oxidative activities may be the most important ( 32 ).
In a hepatic I/R injury animal model, the endocannabinoids AEA and 2-arachidonoylglycerol (2-AG) increased several-fold in the liver and positively correlated with the degree of hepatic damage and the serum levels of TNF-␣ , macrophage infl ammatory protein (MIP)-1 ␣ , and MIP-2. Furthermore, a brief exposure of hepatocytes to various oxidants (hydrogen peroxide, peroxynitrite) or infl ammatory stimuli (endotoxin, TNF-␣ ) also increases endocannabinoid levels. Interestingly, the selective CB2 cannabinoid receptor agonist JWH133 protects against I/R damage by attenuating infl ammatory cell infi ltration, tissue and serum TNF-␣ , MIP-1 ␣ and MIP-2 levels, tissue lipid peroxi- tory cytokine expression in peripheral blood and attenuates LPS-stimulated ERK1/2 and JNK1/2 phosphorylation in monocytes ( 43 ). Also, ␤ -caryophyllene induces a switch from a TH1 to a TH2 immune response by inhibiting the pathway triggered by activation of the toll-like receptor complex CD14/TLR4/MD2 ( 44 ). Many studies have focused on the role of the CB2 receptor in the anti-infl ammatory and anti-oxidative effects of WIN55,212-2 ( 6,7,(32)(33)(34). In the present study, we observed that the selective CB2 receptor antagonist AM630 blocked the effects of WIN55,212-2 on oxLDL-induced TNF-␣ expression, ROS generation, and ERK1/2 phosphorylation. These results support an important role for the CB2 receptor in the antiinfl ammation and anti-oxidation actions of WIN55,212-2. However, AM251 or AM630 did not exert strong effects on the ability of WIN55,212-2 to reduce NF-kappa B activity, which suggests a mechanism other than CB1/CB2 receptors in the regulation of infl ammation and oxidative stress by endocannabinoids.

CONCLUSION
We demonstrated for the fi rst time that the cannabinoid WIN55,212-2 protects against oxLDL-induced infl ammation and oxidative stress in murine macrophages via the CB2 receptor, which suggests that the CB2 receptor might be a promising pharmacological target for atherosclerosis therapy. However, the involvement of other receptors and mechanisms cannot be ruled out at this stage.
The present study should be interpreted within the context of its limitation. WIN55,212-2 is a nonselective cannabinoid receptor agonist; therefore, further studies are needed using a more specifi c CB2 receptor agonist, such as HU308 or JWH-133, to verify our results. activity and IL-1 ␤ and CXC chemokine ligand levels in the injured tissue. This cardioprotective effect was almost abolished by the selective CB2 antagonist AM630, but it was not affected by the selective CB1 antagonist AM251. In addition, JWH133 reduced myocardial infarct size in mice with I/R injury, and the cardioprotective effect was linked with reduced oxidative stress and neutrophil infi ltration in the infarcted myocardium ( 33 ). Taken as a whole, these fi ndings indicate that endocannabinoids exert cardioprotective effects via the CB2 receptor ( 34 ).
In our previous study, we demonstrated that the endocannabinoid system is activated by oxLDL in macrophages. In addition, we observed that WIN55,212-2 promoted cellular cholesterol accumulation in macrophages, which was associated with increased CD36 expression and decreased ABCA1 expression via peroxisome proliferator-activated receptor ␥ (PPAR ␥ ) ( 4 ). To test the hypothesis that the endocannabinoid system is involved in regulating oxLDLinduced infl ammation and oxidative stress, we determined the effect of WIN55,212-2 on oxLDL-induced TNF-␣ expression and ROS generation in RAW264.7 macrophages and in peritoneal macrophages isolated from rats. We observed that elevated TNF-␣ and ROS were dose-dependently attenuated by WIN55,212-2. Our results were consistent with previous studies: WIN55,212-2 decreased IL-1 ␤ -induced TNF-␣ release in human astrocytes ( 35 ) and lipopolysaccharide (LPS)-stimulated TNF-␣ production in human mononuclear cells ( 36 ) in vitro. Additionally, there is increasing evidence that WIN55,212-2 reduces ROS formation in cortical neuron cultures treated with FeCl 2 ( 37 ) and protects against I/R damage by decreasing infl ammation and oxidative stress ( 6,7,(32)(33)(34). Thus, the endocannabinoid system appears to play a role in the pathophysiology of atherosclerosis by attenuating oxLDL-induced infl ammation and oxidative stress in macrophages.
Previous studies have reported that oxLDL activates MAPK signaling to stimulate infl ammation and oxidative stress in macrophages via LOX-1 ( 3,38 ). We therefore hypothesized that MAPK (ERK1/2) signaling may be involved in the attenuation of oxLDL-induced ROS generation and TNF-␣ expression by cannabinoids. We found that WIN55,212-2 inhibited oxLDL-mediated ERK1/2 phosphorylation in RAW264.7 macrophages and in peritoneal macrophages isolated from rats. Further, the MEK inhibitors PD98059 and U0126 suppressed oxLDL-induced TNF-␣ expression and ROS formation in cultured macrophages, which supports the requirement of ERK1/2 in oxLDLinduced infl ammation and oxidative stress. Thus, the MAPK signaling pathway may be involved the WIN55,212-2 regulation of TNF-␣ expression and ROS formation.