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-induced human endothelial cell activation, transendothelial migration of monocytes, and monocyte-endothelial adhesion1Section on Oxidative Stress Tissue Injury, Laboratory of Physiological Studies, National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, Bethesda, Maryland; 2Department of Surgery, New Jersey Medical School, University of Medicine and Dentistry of New Jersey, Newark, New Jersey; 3Department of Intensive Care Medicine, University Hospital, Lausanne, Switzerland; 4Howard L. Hunter Chemistry Laboratory, Clemson University, South Carolina; 5Department of Physiology, New York Medical College, Valhalla, New York; 6Department of Psychological and Brain Sciences, Indiana University, Bloomington, Indiana; and 7Department of Pediatrics, Lipid Research Unit, Johns Hopkins University, Baltimore, Maryland
Submitted 13 June 2007 ; accepted in final form 26 July 2007
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ABSTRACT |
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-induced signal transduction in human coronary artery endothelial cells in vitro and on endotoxin-induced vascular inflammatory response in vivo. TNF- induced NF-
B and RhoA activation and upregulation of adhesion molecules ICAM-1 and VCAM-1, increased expression of monocyte chemoattractant protein, enhanced transendothelial migration of monocytes, and augmented monocyte-endothelial adhesion. Remarkably, all of the above-mentioned effects of TNF- were attenuated by CB2 agonists. CB2 agonists also decreased the TNF-- and/or endotoxin-induced ICAM-1 and VCAM-1 expression in isolated aortas and the adhesion of monocytes to aortic vascular endothelium. CB1 and CB2 receptors were detectable in human coronary artery endothelial cells by Western blotting, RT-PCR, real-time PCR, and immunofluorescence staining. Because the above-mentioned TNF--induced phenotypic changes are critical in the initiation and progression of atherosclerosis and restenosis, our findings suggest that targeting CB2 receptors on endothelial cells may offer a novel approach in the treatment of these pathologies.
endothelial activation; inflammation; RhoA; adhesion molecules
), which mediate, at least in part, their proatherogenic effects by eliciting NF-B activation in endothelial cells (17, 21, 24, 39, 48, 50), are considered to play pivotal roles in vascular inflammation associated with atherosclerosis and development of coronary arterial disease (4, 20, 23, 27, 35, 39, 43, 44). The activation of this pathway leads to induction of adhesion molecules and chemokines, e.g., VCAM-1, ICAM-1 (48), which promote monocyte adhesiveness to the endothelium, and the release of a variety of factors that promote smooth muscle migration and proliferation (from the medium into the intima), which then synthesize and deposit extracellular matrix (10, 14, 28). There is considerable evidence suggesting that disruption of the cytokine-induced NF-B signaling pathway confers significant vasculoprotective effects by attenuating vascular inflammation (24, 36), which delays or prevents atherogenesis in animal models (4, 18, 42) of disease. To date, two cannabinoid (CB) receptors have been identified by molecular cloning: the CB1 receptor, which is highly expressed in the brain but is also present in peripheral tissues including the heart, vascular tissues, and liver, and the CB2 receptor, previously thought to be expressed predominantly by immune and hematopoietic cells (reviewed in Refs. 22, 29). However, more recent studies have also demonstrated CB2 receptors in brain (45), myocardium (26), cardiomyoblasts (26, 34), and endothelial cells of various origins (3, 9, 25, 52) (reviewed in Refs. 19, 29).
The natural ligands of these receptors are lipid-like substances called endocannabinoids, which include arachidonoyl ethanolamide or anandamide and 2-arachidonoylglycerol (22, 29). Endocannabinoids and their synthetic analogs exert various central nervous system, cardiovascular, and anti-inflammatory effects through CB1 and CB2 receptors (19, 29).
A recent study has demonstrated that orally administered cannabis constituent 
9-tetrahydrocannabinol, which is a mixed weak CB1/2-receptor agonist with psychoactive property, inhibited atherosclerosis progression in a mouse model of disease, an effect that could be blocked by a selective CB2-receptor antagonist (38). However, the role of CB2 receptor in vascular endothelial cell activation and inflammatory response is still unexplored.
Herein, we evaluated the effects of CB2-receptor activation on TNF--induced NF-B and RhoA activation, upregulation of adhesion molecules ICAM-1 and VCAM-1, expression of monocyte chemoattractant protein in human coronary artery endothelial cells (HCAECs), transendothelial migration (TEM) of monocytes, monocyte-endothelial adhesion in vitro, and endotoxin-induced vascular inflammatory response in vivo. Because these pathological events are pivotal in the initiation and progression of atherosclerosis and restenosis, our findings may have important clinical implications and underscore the role of CB2 receptors as a novel therapeutic target in various vascular diseases associated with inflammation.
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MATERIALS AND METHODS |
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The selective CB2 receptor agonist JWH-133 was either purchased from Tocris Bioscience (Ellisville, MO) or synthesized as described earlier (13). The selective CB2-receptor agonist HU-308 (11) was from Cayman Europe (Tallinn, Estonia). The CB1-receptor antagonist AM-281 and CB2-receptor antagonist AM-630 were purchased from Tocris Bioscience (Ellisville, MO). The CB1 antagonist SR-141716A and CB2 antagonist SR-144528 were from NIDA Drug Supply (Research Triangle Park, NC). Human recombinant TNF- was obtained from R&D Systems. Sources of all the other reagents are mentioned in the text where appropriate.
Cell Culture
HCAECs and the growth medium were purchased from Cell Applications (San Diego, CA). The human monocytic cell line THP-1 was obtained from American Type Culture Collection; cells were grown in RPMI 1640 medium supplemented with 2 mM L-glutamine, 10 mM HEPES, 10% FBS, 100 U of penicillin, and 100 µg of streptomycin/ml (Invitrogen).
CB1 and CB2 Expression in HCAECs
Immunofluorescence staining. CB1 expression and CB2 expression in the human endothelial cells were determined by immunofluorescence staining technique. In brief, HCAECs were grown to confluence in chamber slides (Nalgene-Nunc, Lab-Tek). Growth medium was aspirated, and cells were washed three times with PBS and then fixed with 4.0% paraformaldehye for 20 min at 4°C. After cells were washed with PBS, cells were permeabilized with 0.2% Triton X-100 in PBS for 15 min at room temperature. Subsequently, CB1 expression and CB2 expression in the human endothelial cells were determined by immunofluorescence staining technique using anti-CB1 (rabbit polyclonal; Cayman Chemical) or anti-CB2 (rabbit polyclonal; Cayman Chemical), respectively, used at 1:100 dilution for 6 h at 4°C. After the cells were rinsed with PBS, the cells were probed with goat anti-rabbit FITC (1:250; Pierce) for 1 h at room temperature. The nucleus was counterstained with 4',6-diamidino-2-phenylindole (Molecular Probes). Images were obtained with a fluorescent microscope (Olympus IX 81) at x20 objective with x1.5 optical zoom. To rule out the nonspecific staining, we used the corresponding blocking peptides for CB1 (catalog no. 10006591, Cayman Chemical) or CB2 (catalog no. 301550, Cayman Chemical). In brief, the blocking peptides were mixed with corresponding antibodies in a 1:1 ratio and incubated for 1 h at room temperature. This preabsorbed antibody was then used for the staining process. This procedure essentially blocks the antibody-antigen (protein) formation during the immunofluorescence staining and aids in determining the specificity of the staining.
Conventional RT-PCR and quantitative real-time RT-PCR.
Total RNA was isolated from the cells using Trizol LS reagent (Invitrogen) according to manufacturer's instruction. The RNA was treated with RNase-free DNase (Ambion) to remove traces of genomic DNA contamination. Total RNA was then reverse transcribed to cDNA using SuperScript II (Invitrogen), and the target genes were amplified with the standard PCR kit (Bio-Rad). The PCR conditions were as follows: after initial denaturation at 95°C for 2 min, 35 cycles were performed at 95°C for 30 s and at 60°C for 30s. Primers used were as follows: for CB1, 5'-TTCCCTCTTGTGAAGGCACTG-3' (forward) and 5'-TCTTGACCGTGCTCTTGATGC-3' (reverse); for CB2, 5'-TTTGCTTTCTGCTCCATGCTG-3' (forward) and 5'-TTCTTTTGCCTCTGACCCAAG-3' (reverse); for 
-actin, 5'-ATTGCCGACAGGATGCAGAAG-3'(forward) and 5'-TAGAAGCATTTGCGGTGGACG-3' (reverse).
The amplified products were separated on 1.5% agarose gels, stained with ethidium bromide, and documented using the Typhoon system (GE Healthcare).
In a separate set of experiments, real-time PCR was performed in identical conditions, except amplification and quantification of the target gene expression were performed with the iTaq Sybr green mix (Bio-Rad) and Bio-Rad chromo4/opticon system (data not shown). Relative quantification was performed with the relative comparative threshold (CT) method.
Western immunoblot assay.
HCAECs were grown to confluence in 100-mm culture dishes coated with 0.2% gelatin. Cells were suspended in RIPA lysis buffer (Pierce) supplemented with protease inhibitors (Roche). Cell lysates were then prepared by sonication 15k for 20 s) on ice. The lysates then were clarified to remove the cellular debris by centrifuging at 10,000 rpm for 15 min at 4°C. Protein content in the lysates was determined with the Lowry assay (Bio-Rad). Protein (30 µg) was resolved in 12% SDS-PAGE and transferred to nitrocellulose membranes (GE Healthcare). Blocking was performed for 2 h at room temperature with 5% nonfat skimmed milk powder prepared in PBS containing 0.1% Tween 20 (PBST; Sigma). After they were washed with PBST, membranes were probed with either rabbit polyclonal CB1 (Cayman Chemical; 1:1,000 dilution or an antibody raised against the last 15 residues of rat CB1) or CB2 antibody (Cayman Chemical; 1:1,000 dilution) overnight at 4°C. After a subsequent washing with PBST, the secondary antibody goat anti-rabbit horseradish peroxidase (Pierce) and incubated at room temperature for 1 h. The membranes were then developed using chemiluminescence detection kit (SuperSignal-west Pico substrate, Pierce). To confirm uniform loading, membranes were stripped and reprobed with -actin (Chemicon). Proteins from mouse brain extract and human monocytic cell line (THP-1) lysate were used as appropriate positive controls for CB1 and CB2 receptors, respectively.
Cell Surface ICAM-1 and VCAM-1 Expression Assay
Cell surface expression of ICAM-1 and VCAM-1 was measured by in situ ELISA as described (2). In brief, HCAECs were grown in 96-well plates coated with 0.2% gelatin. After treatments, in situ ELISA was performed with anti-human ICAM-1 or VCAM-1 monoclonal antibodies (1:1,500 dilution; R&D Systems) and by measuring the absorbance at colorimetrically at 450 nm using the horseradish peroxidase-3,3',5,5'-tetramethylbenzidine developing system (Sigma). Each treatment was performed in triplicate, and the experiments were repeated three times.
Monocyte-Endothelial Cell Adhesion Assay
Monocyte adhesion to endothelial cells was performed as described with modifications (47). In brief, HCAECs were grown to confluence in 24-well plates and treated with TNF- ± CB2 agonists/antagonists. THP-1 monocytes were then labeled with 1.5 µM calcein-AM (Molecular Probes-Invitrogen) for 1 h at 37°C in RPMI 1640 containing 1% FBS. HCAECs were washed twice with HCAEC basal medium and incubated with 400 µl of basal medium. Then 105 cells/100 µl of labeled THP-1 cells were overlaid on HCAECs and incubated for 1 h at 37°C. After incubation, the monolayer was carefully washed with PBS to remove the unbound monocytes. The adherent monocytes to the endothelial cells were documented with an Olympus IX 81 fluorescent microscope using x10 objective with x1.5 optical zoom. Individual treatments were preformed in duplicate, and the entire set of experiments was repeated at least three times.
Monocyte Chemoattractant Protein-1 Expression
Levels of monocyte chemoattractant protein-1 (MCP-1) expression in the HCAECs were determined using an ELISA kit (R&D Systems).
Monocyte Transendothelial Migration Assay
HCAECs were allowed to reach confluence on 0.2% gelatin-coated 3.0-µm polyethylene terepthalate track-etched cell culture inserts (BD Biosciences). TEM assays were then performed essentially as described with modifications (5). In brief, THP-1 cells were labeled with 2 µM Cell Tracker green 5-chloromethylfluorescein diacetate (Molecular Probes). Labeled THP-1 cells (3 x 105/200 µl) were added to the upper compartment of the cell culture insert, and the lower compartment of the insert contained 0.5 ml serum-free RPMI 1640. The monocytes were then allowed to transmigrate for 4 h at 37°C in 5% CO2 incubator. After incubation, fluorescence at the lower chamber was measured with the Victor-Wallac Multilabel counter (Perkin Elmer) at excitation of 492 nm and emission of 571 nm. Each treatment was performed in duplicate, and experiment sets were repeated two times. The monocyte TEM was expressed as percent cell migrated.
RhoA Activation Assay
Rho activation in HCAECs was performed with commercially available kits (Pierce). In brief, RhoA activation assay was performed by pull-down assay, with Rhotekin-Rho binding domain fused with glutathione S-transferase. The active or GTP-RhoA pulled down from the cell lysates was analyzed by Western blot assays using anti-Rho antibody.
NF-B Activation
NF-B activation by TNF- and the effect of CB2 agonists/antagonists were analyzed by Western imunoblot and immunofluorescence assays, respectively.
In Vivo Vascular Inflammation Model
Protocols involving the use of experimental animals were approved by the author's institutional animal care and use committee and were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Male C57Bl/6J mice (6–8 wk) were administered 3 mg/kg LPS (E. coli serotype O197:B8; Sigma) in a single intraperitoneal dose. In some experiments, 1 h before LPS administration, experimental animals were injected with HU-308 or JWH-133 (10 mg/kg ip) ± AM-630 (3 mg/kg ip). After 4 h, animals were euthanized, and aortas were dissected and snap frozen for the determination of ICAM-1 and VCAM-1 expressions.
Monocyte Adhesion to Aortic Vascular Endothelium
Monocyte-enriched peripheral blood mononuclear cells were isolated from rats, and binding of BCECF-labeled (5 µmol/l final concentration; Molecular Probes) monocytes to the vascular endothelium was determined as previously described (6). In brief, rat aortic segments were treated with HU-308 or JWH-133 (3 µM) ± AM-630 (1 µM) ± TNF- (50 ng/ml, for 4 h). The vessels were cut open (en face preparation) and incubated with BCECF-loaded monocytes. After a 1-h incubation at 37°C, unbound monocytes were washed out. Bound monocytes were quantified by counting the cells under a fluorescent microscope. Representative images were captured using a fluorescent microscope (Olympus IX 81; original magnification: x10; endothelial cell's nuclei were counterstained with Hoechst 33258 for orientation).
In another set of experiments, rats were injected with a single dose of LPS (3 mg/kg ip) with or without the pretreatment with HU-308 or JWH-133 (10 mg/kg) ± AM-630 (3 mg/kg) for 1 h; 6 h later aortas were isolated, and monocyte adhesion to vascular endothelium was performed. Monocyte adhesion to the vascular endothelium was then determined as described in the previous paragraph.
Statistical Analysis
All values are means ± SE. Statistical significance of the data was assessed by one-way ANOVA with Tukey's post hoc test (GraphPad-Prism4). P < 0.05 was considered significant.
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RESULTS |
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As shown in Figs. 1 and 2, CB1 and CB2 receptors are expressed in endothelial cells, at basal conditions as demonstrated by immunofluorescence assays (Fig. 1A), conventional RT-PCR (Fig. 1, B and C), and Western blot (Fig. 1, D and E). From quantitative analysis of Western blot, it was revealed that mice brain extracts had 
4.5-fold increased CB1-receptor expression compared with HCAECs (Fig. 1D). CB2 expression was 5.5-fold higher in THP-1 monocytes than in HCAECs (Fig. 1E). Protein from mouse brain extracts and THP-1 cell lysates were used as appropriate positive controls for CB1 and CB2 receptors, respectively. To rule out the nonspecific staining for CB1 or CB2 expression, we preabsorbed either CB1 or CB2 with the corresponding blocking peptides supplied with the primary antibodies. Next, we used the preabsorbed antibody for the detection of CB1 and CB2 in HCAECs simultaneously with the neat antibodies. Our results indicated that preabsorbed antibodies failed to stain the CB1 or CB2 receptors in HCAECs, suggesting that the antibody specifically recognizes CB1 or CB2 receptors in HCAECs (Fig. 1A). Similar results were obtained with Western blot assays (data not shown).
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-Induced ICAM-1 and VCAM-1 Expression In Vitro
TNF- (50 ng/ml) treatment of HCAECs for 6 h, resulted in marked upregulation of ICAM-1 and VCAM-1 expressions, which were dose dependently diminished by HU-308 or JWH-133 (Fig. 2, A and B). These effects of CB2 agonists were attenuated by CB2 (SR-144528 and AM-630) but not by CB1 (SR-141716 and AM-251) antagonists (Fig. 2, C and D). CB1 or CB2 antagonists by themselves had no effect on TNF--induced adhesion molecule expression (Fig. 2, C and D).
CB2 Agonists Attenuate TNF--Induced Monocyte Adhesion and MCP-1 Expression In Vitro and Ex Vivo
Pretreatment of HCAECs with HU-308 or JWH-133 (3 µM) from 1 h before and during the complete TNF- exposure inhibited TNF--induced monocyte adhesion (Fig. 3, A and B) and MCP-1 expression (Fig. 3C), and these effects were attenuated by CB2 antagonists (Fig. 3). HU-308 also inhibited TNF--induced monocyte adhesion in aortas ex vivo (Fig. 4A).
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Endotoxin administration to mice has been shown to induce massive vascular inflammation and characterized by increase in adhesion molecules and NF-B activation (40, 51). Because in vitro CB2-receptor stimulation blunted cytokine-induced adhesion molecule expression and NF-B activation, we tested whether CB2-receptor stimulation could ameliorate LPS-induced vascular inflammation in vivo. We found that LPS administration markedly induced ICAM-1 and VCAM-1 (Fig. 4B) expression in the aortas (5.6- and 4.8-fold increase compared with mice treated with vehicle alone). HU-308 or JWH-133 pretreatment markedly reduced the expression of both ICAM-1 and VCAM-1 (Fig. 4B), which could be attenuated by the CB2 antagonist AM-630.
CB2 agonist HU-308 also significantly inhibited monocyte adhesion to the aortic segments prepared from rats treated with LPS (Fig. 4C). This effect was attenuated by CB2 antagonist AM-630 (Fig. 4C), suggesting that CB2-receptor activation could mitigate the endotoxin-induced vascular inflammation.
CB2 Agonists Inhibit TNF--Induced Monocyte TEM and RhoA Activation in HCAECs
TNF- treatment of HCAECs markedly increased monocyte TEM 4–3 fold vs. control. This was attenuated by HU-308 or JWH-133 (3 µM; Fig. 5A). Furthermore, TNF- treatment induced RhoA activation (4.0-fold vs. control), and this activation was attenuated by treatment with HU-308 or JWH-133 (Fig. 5B). These effects of CB2 agonists were attenuated by CB2 antagonist.
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-Induced NF-B Activation in HCAECs
TNF- treatment leads to marked NF-B activation, characterized by increased translocation to the nucleus as demonstrated by Western blot (Fig. 6A). We observed 2.5-fold increase in nuclear translocation of p65 (NF-B) (Fig. 6A); likewise, a marked increase in immunofluorescence was also observed above the nuclei of cells treated with TNF- (Fig. 6B). These effects were significantly decreased by the pretreatment with HU-308 or JWH-133, and the latter could be attenuated by CB2-antagonist AM-630.
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DISCUSSION |
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-triggered NF-B and RhoA activation, upregulation of adhesion molecules ICAM-1 and VCAM-1, expression of monocyte chemoattractant protein, TEM of monocytic THP-1 cells, and monocyte-endothelial adhesion in HCAECs. Furthermore, CB2-receptor activation attenuates TNF--induced adhesion of monocytes to aortic endothelium ex vivo, the endotoxin-induced ICAM-1 and VCAM-1 expression and adhesion of monocytes to aortic endothelium of isolated aortas of endotoxintreated rats.
Presently, it is thought that CB1 receptors are primarily expressed in the brain and some peripheral tissues, including the heart, vascular tissues, adipocytes, and liver (22, 29). On the other hand, the CB2 receptor was previously considered to be expressed predominantly by immune and hematopoietic cells (22, 29). However, more recent studies have also found CB2 receptors in brain (45), myocardium (26), cardiomyoblasts (26, 34), and endothelial cells of various origins (3, 9, 25, 52). Here, we report expression of CB1 and CB2 receptors in HCAECs under basal/physiological conditions. Interestingly, the expression of CB2 receptors could be enhanced by the proinflammatory cytokine TNF- in endothelial cells (data not shown). A similar phenomenon was previously proposed during the microglia activation (46).
The hallmark for the development of atherosclerosis is the adhesion of monocytes to the endothelium, followed by TEM of monocytes (10). It has been well-established that TNF- and/or endotoxin(s) induces NF-B-dependent upregulation of the expression of ICAM-1, VCAM-1, and MCP-1 in endothelial cells, contributing to the increased adhesion of monocytes to the endothelium migration and TEM (43). The TNF--induced NF-B activation has been shown to involve RhoA activation (21), and RhoA activation was also implicated in monocyte TEM (33). Likewise, MCP-1 has been shown to be involved in monocyte TEM (41) and endothelial barrier disruption through RhoA activation (37). Consistent with the above-mentioned studies, we found that TNF- increased NF-B and RhoA activation, upregulation of adhesion molecules ICAM-1 and VCAM-1, expression of MCP-1, monocyte-endothelial adhesion in HCAECs, and TEM of monocytes. We demonstrate that selective CB2-receptor agonists JWH-133 and HU-308 markedly decrease TNF--increased NF-B activation, upregulation of adhesion molecules ICAM-1 and VCAM-1, expression of MCP-1, monocyte-endothelial adhesion in HCAECs, and TEM of monocytic THP-1 cells in a CB2-dependent manner, since these effects of CB2 agonists could be attenuated by CB2 antagonists. Consistent with our observation, it has been recently demonstrated that JWH-015, another less potent CB2 agonist inhibited the TEM of T lymphocytes stimulated by chemokines such as CXCL12 and CXCR4 (8).
Adhesion molecules also mediate the initial attachment of neutrophils to the activated endothelium, which is a crucial early event in ischemia-reperfusion injury (15, 16). On reperfusion, inflammatory cytokines (e.g., TNF-) act as continuous stimuli for neutrophil infiltration and upregulate the production of chemokines, which may also contribute to the upregulation of cell adhesion molecules and neutrophil activation (15, 16). Numerous recent studies have demonstrated that CB2-receptor activation may also protect against myocardial (7), cerebral (49), and hepatic ischemia-reperfusion (1, 32) injuries by decreasing the endothelial cell activation, the expression of adhesion molecules ICAM-1 and VCAM-1, TNF-, and chemokine (MIP-1 and MIP-2) levels, neutrophil infiltration, lipid peroxidation, and apoptosis.
Collectively, our results suggest that selective CB2-receptor agonists may offer a novel approach in the treatment of a variety of inflammatory diseases, including atherosclerosis. Thus attenuation of TNF signaling, coupled with the absence of psychoactive effects associated with CB2-receptor stimulation, makes this a particularly encouraging therapeutic approach.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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P < 0.05 vs. TNF-
