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, 293 (4), H2210-8

CB2-receptor Stimulation Attenuates TNF-alpha-induced Human Endothelial Cell Activation, Transendothelial Migration of Monocytes, and Monocyte-Endothelial Adhesion

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CB2-receptor Stimulation Attenuates TNF-alpha-induced Human Endothelial Cell Activation, Transendothelial Migration of Monocytes, and Monocyte-Endothelial Adhesion

Mohanraj Rajesh et al. Am J Physiol Heart Circ Physiol.

Abstract

Targeting cannabinoid-2 (CB(2)) receptors with selective agonists may represent a novel therapeutic avenue in various inflammatory diseases, but the mechanisms by which CB(2) activation exerts its anti-inflammatory effects and the cellular targets are elusive. Here, we investigated the effects of CB(2)-receptor activation on TNF-alpha-induced signal transduction in human coronary artery endothelial cells in vitro and on endotoxin-induced vascular inflammatory response in vivo. TNF-alpha induced NF-kappaB 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-alpha were attenuated by CB(2) agonists. CB(2) agonists also decreased the TNF-alpha- and/or endotoxin-induced ICAM-1 and VCAM-1 expression in isolated aortas and the adhesion of monocytes to aortic vascular endothelium. CB(1) and CB(2) 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-alpha-induced phenotypic changes are critical in the initiation and progression of atherosclerosis and restenosis, our findings suggest that targeting CB(2) receptors on endothelial cells may offer a novel approach in the treatment of these pathologies.

Figures

Fig. 1
Fig. 1
Cannabinoid-1 (CB1) and cannabinoid-2 (CB2) receptors are expressed in human coronary artery endothelial cells (HCAECs). A: expression of CB1 and CB2 receptors in HCAECs demonstrated by immunofluorescence staining. CB1 expression and CB2 expression in the endothelial cells were detected with rabbit polyclonal anti-CB1 and anti-CB2 antibodies (Cayman Chemical) and detected by secondary antibody goat anti-rabbit FITC (Pierce); the nucleus was counterstained with 4′,6-diamidino-2-phenylindole (Molecular Probes, Invitrogen). B and C: RT-PCR analysis of CB1 (B) and CB2 (C) receptor expression in endothelial cells. D and E, left: Western immunoblot demonstrating expression of CB1 (D) and CB2 (E) in endothelial cells. Six samples from 3–5 passages from HCAECs or THP-1 and lysates from six brain tissues from wild-type C57Bl/6J mice were used to evaluate CB1 and CB2 expression in human endothelial cells. D and E, right: the quantification of CB1 and CB2 expression in HCAECs; n = 6 samples. *P < 0.05 vs. brain extract or THP-1 cells.
Fig. 2
Fig. 2
CB2 agonists decrease TNF-α-induced ICAM-1 and VCAM-1 expression in HCAECs. A and B: ICAM-1 (A) and VCAM-1 (B) expression. Cells were treated with either TNF-α or CB2 agonists for 6 h or with CB2 agonists with the indicated concentrations followed by treatment with TNF-α for 6 h. Cell surface ELISA was then performed by measuring the absorbance at 450 nm as described in MATERIALS AND METHODS to determine cell surface ICAM-1 or VCAM-1 expression; n = 9 samples. *P < 0.05 vs. control; #P < 0.05 vs. TNF-α. C and D: cells were pretreated with CB1 or CB2 antagonists followed by treatment with TNF-α ± CB2 agonists as indicated for 6 h or pretreated with CB1 or CB2 antagonists (1 μM each) followed by treatment with either TNF-α alone or in combination with CB2 agonists. Cell surface ELISA was then performed; n = 9 samples. OD, optical density. *P < 0.05 vs. control; #P < 0.05 vs. TNF-α; †P < 0.05 vs. TNF-α + HU-308 or JWH-133.
Fig. 3
Fig. 3
CB2 agonists decrease TNF-α-induced monocyte chemoattractant protein-1 (MCP-1) expression in HCAECs and monocyte adhesion to HCAECs. SR2, SR-144528; T, TNF-α. A: representative fields of monocytes adhered to HCAECs with representative treatments as indicated. B: quantification of data for monocyte adherence to endothelial cells when incubated with TNF-α ± HU-308 or JWH-133 (4 μM) for 6 h or pretreated with CB2 antagonists (1 μM) for 1 h followed by treatment with TNF-α ± HU-308 or JWH-133 for 6 h; n = 9 samples. *P < 0.05 vs. control; #P < 0.05 vs. TNF-α; †P < 0.05 vs. TNF-α + HU-308 or JWH-133. C: MCP-1 expression in HCAECs for treatments indicated; n = 6 samples. *P < 0.05 vs. control; #P < 0.05 vs. TNF-α; †P < 0.05 vs. TNF-α + HU-308 or JWH-133.
Fig. 4
Fig. 4
CB2 agonists mitigate TNF-α- and/or endotoxin (LPS)-induced ICAM-1 and VCAM-1 expression and monocyte adhesion to aortic endothelium of rats ex vivo or in vivo. A: CB2 agonist HU-308 mitigates adhesion of monocytes to aortic vascular endothelium of isolated aortas pretreated with TNF-α; n = 4 samples. *P < 0.05 vs. vehicle [control (Co)]; #P < 0.05 vs. TNF-α; †P < 0.05 vs. LPS + HU-308 or JWH-133. B: mice were treated with LPS ± HU-308 or JWH-133 ± AM-630 as indicated; n = 6 samples. Aortas were then isolated, and ICAM-1 or VCAM-1 expressions were determined by ELISA (R&D Systems).*P < 0.05 vs. vehicle-treated (Co) mice; #P < 0.05 vs. LPS; †P < 0.05 vs. LPS + HU-308 or JWH-133. Protein estimation in the tissue lysates was determined by Lowry assay (Bio-Rad). Values are expressed as ng/mg protein. C: CB2 agonist HU-308 mitigates endotoxin-induced monocyte adhesion to aortic vascular endothelium of isolated aortas of rats treated with endotoxin; n = 4 samples. *P < 0.05 vs. vehicle-treated (Co) mice; #P < 0.05 vs. LPS-treated rats; †P < 0.05 vs. LPS + HU-308 or JWH-133.
Fig. 5
Fig. 5
CB2 agonists decrease TNF-α-induced monocyte transendothelial migration (TEM) and RhoA activation in HCAECs. A: HCAECs were treated as indicated, and then monocyte TEM assays were performed as described in MATERIALS AND METHODS; n = 4 samples. *P < 0.05 vs. controls; #P < 0.05 vs. TNF-α; †P < 0.05 vs. TNF-α + HU-308 or JWH-133. B: HCAECs were grown in 100-mm culture dishes and treated in identical manner as described above, 500 μg cell lysates were incubated with Rhotekin-Rho binding domain-glutathione S-transferase fusion protein, and pull-down assays were performed (Pierce), with active RhoA detected with the use of anti-RhoA antibody; n = 3 samples. *P < 0.05 vs. controls; #P < 0.05 vs. TNF-α; †P < 0.05 vs. TNF-α + HU-308 or JWH-133.
Fig. 6
Fig. 6
CB2 agonists decrease TNF-α-induced NF-κB activation in HCAECs. A: cells were treated with TNF-α ± HU-308 or JWH-133 (3 μM) for 6 h or pretreated with CB2 antagonists (1 μM) followed by treatment with TNF-α ± HU-308 or JWH-133 for 6 h. Nuclear extracts were then prepared, and NF-κB expression was determined by Western immunoblot assay; n = 3 samples. *P < 0.05 vs. control; #P < 0.05 vs. TNF-α; †P < 0.05 vs. TNF-α + HU-308 or JWH-133. B: activation of NF-κB by TNF-α in HCAECs, and CB2 agonists inhibit NF-κB activation. Note the intense nuclear staining of NF-κB (p65) in cells exposed to TNF-α and the faint staining indicating that CB2 agonists diminished TNF-α activation of NF-κB. Shown are representative images from 3 separate experiments.

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