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. 2007 Jul;293(1):H610-9.
doi: 10.1152/ajpheart.00236.2007. Epub 2007 Mar 23.

Cannabidiol Attenuates High Glucose-Induced Endothelial Cell Inflammatory Response and Barrier Disruption

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Free PMC article

Cannabidiol Attenuates High Glucose-Induced Endothelial Cell Inflammatory Response and Barrier Disruption

Mohanraj Rajesh et al. Am J Physiol Heart Circ Physiol. .
Free PMC article

Abstract

A nonpsychoactive cannabinoid cannabidiol (CBD) has been shown to exert potent anti-inflammatory and antioxidant effects and has recently been reported to lower the incidence of diabetes in nonobese diabetic mice and to preserve the blood-retinal barrier in experimental diabetes. In this study we have investigated the effects of CBD on high glucose (HG)-induced, mitochondrial superoxide generation, NF-kappaB activation, nitrotyrosine formation, inducible nitric oxide synthase (iNOS) and adhesion molecules ICAM-1 and VCAM-1 expression, monocyte-endothelial adhesion, transendothelial migration of monocytes, and disruption of endothelial barrier function in human coronary artery endothelial cells (HCAECs). HG markedly increased mitochondrial superoxide generation (measured by flow cytometry using MitoSOX), NF-kappaB activation, nitrotyrosine formation, upregulation of iNOS and adhesion molecules ICAM-1 and VCAM-1, transendothelial migration of monocytes, and monocyte-endothelial adhesion in HCAECs. HG also decreased endothelial barrier function measured by increased permeability and diminished expression of vascular endothelial cadherin in HCAECs. Remarkably, all the above mentioned effects of HG were attenuated by CBD pretreatment. Since a disruption of the endothelial function and integrity by HG is a crucial early event underlying the development of various diabetic complications, our results suggest that CBD, which has recently been approved for the treatment of inflammation, pain, and spasticity associated with multiple sclerosis in humans, may have significant therapeutic benefits against diabetic complications and atherosclerosis.

Figures

Fig. 1
Fig. 1
Effect of cannabidiol (CBD) on high glucose (HG)-induced increased ICAM-1 and VCAM-1 expressions in human coronary artery endothelial cells (HCAECs). Cells were treated with either normal glucose (5 mM) or HG (30 mM) alone for 48 h or pretreated with CBD at indicated concentrations followed by treatment with HG for 48 h, and cell-surface ELISA was then performed by measuring absorbance at 450 nm as described in MATERIALS AND METHODS. A: effect of CBD on HG-induced increased ICAM-1 expression. *P < 0.001 vs. 5 mM glucose; #P < 0.001 vs. HG; ‡P < 0.05 vs. HG. OD, optical density (n = 9). B: effect of CBD on HG-induced increased VCAM-1 expression. *P < 0.001 vs. 5 mM glucose; #P < 0.001 vs. HG; ‡P < 0.05 vs. HG (n = 9). Cells were treated with either normal glucose, HG, or CBD alone for 48 h or pretreated with either cannabinoid-1 or -2 (CB1 or CB2, respectively) antagonists (1 μM each) followed by treatment with either HG alone or in combination with CBD, and cell surface ELISA was performed for ICAM-1 (C) or VCAM-1 (D) expression (n = 9). *P < 0.0001 vs. normal glucose; #P < 0.001 vs. HG (n = 3). E: representative ICAM-1 immunoblot from 3 identical blots depicting the effect of indicated treatments on ICAM-1 expression. *P < 0.001 vs. 5 mM glucose or CBD (4 μM alone); #P < 0.01 vs. HG. F: representative VCAM-1 immunoblot from 3 identical blots depicting the effect of indicated treatments on VCAM-1 expression. *P < 0.01 vs. 5mM glucose or CBD (4 μM alone); #P < 0.01 vs. HG (n = 3).
Fig. 2
Fig. 2
Effect of CBD on HG-induced increased monocyte adhesion to endothelial cells. A: representative pictures of monocytes adhered to HCAECs. HCAECs were either treated with normal glucose, HG for 48 h, or pretreated with CBD (4 μM) or either CB1 or CB2 antagonists followed by incubation with HG alone or in combination with CBD, and adhesion assays were performed as described in the MATERIALS AND METHODS. B: quantification data for monocyte adhered to endothelial cells from 3 independent experiments. *P < 0.001 vs. normal glucose; #P < 0.001 vs. HG.
Fig. 3
Fig. 3
Effect of CBD on HG-induced increased monocyte transendothelial migration (TEM). HCAECs were treated with either normal glucose, HG, or CBD for 48 h or pretreated with CBD or either CB1 or CB2 antagonists followed by incubation with HG ± CBD and continued incubation for 48 h, and monocyte adhesion TEM assays were performed as outlined in MATERIALS AND METHODS. *P < 0.001 vs. control or normal glucose; #P < 0.001 vs. HG (n = 6).
Fig. 4
Fig. 4
Effect of CBD on HG-induced barrier dysfunction and mitochondrial superoxide production. A: representative images of immunofluorescence staining for vascular endothelial cadherin expression in HCAECs from 3 independent experiments with similar results. B: quantitative data for in vitro transcellular hyperpermeability of FITC-dextran induced by HG and the effect of CBD on HG-induced barrier dysfunction in HCAECs. *P < 0.001 vs. normal glucose; #P < 0.001 vs. HG (n = 6). C: effect of CBD on HG induced mitochondrial superoxide production. Mitocondrial superoxide generation was then determined by FACS calibur system using MitoSOX as described in MATERIALS AND METHODS.Shown are representative flow cytometry data of mitochondrial superoxide formation by HG and partial attenuation CBD. Quantifications were performed from mean intensity of MitoSOX fluorescence from 3 independent experiments. Error bars represent standard deviation. C, left: numbers in graphs represent mean fluorescence intensity. *P < 0.001 vs. normal glucose; #P < 0.05 vs. HG (n = 3).
Fig. 5
Fig. 5
Effect of CBD on HG-induced NF-κB activation. A, top: HCAECs were treated as indicated, cytoplasmic extracts were prepared, and Western immunoblot assays were performed to determine inhibitory κB-α factor (IκB-α) degradation. Shown is the representative blot from 3 independent experiments with similar results. A, bottom: quantification of IκB-α degradation in the cytosol. *P < 0.001 vs. control; #P < 0.001 vs. HG. B, top: representative blot for NF-κB (p65) expression in nuclear extracts prepared after treatments as indicated. B, bottom: quantification data for NF-κB expression in nucleus after normalization with histone. *P < 0.01 vs. control; #P < 0.01 vs. HG (n = 3). C: HG-induced nuclear translocation of NF-κB (p65), indicating its activation and its inhibition by CBD. Note intense nuclear staining of p65 in HG-treated cells. Shown are representative images from 5 independent experiments yielding similar results.
Fig. 6
Fig. 6
Effect of CBD on HG-induced inducible nitric oxide (NO) synthase (iNOS) expression and 3-nitrotyrosine (3-NT) formation, proposed protective mechanisms of CBD. A: HCAECs were treated with either normal glucose, CBD (4 μM), HG alone for 48 h or pretreated with CBD followed by HG treatment for 48 h. Total cell lysates were prepared, and 25 μg of protein were resolved on 12% SDS-PAGE gels and probed with anti-human iNOS (monoclonal, used at 1:1,000 dilution; BD Biosciences). Shown is the representative image from 3 separate experiments with identical results. *P < 0.001 vs. 5 mM glucose; #P < 0.01 vs. HG. B: HCAECs were treated as described above, and iNOS expression was analyzed by immunofluorescene staining (mouse monoclonal anti-human iNOS at 1:150 dilution). The nuclei were counterstained with 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI; Molecular Probes, Invitrogen). Shown are representative images from 3 separate experiments. C: 3-NT formation in HG-treated cells and effects of CBD by immunofluorescence assay. Nuclei were counterstained with DAPI. Shown are images from 3 independent experiments with identical results. D: proposed protective mechanisms of CBD against HG-induced endothelial cell inflammatory response and barrier disruption. Mitochondrion is considered to be the major source of hyperglycemia-induced increased superoxide anion production; however, other sources such as xanthine and NAD(P)H oxidases, cyclooxygenase, and uncoupled NOS may also contribute to this process under certain conditions. Hyperglycemia-induced superoxide generation might also favor increased expression of iNOS through activation of NF-κB, which increases the generation of NO. Superoxide anion quenches NO, thereby reducing the efficacy of a potent endothelium-derived vasodilator system. Superoxide can also be converted to hydrogen peroxide by SOD and interact with NO to form a reactive oxidant peroxynitrite (ONOO–), which induces cell damage via lipid peroxidation, inactivation of enzymes and other proteins by oxidation and nitration, and activation of nuclear enzyme poly(ADP-ribose) polymerase (PARP-1). Red arrows/lines indicate activation, and black lines indicate inhibition.

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