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. 2023 Sep 30;12(19):2389.
doi: 10.3390/cells12192389.

Non-Psychoactive Phytocannabinoids Inhibit Inflammation-Related Changes of Human Coronary Artery Smooth Muscle and Endothelial Cells

Elisa Teichmann  1 , Elane Blessing  1 , Burkhard Hinz  1
Affiliations

Non-Psychoactive Phytocannabinoids Inhibit Inflammation-Related Changes of Human Coronary Artery Smooth Muscle and Endothelial Cells

Elisa Teichmann et al. Cells. .

Abstract

Atherosclerosis is associated with vascular smooth muscle cell proliferation, chronic vascular inflammation, and leukocyte adhesion. In view of the cardioprotective effects of cannabinoids described in recent years, the present study investigated the impact of the non-psychoactive phytocannabinoids cannabidiol (CBD) and tetrahydrocannabivarin (THCV) on proliferation and migration of human coronary artery smooth muscle cells (HCASMC) and on inflammatory markers in human coronary artery endothelial cells (HCAEC). In HCASMC, CBD and THCV at nontoxic concentrations exhibited inhibitory effects on platelet-derived growth factor-triggered proliferation (CBD) and migration (CBD, THCV). When interleukin (IL)-1β- and lipopolysaccharide (LPS)-stimulated HCAEC were examined, both cannabinoids showed a concentration-dependent decrease in the expression of vascular cell adhesion molecule-1 (VCAM-1), which was mediated independently of classical cannabinoid receptors and was not accompanied by a comparable inhibition of intercellular adhesion molecule-1. Further inhibitor experiments demonstrated that reactive oxygen species, p38 mitogen-activated protein kinase activation, histone deacetylase, and nuclear factor κB (NF-κB) underlie IL-1β- and LPS-induced expression of VCAM-1. In this context, CBD and THCV were shown to inhibit phosphorylation of NF-κB regulators in LPS- but not IL-1β-stimulated HCAEC. Stimulation of HCAEC with IL-1β and LPS was associated with increased adhesion of monocytes, which, however, could not be significantly abolished by CBD and THCV. In summary, the results highlight the potential of the non-psychoactive cannabinoids CBD and THCV to regulate inflammation-related changes in HCASMC and HCAEC. Considering their effect on both cell types studied, further preclinical studies could address the use of CBD and THCV in drug-eluting stents for coronary interventions.

Keywords: cannabidiol; endothelial cells; histone deacetylases; human coronary artery; nuclear factor κB; smooth muscle cells; tetrahydrocannabivarin; vascular cell adhesion molecule-1.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Effect of CBD and THCV on PDGF-induced metabolic activity, cell number, proliferation, and migration of HCASMC. To analyze metabolic activity (A,B), cell number (C,D), and proliferation (E,F), HCASMC were incubated with 25 ng/mL PDGF or its vehicle and increasing concentrations of CBD or THCV or its vehicle for 144 h. Thereafter, metabolic activity was determined by WST-1 colorimetric assay, cell number by crystal violet staining, and proliferation by BrdU incorporation assay. For analysis of migration (G,H), a scratch wound was made in confluent HCASMC. Cells were then incubated with 25 ng/mL PDGF or its vehicle and increasing concentrations of CBD and THCV or its vehicle. Scratch wounds were analyzed after 0 h and 24 h of incubation. Representative images of crystal violet staining (G,H) show the scratch wound after 24 h (scale bar, 200 µm). Vehicle-treated cells were used as control (100%), except for migration experiments, where the wound area after 24 h was set in relation to the wound area after 0 h. Data are presented as means ± SEM of n = 9–12 (3 independent experiments, (A–D)), n = 9 (3 independent experiments, (E,F)), and n = 6–7 (3–4 independent experiments, (G,H)). * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 vs. vehicle control; # p ≤ 0.05, ## p ≤ 0.01, ### p ≤ 0.001 vs. PDGF-stimulated cells; one-way ANOVA plus Bonferroni post hoc test.
Figure 2
Figure 2
Effect of CBD and THCV on metabolic activity and cell number of HCAEC. HCAEC were incubated with increasing concentrations of CBD (A,C) or THCV (B,D) or with vehicle for 24 h. Thereafter, metabolic activity was determined by WST-1 assay (A,B) and cell number by crystal violet staining (C,D). Vehicle-treated cells were used as controls (100%). Data are presented as means ± SEM of n = 9 (3 independent experiments). ** p ≤ 0.01, *** p ≤ 0.001 vs. vehicle control; one-way ANOVA plus Dunnett post hoc test.
Figure 3
Figure 3
Effect of CBD and THCV on VCAM-1 and ICAM-1 mRNA expression in HCAEC under basal, IL-1β- and LPS-induced conditions. HCAEC were incubated for 24 h with 10 ng/mL IL-1β or its vehicle and increasing concentrations of CBD (A,C), THCV (B,D), or with vehicle. For LPS-stimulated cells, HCAEC were preincubated with increasing concentrations of CBD (E,G), THCV (F,H), or vehicle for 1 h and then co-incubated with 1 µg/mL LPS or its vehicle for 24 h. Thereafter, mRNA expression was determined by qRT-PCR. Cells treated with vehicle in combination with IL-1β or LPS were set 100%. Data are presented as means ± SEM of n = 3 (3 independent experiments, (B,D,F,H)) or n = 4 (4 independent experiments, (A,C,E,G)). * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 vs. non-stimulated vehicle control group; ### p ≤ 0.001 vs. IL-1β- or LPS-stimulated cells; one-way ANOVA plus Bonferroni post hoc test.
Figure 4
Figure 4
Effect of CBD and THCV on VCAM-1 and ICAM-1 protein expression in HCAEC under basal, IL-1β-, and LPS-induced conditions. For analysis under basal conditions, HCAEC were incubated with increasing concentrations of CBD (A,D), THCV (G,J), or vehicle control for 24 h. For analysis under IL-1β-induced conditions, HCAEC were incubated with 10 ng/mL IL-1β or its vehicle and increasing concentrations of CBD (B,E) or THCV (H,K) or its vehicle for 24 h. For analysis under LPS-induced conditions, HCAEC were preincubated with increasing concentrations of CBD (C,F) or THCV (I,L) or its vehicle for 1 h, followed by the addition of 1 µg/mL LPS or its vehicle and further incubation for 24 h. Protein expression was determined by Western blot analysis, with representative blots shown here. In (C,F), in (G,J), as well as in (I,L), the same β-actin blots are shown, since the proteins analyzed in these Western blots were separated on the same gel. Cells treated with vehicle (A,D,G,J) or vehicle in combination with IL-1β (B,E,H,K) or LPS (C,F,I,L) served as control (100%). Data are expressed as means ± SEM of n = 3 (3 independent experiments, (A–C,E–L)) or n = 4 (4 independent experiments, D). * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 vs. corresponding control; # p ≤ 0.05, ## p ≤ 0.01, ### p ≤ 0.001 vs. IL-1β- or LPS-stimulated cells; one-way ANOVA plus Bonferroni post hoc test.
Figure 5
Figure 5
Effect of antagonists against cannabinoid receptors CB1 and CB2 and TRPV1 on VCAM-1 protein levels reduced by CBD and THCV in IL-1β- and LPS-stimulated HCAEC. HCAEC were preincubated with 1 µM AM251, 1 µM AM630, and 1 µM capsazepine for 1 h followed by the addition of 10 ng/mL IL-1β, 6 µM CBD (A), 10 µM THCV (C), or vehicles and subsequent 24 h co-incubation of cells with the compounds or their vehicles. For LPS-stimulated cells, HCAEC were preincubated with receptor antagonists for 1 h and then preincubated with 6 µM CBD (B), 10 µM THCV (D), or vehicle for an additional 1 h. This was followed by the addition of 1 µg/mL LPS or its vehicle and a further 24 h co-incubation of cells with the compounds or their vehicles. As a control, the effects of the receptor antagonists were analyzed upon co-incubation with IL-1β (E) and LPS (F) for 24 h without cannabinoid addition. Thereafter, protein expression was determined by Western blot analysis. The blots shown are representative. Cells treated with vehicle in combination with IL-1β or LPS were set 100%. Data are presented as means ± SEM of n = 3 (3 independent experiments). * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 vs. non-stimulated vehicle control group (open columns); # p ≤ 0.05, ## p ≤ 0.01, ### p ≤ 0.001 vs. the 100% IL-1β- or LPS stimulated control group; one-way ANOVA plus Bonferroni post hoc test.
Figure 6
Figure 6
Effect of MAPK, NF-κB (A,B), HDAC (C,D) and ROS inhibitors (E,F) on IL-1β- and LPS-induced VCAM-1 and ICAM-1 protein expression in HCAEC. HCAEC were preincubated with the respective inhibitor (SB203580, PD98059, SP600125: 10 µM; BAY 11-7082: 1 µM; TSA: 0.1 µM, 10 µM; NAC: 1 mM) or vehicle for 1 h, followed by the addition of 10 ng/mL IL-1β (A,C,E) or 1 µg/mL LPS (B,D,F) or vehicle and further co-incubation of the substances for 24 h. Protein expression was then determined by Western blot analysis. The blots shown are representative. Cells treated with inhibitor vehicle and IL-1β or LPS served as controls (set as 100%). Data are shown as means ± SEM of n = 3 (3 independent experiments). * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 vs. non-stimulated vehicle control group; # p ≤ 0.05, ## p ≤ 0.01, ### p ≤ 0.001 vs. the 100% IL-1β- or LPS stimulated control group; one-way ANOVA plus Bonferroni post hoc test.
Figure 7
Figure 7
Effect of CBD and THCV on the phosphorylation of p38 MAPK in IL-1β- and LPS-stimulated HCAEC. HCAEC were incubated for the indicated times with 10 ng/mL IL-1β in the presence of 6 µM CBD (A), 10 µM THCV (C), or vehicle. For LPS-stimulated cells, HCAEC were preincubated with 6 µM CBD (B), 10 µM THCV (D), or vehicle for 1 h and then co-incubated with 1 µg/mL LPS for the indicated time points. At the 0 h time point, the HCAEC were treated with the test substances, and the supernatants were then immediately collected and the cells processed for subsequent analysis. Phosphorylation of proteins was then determined by Western blot analysis. The blots shown are representative. Cells treated with IL-1β or LPS for 0 h were used as controls (100%). Data are shown as means ± SEM of n = 3 (3 independent experiments). Statistical significance between the two groups of an incubation period was excluded using Student’s unpaired two-tailed t test.
Figure 8
Figure 8
Effect of CBD and THCV on regulators of the NF-κB signaling pathway in IL-1β- and LPS-stimulated HCAEC. HCAEC were incubated with 10 ng/mL IL-1β and 6 µM CBD (A,B), 10 µM THCV (C,D), or vehicle for the indicated times. For LPS-stimulated cells, HCAEC were preincubated with 6 µM CBD (E,F), 10 µM THCV (G,H), or vehicle for 1 h and co-incubated with the appropriate compounds after the addition of 1 µg/mL LPS for the indicated times. For analysis of phospho-p65 NF-κB in the cytosolic (I) and nuclear fractions (J), HCAEC were preincubated with 6 µM CBD, 10 µM THCV, or vehicle for 1 h, followed by the addition of 1 µg/mL LPS and co-incubation with the compounds for 2 h. The blots shown are representative. At the 0 h time point, the HCAEC were treated with the test substances, and the supernatants were then immediately collected and the cells processed for subsequent Western blot analysis. Cells treated with IL-1β for 1 h or with LPS for 2 h were used as controls (100%). Data are presented as means ± SEM of n = 3 (3 independent experiments, (A–D,F,I,J)) or n = 4 (4 independent experiments, (E,G,H)). * p ≤ 0.05, ** p ≤ 0.01 vs. corresponding vehicle control; Student’s unpaired two-tailed t test (A–H). * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 vs. IL-1β- or LPS-stimulated cells; one-way ANOVA plus Bonferroni post hoc test (I,J).
Figure 9
Figure 9
Effect of CBD and THCV on phosphorylation of class II HDAC in HCAEC under basal conditions or under IL-1β or LPS stimulation. For analysis under basal conditions, HCAEC were incubated with 6 µM CBD, 10 µM THCV, or vehicle for 24 h (A,B). For analysis under IL-1β-induced conditions, HCAEC were incubated with 10 ng/mL IL-1β together with 6 µM CBD, 10 µM THCV, or vehicle control for 24 h (C,D). For analysis under LPS-induced conditions, HCAEC were preincubated with 6 µM CBD, 10 µM THCV, or vehicle for 1 h, followed by the addition of 1 µg/mL LPS or its vehicle and further incubation for 24 h (E,F). Thereafter, protein expression was determined by Western blot analysis. The blots shown are representative. Vehicle-treated cells were used as controls (100%). Data are shown as means ± SEM of n = 3 (3 independent experiments, (C–F)) or n = 4 (4 independent experiments, (A,B)). * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 vs. corresponding vehicle control; # p ≤ 0.05, ## p ≤ 0.01, ### p ≤ 0.001 vs. IL-1β- or LPS-stimulated cells; one-way ANOVA plus Dunnett (A,B) or Bonferroni post hoc test (C–F).
Figure 10
Figure 10
Effect of CBD and THCV on adhesion of THP-1 monocytes to IL-1β- and LPS-stimulated HCAEC. HCAEC were incubated for 24 h with 10 ng/mL IL-1β or its vehicle and 6 µM CBD, 10 µM THCV or its vehicle (A). For LPS-stimulated cells, HCAEC were preincubated with 6 µM CBD or 10 µM THCV or vehicle for 1 h, followed by the addition of 1 µg/mL LPS or its vehicle and further co-incubation with the compounds or vehicles for 24 h (B). Calcein-AM-labeled THP-1 cells (green) were then attached to endothelial cells for 30 min before analysis by fluorescence microscopy. The nuclei of all cells are shown in blue. The images shown are representative (scale bar, 50 µm). Cells treated with IL-1β or LPS were used as controls (100%). Data are presented as means ± SEM of n = 7–8 (4 independent experiments). *** p ≤ 0.001 vs. vehicle control; one-way ANOVA plus Bonferroni post hoc test.
Figure 11
Figure 11
Summary of the effect of CBD and THCV on proatherosclerotic and proinflammatory properties of HCASMC and HCAEC (Created with BioRender.com, accessed on 18 August 2023). Common abbreviations used have been explained in the manuscript when first mentioned.
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This research was funded in part by the joint research project RESPONSE FV18 (reference: 03ZZ0928A) of the Federal Ministry of Education and Research (BMBF), Germany.

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