Cannabidiol attenuates pulmonary arterial hypertension by improving vascular smooth muscle cells mitochondrial function

Figures

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  Figure 1. Inhibition of CBD in mice PASMCs' hyperproliferation without cytotoxicity. A, Purity of mice PASMCs assessed by immunohistochemistry with the α-SMA antibody. Scale bar = 100 µm B, mRNA levels of Il6 in mice PASMCs, incubation with different concentrations of CBD and/or CoCl2 at 200 µM for stimulating the hypoxia condition, n = 6 per group. C and D, Level of LDH assessed with LDH detection assay in both extracellular (death rate) and intracellular (cell viability) (n = 6 per group). E, Quantitative assessment of BrdU antibody to calculate the ratio of PASMCs proliferation. F, Immunofluorescence of BrdU positive ratio of the PASMCs (n = 7 per group), the nuclei of cells were stained with 4',6-diamidino-2-phenylindole (DAPI). The results were analyzed by one-way ANOVA followed by Bonferroni's multiple comparison test, *P < 0.05, **P < 0.01, ***P < 0.001 vs. the control group, and #P < 0.05, ##P < 0.01, ###P < 0.001 vs. the CoCl2 group or mice PAH-PASMCs.

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  Figure 2. CBD reversed pathological progression of Sugen-hypoxia induced PAH in both preventive and therapeutic mouse models. A-F, Data from PAH preventive mice model. G-L, Data from PAH therapeutic mice model. A and G, Assessments of RVSP. B and H, Assessments of RVH. C and I, Representative images of pulmonary arteries stained with H&E, and representative images of vascular remodeling in the distal arterioles stained with elastin, immunostained for PCNA and α-SMA. Scale bar = 20 µm. D-F and J-L, Pulmonary vascular remodeling rate in the Sugen-hypoxia mice, including the quantification of the relative number of PCNA+/nuclei, the degree of medial wall thickness as a ratio of total vessel size (Media/CSA), and the proportion of non-, partially-, or fully- muscularized pulmonary arterioles (25 to 75 μm in diameter) from PAH mouse model, n = 10 per group. The results were analyzed by one-way ANOVA followed by Bonferroni's multiple comparison test, *P < 0.05, **P < 0.01, ***P < 0.001 vs. the control group, and #P < 0.05, ##P < 0.01, ###P < 0.001 vs. the hypoxia group.

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  Figure 3. CBD inhibited inflammation in PAH without canonical cannabinoid receptors involvement. A-B, mRNA levels of Il6 and Tnfα assessed in the lungs of hypoxia-induced preventive PAH mouse model. C, mRNA levels of Ccl2 and Cxcl10 assessed in the mice PASMCs with or without CoCl2 treatment. D-F, mRNA level of Il6 in mice PASMCs treated with CoCl2, and the effect of antagonists of cannabinoid receptors (SR144528, AM630 and both), the concentration of the antagonists was 10 µM, which was equal to the concentration of CBD, the antagonists were pre-treated for 30 min before CBD added, n = 6 per group. G-H, Assessments of RVSP, RVH in the PAH Cnr2-/- mice, n = 10 per group. I, Representative images of pulmonary arteries stained with H&E and elastin of Cnr2-/- and WT littermate controls with or without Sugen hypoxia-induced or daily CBD-treated, scale bar = 20 µm. The results were analyzed by one-way ANOVA followed by Bonferroni's multiple comparison test, *P < 0.05, **P < 0.01, ***P < 0.001 vs. the control group, and #P < 0.05, ##P < 0.01, ###P < 0.001 vs. the hypoxia or CoCl2 treatment group, $P < 0.05 vs. the CoCl2 with CBD group.

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  Figure 4. CBD improved mitochondrial function in mice PAH-PASMCs. A, Representative images of PASMCs isolated from the control and PAH mice labeled with MitoTracker after the treatment with CBD (10 µM) or the control vehicle for 2 h. Nuclei were counterstained using Hoechst 33342, scale bar = 10 µm. B and C, Quantification of mitochondrial proportion of lineage that longer than 1 µm (40 pixels, about 1 µm) and mean length of mitochondria in control PASMCs and PAH-PASMCs labeled with Mitotracker after treatment with or without CBD for 2 h, n = 20 per group. D, Immunoblotting for DRP1, FIS1, MFN2 proteins in mice PAH-PASMCs with CBD (10 µM) or vehicle for 12 h. All blots were re-probed for β-actin as control. The results were analyzed by one-way ANOVA followed by Bonferroni's multiple comparison test, *P < 0.05, **P < 0.01, ***P < 0.001 vs. the control group, and #P < 0.05, ##P < 0.01, ###P < 0.001 vs. the mice PAH-PASMCs.

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  Figure 5. Analysis of the protection effect of CBD on oxidant stress. A-B, Quantification of enzyme activities of GR and GSH-PX in the lungs of hypoxia-induced preventive PAH mice, n = 6 per group. C, MDA content in the blood of preventive hypoxia-induced PAH mice, n = 6 per group. D-H, mRNA levels of Keap1, Nfe2l2, Nqo1, Hmox1 and Sod1 quantified using RT-PCR in PASMCs, which were isolated from control and PAH mice after the treatment with CBD (10 µM) or control vehicle, were n = 5 per group. I, Quantification of ROS with a fluorescence 96-plate by a fluorescence microplate reader, DCFH-DA fluorescence intensity in PASMCs isolated from control and PAH mice after the treatment with CBD (10 µM) or control vehicle or rosup (provided in the kit as a positive control for ROS) for 2 h, n = 8 per group. J, Representative images of ROS fluorescence in PASMCs isolated from control and PAH mice, assessed by laser scanning microscope. Nuclei was counterstained using DAPI, n≥7 per group. Scale bar = 100 µm. The results were analyzed by one-way ANOVA followed by Bonferroni's multiple comparison test, *P < 0.05, **P < 0.01, ***P < 0.001 vs. the control group, and #P < 0.05, ##P < 0.01, ###P < 0.001 vs.…

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  Figure 6. CBD prevented the transition from oxidative phosphorylation to glycolysis in PAH. A-B, Quantification of the mitochondrial OCR and ECAR of control human PASMCs and hypoxia-induced human PASMCs after treatment with CBD or the control vehicle for 12 h, n = 5 per group. C, Gene expression of PDK1 in the lungs of patients with PAH (n = 15) and control subjects (n = 11), analyzed by an unpaired two-tailed Student's t-test. D, Quantification of Pdk1 in PASMCs isolated from control and PAH mice after the treatment with or without CBD for 12 h, n = 5 per group. E, Contents of lactate in the blood of hypoxia-induced preventive PAH mice detected with the lactate assay, n = 6 per group. F, mRNA level of Pfkfb3 quantified in PAH mice lungs with or without CBD treatment, n = 5 per group. G, Immunoblotting for PFKFB3 protein in hypoxia-induced preventive PAH mice lungs (up), control human PASMCs and hypoxia-induced human PASMCs after the treatment with or without CBD (middle) and PASMCs isolated from control and PAH mice after the treatment with CBD (below). All blots were re-probed for β-actin as control. The results were analyzed by one-way ANOVA followed by Bonferroni's multiple comparison…