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The Endocannabinoid/CB1R System Is Overactive in Bleomycin-Induced Pulmonary Fibrosis in Pale Ear (HPS1) Mice
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A5757 - The Endocannabinoid/CB1R System Is Overactive in Bleomycin-Induced Pulmonary Fibrosis in Pale Ear (HPS1) Mice
Author Block: R. Cinar1, T. Yokoyama2, N. J. Coffey1, J. K. Park1, M. R. Iyer1, B. R. Gochuico2, W. A. Gahl2, M. C. Malicdan2, G. Kunos1; 1Laboratory of Physiological Studies, National Institute on Alcohol Abuse and Alcoholism, Rockville, MD, United States, 2Medical Genetics Branch, National Human Genome Research Institute, Bethesda, MD, United States.
RATIONALE: Hermansky-Pudlak syndrome (HPS) is a rare autosomal recessive disorder, which in its most common and severe form (HPS-1) leads to adult-onset fatal PF with no effective treatment. Considering the complex disease pathogenesis, targeting multiple pathways may improve therapeutic efficacy. Endocannabinoids acting via CB1 receptors (CB1R) promote tissue fibrosis, including PF. The activity of inducible nitric oxide synthase (iNOS) is also increased in fibrosis. Using C57BL6/J mice with bleomycin (Bleo)-induced PF, we showed that simultaneous targeting of CB1R and iNOS by a hybrid inhibitor (MRI-1867) yielded greater anti-fibrotic efficacy than inhibiting either target alone (Cinar et al., JCI Insight 2017 2(8):92281). We also found that CB1R and iNOS are overexpressed in the lung of HPS-PF patients. Here, we evaluated the status of the ECS and iNOS in bleomycin-induced PF in a mouse model of HPS, Pale-ear.
METHODS: PF was induced in 2-3-month-old Pale-ear mice by oropharyngeal aspiration of Bleo (0.3U/kg). The status of the ECS and iNOS was assessed by endocannabinoid measurements, gene expression profile, hydroxyproline (Hyp) content and fluorescent in-situ hybridization studies in lung tissue. Bleo-challenged mice were treated daily for 14 days by oral gavage of vehicle or MRI-1867 (10 mg/kg/day), a peripherally restricted dual inhibitor of CB1R/iNOS.
RESULTS: Bleo treatment of Pale-ear mice increased lung Hyp content by ~2-fold without causing significant mortality. Bleo treatment also increased lung levels of the endocannabinoids anandamide and 2-arachidonylglycerol by 2-3-fold and Cnr1 and Nos2 expression by ~2-fold, relative to levels in vehicle-treated controls. Bleo-treatment also robustly induced iNOS expression in the lung. Cnr1 and Nos2 mRNA were localized in distinct areas of the fibrotic lung, which suggests their specific pathogenic functions and further justifies their joint therapeutic targeting. Fibrosis development and increased gene expression of fibrogenic markers were partially but significantly attenuated by MRI-1867 treatment, whereas iNOS expression was unaffected. Following a single oral dose, the concentration of MRI-1867 in the lung was much lower in Bleo-treated (1.5 µM) than in control mice (20 µM), which may explain the lack of efficacy against iNOS. As MRI-1867 is a P-glycoprotein substrate, induction of P-glycoprotein by Bleo may limit target organ exposure and efficacy of MRI-1867.
CONCLUSION: The ECS is activated in the lung in a mouse model of HPS-1 PF, and the hybrid CB1R/iNOS inhibitor MRI-1867 partially attenuates PF. Bleomycin reduces target organ exposure to MRI-1867, likely by inducing P-glycoprotein. Further studies are needed to refine mouse model for HPS-PF.
Author Block: R. Cinar1, T. Yokoyama2, N. J. Coffey1, J. K. Park1, M. R. Iyer1, B. R. Gochuico2, W. A. Gahl2, M. C. Malicdan2, G. Kunos1; 1Laboratory of Physiological Studies, National Institute on Alcohol Abuse and Alcoholism, Rockville, MD, United States, 2Medical Genetics Branch, National Human Genome Research Institute, Bethesda, MD, United States.
RATIONALE: Hermansky-Pudlak syndrome (HPS) is a rare autosomal recessive disorder, which in its most common and severe form (HPS-1) leads to adult-onset fatal PF with no effective treatment. Considering the complex disease pathogenesis, targeting multiple pathways may improve therapeutic efficacy. Endocannabinoids acting via CB1 receptors (CB1R) promote tissue fibrosis, including PF. The activity of inducible nitric oxide synthase (iNOS) is also increased in fibrosis. Using C57BL6/J mice with bleomycin (Bleo)-induced PF, we showed that simultaneous targeting of CB1R and iNOS by a hybrid inhibitor (MRI-1867) yielded greater anti-fibrotic efficacy than inhibiting either target alone (Cinar et al., JCI Insight 2017 2(8):92281). We also found that CB1R and iNOS are overexpressed in the lung of HPS-PF patients. Here, we evaluated the status of the ECS and iNOS in bleomycin-induced PF in a mouse model of HPS, Pale-ear.
METHODS: PF was induced in 2-3-month-old Pale-ear mice by oropharyngeal aspiration of Bleo (0.3U/kg). The status of the ECS and iNOS was assessed by endocannabinoid measurements, gene expression profile, hydroxyproline (Hyp) content and fluorescent in-situ hybridization studies in lung tissue. Bleo-challenged mice were treated daily for 14 days by oral gavage of vehicle or MRI-1867 (10 mg/kg/day), a peripherally restricted dual inhibitor of CB1R/iNOS.
RESULTS: Bleo treatment of Pale-ear mice increased lung Hyp content by ~2-fold without causing significant mortality. Bleo treatment also increased lung levels of the endocannabinoids anandamide and 2-arachidonylglycerol by 2-3-fold and Cnr1 and Nos2 expression by ~2-fold, relative to levels in vehicle-treated controls. Bleo-treatment also robustly induced iNOS expression in the lung. Cnr1 and Nos2 mRNA were localized in distinct areas of the fibrotic lung, which suggests their specific pathogenic functions and further justifies their joint therapeutic targeting. Fibrosis development and increased gene expression of fibrogenic markers were partially but significantly attenuated by MRI-1867 treatment, whereas iNOS expression was unaffected. Following a single oral dose, the concentration of MRI-1867 in the lung was much lower in Bleo-treated (1.5 µM) than in control mice (20 µM), which may explain the lack of efficacy against iNOS. As MRI-1867 is a P-glycoprotein substrate, induction of P-glycoprotein by Bleo may limit target organ exposure and efficacy of MRI-1867.
CONCLUSION: The ECS is activated in the lung in a mouse model of HPS-1 PF, and the hybrid CB1R/iNOS inhibitor MRI-1867 partially attenuates PF. Bleomycin reduces target organ exposure to MRI-1867, likely by inducing P-glycoprotein. Further studies are needed to refine mouse model for HPS-PF.
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