Bleomycin Induces Drug Efflux in Lungs. A Pitfall for Pharmacological Studies of Pulmonary Fibrosis

Park, Joshua K.; Coffey, Nathan J.; Bodine, Steven P.; Zawatsky, Charles N.; Jay, Lindsey; Gahl, William A.; Kunos, George; Gochuico, Bernadette R.; Malicdan, May Christine V.; Çınar, Reşat

doi:10.1165/rcmb.2018-0147oc

Cited by 20 publications

(29 citation statements)

References 34 publications

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“…Proinflammatory cytokines and the related were also significantly overrepresented in day 14 of pulmonary fibrosis lung tissues, showing that the changes in the lung transcriptome during the inflammatory-to-fibrotic transition phase after bleomycin injection recapitulate those pulmonary fibroblasts stimulated with eCIRP. This is in line with previous studies showing an increase in the lung infiltration by total white blood cells, neutrophils, and macrophages peaks at day 14, the expressions of profibrotic markers peak at day 21-28 (53,54,80,81). We have previously shown that eCIRP promotes acute lung injury via activation of macrophages, neutrophils, pneumocytes, and lung vascular endothelial cells in the contexts of sepsis, hemorrhagic shock, and intestinal ischemia/ reperfusion injury (1,5,6,(82)(83)(84).…”

Section: Discussionsupporting

confidence: 92%

“…The tissues were collected on 7, 14, 21, 28, and 42 days after the first injection (51). While the exact timing of peak inflammation and fibrosis in the lung, in this model, is not clear, based on previous studies, inflammation occurs between days 7 and 14, and peak fibrosis likely happens between days 21 and 28 (52)(53)(54). Day 14, which we call the inflammatory-to-fibrotic transition phase of exposure to bleomycin, is a time when both processes are active.…”

Section: Analysis Of Gene Expression Omnibus Datasetmentioning

confidence: 96%

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Extracellular CIRP Induces an Inflammatory Phenotype in Pulmonary Fibroblasts via TLR4

2021

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Extracellular cold-inducible RNA-binding protein (eCIRP), a new damage-associated molecular pattern (DAMP), has been recently shown to play a critical role in promoting the development of bleomycin-induced pulmonary fibrosis. Although fibroblast activation is a critical component of the fibrotic process, the direct effects of eCIRP on fibroblasts have never been examined. We studied eCIRP’s role in the induction of inflammatory phenotype in pulmonary fibroblasts and its connection to bleomycin-induced pulmonary fibrosis in mice. We found that eCIRP causes the induction of proinflammatory cytokines and differentially expression-related pathways in a TLR4-dependent manner in pulmonary fibroblasts. Our analysis further showed that the accessory pathways MD2 and Myd88 are involved in the induction of inflammatory phenotype. In order to study the connection of the enrichment of these pathways in priming the microenvironment for pulmonary fibrosis, we investigated the gene expression profile of lung tissues from mice subjected to bleomycin-induced pulmonary fibrosis collected at various time points. We found that at day 14, which corresponds to the inflammatory-to-fibrotic transition phase after bleomycin injection, TLR4, MD2, and Myd88 were induced, and the transcriptome was differentially enriched for genes in those pathways. Furthermore, we also found that inflammatory cytokines gene expressions were induced, and the cellular responses to these inflammatory cytokines were differentially enriched on day 14. Overall, our results show that eCIRP induces inflammatory phenotype in pulmonary fibroblasts in a TLR4 dependent manner. This study sheds light on the mechanism by which eCIRP induced inflammatory fibroblasts, contributing to pulmonary fibrosis.

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Section: Discussionsupporting

confidence: 92%

Section: Analysis Of Gene Expression Omnibus Datasetmentioning

confidence: 96%

Extracellular CIRP Induces an Inflammatory Phenotype in Pulmonary Fibroblasts via TLR4

2021

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“…Fluorescent in situ hybridization (FISH) was performed using the RNAscope R Multiplex Fluorescent Kit v2 (Advanced Cell Diagnostics [ACD], Newark, CA, USA, Cat. 323110) as previously described 39…”

Section: Fluorescent In Situ Hybridizationmentioning

confidence: 99%

CB₁R and iNOS are distinct players promoting pulmonary fibrosis in Hermansky–Pudlak syndrome

Çınar

Park

Zawatsky

et al. 2021

Clinical & Translational Med

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Overactivity of CB 1 R and iNOS contributes to HPSPF development and progression via multiple critical pathological processes including dysregulation of mitochondrial function and IL-11. Simultaneous inhibition of lung CB 1 R and iNOS by a hybrid inhibitor (MRI-1867) is a rational therapeutic strategy for achieving therapeutic efficacy in HPSPF with attenuating fibrosis, improving pulmonary function and survival.

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“…In this study, skin fibrosis was induced by daily subcutaneous injection of bleomycin for 28 days as detailed in the methods and as depicted in Figure 1A . We recently demonstrated that bleomycin induces drug efflux transporters in murine lungs and that this mechanism compromises lung exposure to chemical compounds that happen to be substrates ( 27 ). To determine whether a similar mechanism is triggered by bleomycin in the skin, we first assessed the skin levels of the peripherally restricted hybrid CB 1 R/iNOS inhibitor MRI-1867, a substrate of P-gp ( 16 ), in a bleomycin-induced murine model of skin fibrosis.…”

Section: Resultsmentioning

confidence: 99%

“…Immunohistochemistry was performed as previously described ( 27 ). Tissue sections were incubated overnight at 4°C in an optimized blocking solution: 5% blotting-grade milk (Bio-Rad, Hercules, CA) and 5% horse serum (Vector Laboratories, Burlingame, CA, USA) diluted in double distilled H 2 O.…”

Section: Methodsmentioning

confidence: 99%

Peripheral Hybrid CB1R and iNOS Antagonist MRI-1867 Displays Anti-Fibrotic Efficacy in Bleomycin-Induced Skin Fibrosis

et al. 2021

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Scleroderma, or systemic sclerosis, is a multi-organ connective tissue disease resulting in fibrosis of the skin, heart, and lungs with no effective treatment. Endocannabinoids acting via cannabinoid-1 receptors (CB1R) and increased activity of inducible NO synthase (iNOS) promote tissue fibrosis including skin fibrosis, and joint targeting of these pathways may improve therapeutic efficacy. Recently, we showed that in mouse models of liver, lung and kidney fibrosis, treatment with a peripherally restricted hybrid CB1R/iNOS inhibitor (MRI-1867) yields greater anti-fibrotic efficacy than inhibiting either target alone. Here, we evaluated the therapeutic efficacy of MRI-1867 in bleomycin-induced skin fibrosis. Skin fibrosis was induced in C57BL/6J (B6) and Mdr1a/b-Bcrp triple knock-out (KO) mice by daily subcutaneous injections of bleomycin (2 IU/100 µL) for 28 days. Starting on day 15, mice were treated for 2 weeks with daily oral gavage of vehicle or MRI-1867. Skin levels of MRI-1867 and endocannabinoids were measured by mass spectrometry to assess target exposure and engagement by MRI-1867. Fibrosis was characterized histologically by dermal thickening and biochemically by hydroxyproline content. We also evaluated the potential increase of drug-efflux associated ABC transporters by bleomycin in skin fibrosis, which could affect target exposure to test compounds, as reported in bleomycin-induced lung fibrosis. Bleomycin-induced skin fibrosis was comparable in B6 and Mdr1a/b-Bcrp KO mice. However, the skin level of MRI-1867, an MDR1 substrate, was dramatically lower in B6 mice (0.023 µM) than in Mdr1a/b-Bcrp KO mice (8.8 µM) due to a bleomycin-induced increase in efflux activity of MDR1 in fibrotic skin. Furthermore, the endocannabinoids anandamide and 2-arachidonylglycerol were elevated 2-4-fold in the fibrotic vs. control skin in both mouse strains. MRI-1867 treatment attenuated bleomycin-induced established skin fibrosis and the associated increase in endocannabinoids in Mdr1a/b-Bcrp KO mice but not in B6 mice. We conclude that combined inhibition of CB1R and iNOS is an effective anti-fibrotic strategy for scleroderma. As bleomycin induces an artifact in testing antifibrotic drug candidates that are substrates of drug-efflux transporters, using Mdr1a/b-Bcrp KO mice for preclinical testing of such compounds avoids this pitfall.

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Bleomycin Induces Drug Efflux in Lungs. A Pitfall for Pharmacological Studies of Pulmonary Fibrosis

Cited by 20 publications

References 34 publications

Extracellular CIRP Induces an Inflammatory Phenotype in Pulmonary Fibroblasts via TLR4

Extracellular CIRP Induces an Inflammatory Phenotype in Pulmonary Fibroblasts via TLR4

CB₁R and iNOS are distinct players promoting pulmonary fibrosis in Hermansky–Pudlak syndrome

Peripheral Hybrid CB1R and iNOS Antagonist MRI-1867 Displays Anti-Fibrotic Efficacy in Bleomycin-Induced Skin Fibrosis

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Bleomycin Induces Drug Efflux in Lungs. A Pitfall for Pharmacological Studies of Pulmonary Fibrosis

Cited by 20 publications

References 34 publications

Extracellular CIRP Induces an Inflammatory Phenotype in Pulmonary Fibroblasts via TLR4

Extracellular CIRP Induces an Inflammatory Phenotype in Pulmonary Fibroblasts via TLR4

CB1R and iNOS are distinct players promoting pulmonary fibrosis in Hermansky–Pudlak syndrome

Peripheral Hybrid CB1R and iNOS Antagonist MRI-1867 Displays Anti-Fibrotic Efficacy in Bleomycin-Induced Skin Fibrosis

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CB₁R and iNOS are distinct players promoting pulmonary fibrosis in Hermansky–Pudlak syndrome