Highly Calibrated Relationship Between Bleomycin Concentrations and Facets of the Active Phase Fibrosis in Classical Mouse Bleomycin Model.

Abstract

The mouse bleomycin model is useful in pre-clinical IPF research to understand pathophysiological mechanisms and pharmacological interventions. In the present study, we systematically investigated the effects of bleomycin at a 60-fold dose range on experimental features of lung fibrosis in the mouse bleomycin model. We analyzed the effect of intratracheal (i.t.) dosing of 0.05-3 U/kg bleomycin on disease phenotypes, including weight loss, morbidity and mortality, pulmonary inflammation, lung collagen content, various BALF biomarkers, and histology in a 14-day mouse model when the animals are in the active phase of fibrosis. In mice, challenge with 1-2 U/kg bleomycin doses induced significant and saturated responses on fibrotic endpoints, confirmed by collagen content, BALF biomarker levels, and marked weight loss compared to the normal control (NC). We observed 100% mortality in 3 U/kg of bleomycin-treated mice. In contrast, 0.05-0.5 U/kg bleomycin doses induced a dose-dependent fibrotic phenotype. The mice challenged with doses of 0.25-0.5 U/kg bleomycin showed optimum body weight loss, a significant increase in pulmonary inflammation, and the fibrotic phenotype compared to NC. Furthermore, we showed 0.25-0.5 U/kg bleomycin increases expression levels of (pro-) fibrotic cytokines, which are the mediators involved in the activation of myofibroblast during fibrogenesis (TGF-β1, IL-13, IL-6, WISP-1, VEGF), angiogenesis (VEGF), matrix remodeling (TIMP-1), and non-invasive lung function biomarker (CRP) compared to NC. A modified Ashcroft scale quantified that the fibrotic changes in the lungs were significantly higher in the lung of mice dosed at 0.25-0.5 U/kg > 0.1 U/kg bleomycin and non-significant in mice lung dosed at 0.05 U/kg bleomycin compared to NC. We demonstrated that the changes due to 0.25-0.5 U/kg i.t. bleomycin on protein biomarkers are enough to drive robust and detectable fibrotic pathology without mortality. The 0.1 U/kg has a moderate phenotype, and 0.05 U/kg had no detectable phenotype. The Goodness of Fit (r2) and Pearson correlation coefficient (r) analyses revealed a positive linear association between change evaluated in all experimental features of fibrosis and bleomycin concentrations (0.05-0.5 U/kg). Here, we provide an examination of a highly calibrated relationship between 60-fold bleomycin concentrations and a set of in vivo readouts that covers various facets of experimental fibrosis. Our study shows that there is a dose-dependent effect of bleomycin on the features of experimental fibrosis at <1 U/kg, whereas saturated responses are achieved at >1 U/kg. Our careful experimental observations, accuracy, and comprehensive data set provided meaningful insights into the effect of bleomycin dose(s) on the fibrotic phenotype, which is valuable in preclinical drug development and lung fibrosis research. In addition, we have presented a set of reproducible frameworks of endpoints that can be used for reliable assessment of the fibrotic phenotype, and in vivo therapeutic intervention(s) with improved accuracy.