2021-2022
Characterizing MUC5AC upregulating and morphology restoring capacity of the synthetic steroid VBP15 in Cystic Fibrosis airway models
Abstract
Cystic Fibrosis (CF) is the most common autosomal recessive disease in the world. It is characterized by a chronic muco-inflammation cycle resulting in hypersecretion of mucins MUC5AC and MUC5B via activation of NF-kB. This contributes to a thick and more viscous mucus. The current treatments available are only effective in 85% of the CF patients, which creates a need for a universal CF medicine. VBP15, a new drug for Duchenne muscular dystrophy and asthma, is expected to reduce MUC5AC expression through inhibition of NF-kB. The final purpose of this study is to investigate whether VBP15 results in a lower mucus viscosity in CF nasal epithelial cells in vitro. We are going to fully characterize this, firstly, by examining the VBP15 toxicity at a range of micromolar concentrations by performing an Alamar blue assay and flow cytometry. In addition, intracellular expression of MUC5AC and MUC5B was determined on mRNA level using qPCR and on protein level using western blot and flow cytometry. The effect of long term VBP15 exposure on airway epithelial differentiation was examined using qPCR and immuno-fluorescence (IF). Lastly, our final goal, mucus viscosity, was investigated by rheology assays and Scanning Electron Microscopy. The toxicity assay demonstrated that neither CF nor WT cells were affected by VBP15. Intracellular there was more MUC5AC detected in WT than CF. In addition, western blot revealed that VBP15 treatment decreased the glycosylation of MUC5AC. qPCR and IF confirmed there were fewer ciliated cells in CF ALI's. After long term VBP15 treatment, qPCR showed an increase in CFTR expression over time. SEM revealed that CF cells show a morphology closer resembling WT cells after VBP15 exposure. Altogether, these data suggest that in ALI cultures, VBP15 can stimulate towards a more WT morphology, increase CFTR expression over time and decrease the diversity in MUC5AC glycosylated states possibly resulting in lower viscous mucus. These implications suggest that VBP15 is a suitable steroid to improve the CF phenotype and could possibly, when used in combination with current treatments, drastically improve the quality of life for all CF patients.
Graphical abstract
Students involved:
Jo-Ann Bonapart, Vera ter Braak, Kim Clevers, Leonie Duin, Ann Haaksman, Dani Holla, Jamilla Huizer, Anne de Jong, Marit Koopman, Luc Krebbeks, Nikki Scheen, Tristan Willaarts, Susanne Covers, Evelien Smits, Milou Verburg and Jeevah Bains
Experts Involved
Prof. Dr. Jeffrey Beekman
Full Professor
Prof. Dr. Kors van der Ent
Full Professor
Sacha Spelier MSc
PhD Student
Juliet Lefferts MSc
PhD Student
Danja Muilwijk MSc
PhD Student
Dr. Gimano Amatngalim
Post-Doc