Lack of cystic fibrosis transmembrane conductance regulator disrupts fetal airway development in pigs

Abstract

Loss of cystic fibrosis transmembrane conductance regulator (CFTR) function causes cystic fibrosis (CF), predisposing the lungs to chronic infection and inflammation. In young infants with CF, structural airway defects are increasingly recognized before the onset of significant lung disease, which suggests a developmental origin and a possible role in lung disease pathogenesis. The role(s) of CFTR in lung development is unclear and developmental studies in humans with CF are not feasible. Young CF pigs have structural airway changes and develop spontaneous postnatal lung disease similar to humans, therefore, we studied lung development in the pig model (non-CF and CF). CF trachea and proximal airways had structural lesions detectable as early as pseudoglandular development. At this early developmental stage, budding CF airways had smaller, hypo-distended lumens compared to non-CF airways. Non-CF lung explants exhibited airway lumen distension in response to forskolin/IBMX as well as to fibroblast growth factor (FGF)-10, consistent with CFTR-dependent anion transport/secretion, but this was lacking in CF airways. We studied primary pig airway epithelial cell cultures and found that that FGF10 increased cellular proliferation (non-CF and CF) and CFTR expression/function (in non-CF only). In pseudoglandular stage lung tissue, CFTR protein was exclusively localized to the leading edges of budding airways in non-CF (but not CF) lungs. This discreet microanatomic localization of CFTR is consistent with the site, during branching morphogenesis, where airway epithelia are responsive to FGF10 regulation. In summary, our results suggest that the CF proximal airway defects originate during branching morphogenesis and that the lack of CFTR-dependent anion transport/liquid secretion likely contributes to these hypo-distended airways.

Mutant CFTR Drives TWIST1 mediated epithelial–mesenchymal transition

“…Functional CFTR is also required for rapid regeneration of human airway surface epithelium after injury, and wound healing is delayed in CF epithelia compared with non-CF controls 7 , 8 . CF vs non-CF airway epithelia also exhibit an overall delay in differentiation 3 and dysfunctional CFTR leads to morphological abnormalities in proximal airways during early foetal airway development 9 . Indeed, mice, pigs and young children lacking functional CFTR display defects in tracheal development namely, malformations of the cartilaginous rings, reduced breathing rate and decreased contractile response 10 , 11 .…”

Principles and approaches for reproducible scoring of tissue stains in research

“…There are important analytical factors to initially consider when using quantitative analysis, including the choice of label (e.g., chromogen or fluorescent dye) for detection. Chromogens are frequently used as detection agents in anatomic pathology applications and are available in a range of colors, with the most commonly used compound being Nuclei [75] Area [75] Pixels [2] Membrane [91,92] Counts Number (cell, nuclei, etc.) per field/area [76,93] Automated a Circularity [94,95] Optical density [11,96] Stereology Stereology [83,97] a Several options are available based for each software platform, but only a selected few examples of automated outputs are reported here DAB, which results in brown staining [78].…”

Fundamental Concepts for Semiquantitative Tissue Scoring in Translational Research

“…Any variable that is not randomized across treatment groups has the potential to confound the data. 13,20,41,44 Stains (incidence; intensity) 3,12,13,20 Scoring methods 4,43,45 Evaluation Biological validation 3,4,14,48 Validation of repeatability [52][53][54] Group comparisons 32,43,[55][56][57][58] Expertise Bias may also be introduced into translational research in studies conducted without the support of expertise-specific collaborators to help plan, execute, and appropriately interpret the study. 33,34 Specifically, statistical and pathological analyses are common components in translational studies, but trained statisticians and board-certified pathologists are often omitted from these multidisciplinary teams, leading to data interpretations that are more prone to errors.…”

“…of human diseases/conditions, semiquantitative scoring is regularly used to compare experimental groups. 1,2,11,20 In the clinical area, semiquantitative scoring of human tissues (eg, cancers, tissue/cell cultures, etc.) is often used to help define disease diagnosis, pathogenesis, biomarkers, and clinical prognosis.…”