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Dissertation
Understanding airway smooth muscle molecular physiology using the cystic fibrosis pig
University of Iowa
Doctor of Philosophy (PhD), University of Iowa
Spring 2018
DOI: 10.17077/etd.005612
Abstract
Airway hyper-responsiveness is a clinical feature with high prevalence (40-70%) in people with cystic fibrosis (CF) that contributes to morbidity and mortality in CF and other airway narrowing diseases. Whether airway smooth muscle dysfunction occurs as an intrinsic defect due to loss of the cystic fibrosis transmembrane conductance regulator (CFTR), the anion channel that is altered in CF, or secondarily to inflammation and infection common to the disease is unknown. To better understand CF lung disease, we produced CFTR null pigs. CFTR−/− pigs develop lung disease similar in scope and course to human disease. We found that CF pigs have increased airway smooth muscle tone at birth before the onset of any inflammation or infection. Thus, we hypothesize that CFTR is critical for proper airway smooth muscle function and that loss of the channel contributes to airway smooth muscle dysfunction in CF. Using a variety of biochemical and functional assays we demonstrate that newborn CFTR−/− pig displays airway smooth muscle abnormalities prior to the onset of inflammation or infection including increased basal tone, increased bronchodilator response, and decreased calcium reuptake. These functional defects are due, in part, to loss of CFTR localization in the sarcoplasmic reticulum. These studies propose a unique trafficking motif for CFTR in airway smooth muscle to ensure proper localization.
In addition to these functional studies, total RNA sequencing of newborn WT (CFTR+/+) and CFTR−/− airway smooth muscle revealed differential and significant changes in muscle contraction related genes, ontologies, and pathways. High-throughput ELISA protein comparisons between WT and CFTR−/− airway smooth muscle cells revealed complimentary pathway dysregulation. The genomic and proteomic signatures were used to perform pathway analysis. Large-scale changes in transcript, protein abundance, and phosphorylation status were significantly different in phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) controlled pathways in CFTR−/− samples. We then used connectivity mapping to match small molecules that cause transcriptional signatures opposite of that of CFTR−/− airway smooth muscle. We hypothesized that by scanning for compounds that elicit a negative signature, we would enrich for therapeutic drugs in CF airway reactivity and other airway narrowing diseases. We found 90 compounds that were significantly enriched that represent potential compounds that alleviate airway contractility and encompass many known modulators of airway hyper-responsiveness.
Based upon these findings, we then studied a small molecule inhibitor of protein tyrosine kinase 2 (PYK2). PYK2 is a calcium-sensitive tyrosine kinase known to be an upstream regulator of both PI3K/AKT and MAPK pathways and we tested whether blocking PYK2 activation could alter airway contractility. Using multiple molecular and functional approaches we found PYK2 inhibition can modulate airway smooth muscle function. Collectively, these studies show that loss of CFTR in airway smooth muscle causes changes in Ca2+ homeostasis that lead to whole genome-wide transcriptional and proteomic changes, including alterations in muscle contraction transcripts and proteins. By recognizing the newborn CFTR−/− pig as a model of airway smooth muscle hyper-reactivity absent inflammation, we can enhance our molecular understanding of airway smooth muscle biology and pathophysiology in addition to discovering novel therapeutics for airflow limitation.
Details
- Title: Subtitle
- Understanding airway smooth muscle molecular physiology using the cystic fibrosis pig
- Creators
- Daniel Paul Cook
- Contributors
- David A Stoltz (Advisor)Isabella M Grumbach (Committee Member)William S Moye-Rowley (Committee Member)Janice L Robertson (Committee Member)Mark A Stamnes (Committee Member)Peter M Snyder (Committee Member)
- Resource Type
- Dissertation
- Degree Awarded
- Doctor of Philosophy (PhD), University of Iowa
- Degree in
- Molecular Physiology and Biophysics
- Date degree season
- Spring 2018
- Publisher
- University of Iowa
- DOI
- 10.17077/etd.005612
- Number of pages
- xvi, 218 pages
- Copyright
- Copyright 2017 Daniel Paul Cook
- Comment
- This thesis has been optimized for improved web viewing. If you require the original version, contact the University Archives at the University of Iowa: https://www.lib.uiowa.edu/sc/contact/
- Language
- English
- Description illustrations
- illustrations (some color)
- Description bibliographic
- Includes bibliographical references (page 156-183).
- Academic Unit
- Molecular Physiology and Biophysics
- Record Identifier
- 9983987797102771
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