The Involvement of Phosphoinositide 3-kinase in Murine Lung Branching Morphogenesis and Insights into Protein Mediated Drug Delivery

Abstract

Epithelial branching morphogenesis is a critical step in the development of manyepithelial organs including the lungs and the salivary glands. Many of the signallingpathways that orchestrate this process show a resurgence in the diseased state.We sought to determine the role of a common disease pathway, thephosphoinositide 3-kinase (PI3K) pathway, in the branching program of the lung. We utilised ex-vivo cultures of embryonic murine lungs that best recapitulates thedevelopmental process. Inhibition of PI3Kα, or its downstream signallingcomponents Akt and mammalian target of rapamycin, was able to greatly enhancethe branching potential of embryonic lungs, implying a negative role for PI3K in thelung branching morphogenesis. Moreover inhibition of PI3K was sufficient to alterthe morphogenic properties of fibroblast growth factor 7 on isolated murine lungepithelium from a cystic to a branching response.We also investigated protein mediated drug delivery tools to provide a means ofenhancing the delivery of PI3K based therapeutics. Cell-penetrating peptides(CPPs) are small proteins that are able to transport a drug cargo across themembrane of a cell. However, given that many CPPs are derived from viralproteins and venoms we sought to determine if common CPPs could induce animmune response. CPPs we capable of delivering a protein cargo into the interiorof epithelial cells without inducing an immune response as measured by a lack ofNFκB activation and no observable increase in interleukin-6 or -8 secretion.We finally explored the potential of repurposing bacterial toxins for therapeuticapplications. The toxins Pseudomonas aeruginosa exotoxin A (PEx) and Cholix(Chx) are natively capable of entering cells and subsequently being transported toeither the cytoplasm of non-polarised cells, or undergoing transcytosis acrosspolarised cells. We were able to identify that the C16:1 ceramide chain variant ofthe ganglioside GM1 is capable of enhancing the intracellular transport of PEx tothe Golgi network. Moreover, we demonstrated that PEx is able to transport ansiRNA cargo across polarised monolayers of Caco-2 cells and deliver said cargointo cells present in the basolateral compartment.

Date of Award	13 Jun 2014
Original language	English
Awarding Institution	University of Bath
Supervisor	Stephen Ward (Supervisor), David Tosh (Supervisor) & Malcolm Watson (Supervisor)