Effect of Airway Microenvironment on TGF-beta driven Epithelial to Mesenchymal Cell Transition in the Transplanted Lung

Some patients dying of lung disease are lucky enough to get a new lease of life with a lung transplant. Tragically, half of them will die within 5 years of having their transplant because their new lungs fail. The new lungs are destroyed by a scarring process which blocks the small bronchial tubes. This process is called obliterative bronchiolitis or OB.
We will try to find out if the lining cells of the lungs’ bronchial tubes, (called epithelial cells), are vital in developing OB. Damaged lining cells are normally repaired in healthy individuals. However, if the damage is frequent or continuous, these cells can change their behaviour to become specialist scarring cells (called mesenchymal cells). Mesenchymal cells are responsible for depositing scar tissue. We will test if repeated damage to the lining cells after lung transplant makes them change into specialist cells which deposit scar tissue which could block the small bronchial tubes.
In this study, we will take lining cells from patients who have had a lung transplant and grow them in the laboratory. We will then investigate if growth factors in the lung when combined with danger signals released at times of injury can drive the change from lining cells to scarring cells.
This is important because if we understand how this change occurs, it may be possible to block it and prevent lining cells from changing behaviour. The hope would be to develop new treatments which protect the bronchial tubes from scarring. This would greatly improve long term survival in patients who have had a lung transplant.

Survival after lung transplantation at only 50% at 5 years is much poorer than after other solid organ transplants. The lung allograft fails due to the development of an obliterative bronchiolitis (OB), a progressive condition characterised by airways obstruction. Associations between OB and inflammatory insults to the lung have been identified, yet the mechanism linking airway epithelial cell injury to airway fibrosis and remodelling after lung transplantation remains elusive.
This proposal will evaluate if epithelial to mesenchymal transition (EMT), a change in epithelial cell phenotype and function driven by TGFbeta, might represent a final common pathway to fibrosis in the injured airway and act as a valuable therapeutic target.The specific aims are:
(1)To characterise TGFbeta-driven EMT in primary airway epithelial cells cultured from lung transplant recipients. Morphological, protein expression and functional changes in cells undergoing EMT will be characterised using confocal microscopy, western blotting and realtime PCR for our panel of EMT markers. Zymography and matrigel invasion assays will assess functional matrix metalloproteinase activity.
(2)To explore how TNFalpha augments TGFbeta driven EMT by assessing change in cell surface expression of the alphaVbeta6 integrin and the decoy TGFbeta receptor BAMBI.
(3)To examine intracellular signalling crosstalk between the MAPkinase and Smad signalling pathways in cells undergoing EMT with specific attention to the TAK-1 ? p38 ? NFkB pathway. Using phosphospecific antibodies and selective blockade with peptide inhibitors, SiRNA and RNAi, I will dissect out the relative contributions from each pathway to the EMT phenotype.
(4)To conduct a longitudinal observational study in 60 lung recipients from the time of transplant by measuring EMT markers on epithelial cells harvested by brushing at serial bronchoscopies. Bronchoalveolar lavage (BAL) fluid from recipients with and without OB will be assessed for its ability to drive EMT in normal airway epithelium. By selective blockade of growth factors and pro-inflammatory signals in the BAL we will determine their relative contribution to the process.
(5)To establish co-cultures of stimulated alveolar macrophages and airway epithelial cells to determine if interaction between these cells is important in driving EMT. The effect of anti-TNFalpha approaches and exogenous latent TGFbeta will be assessed in the model.
The scientific expertise in my institution together with my clinical links to the largest lung transplant programme in UK places me in a unique position to address these aims effectively and move towards improving survival in lung transplant recipients.