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Supervisor: Dr Rob Hynds, J.P. Martinez-Barbera
Project Description:
Background
Airway stenosis (narrowing) is a major clinical problem, occurring as a complication in patients who have undergone endotracheal intubation, tracheostomy, laryngotracheal reconstruction surgery and radiation therapy. In children, stenosis can be congenital or follow tracheo-oesophageal fistula surgery. Common to all of these disease aetiologies is the central role of chronic epithelial injury. Disrupted epithelial-immune and epithelial-stromal interactions drive inflammation, scarring and ultimately stenosis, which can be life-threatening. Unfortunately, we lack model systems in which to explore disease pathogenesis and potential therapies.
Aims, Objectives and Methods
Aim 1: Investigate epithelial senescence in a diverse cohort of airway stenosis patients.
Using an existing biobank of biopsy tissue from patients with airway stenosis, the candidate will perform an immunohistochemical study to assess airway epithelial senescence in a range of airway stenosis aetiologies.
Aim 2: Model airway stenosis using novel mouse modelling approaches.
The candidate will create the first genetically engineered mouse model of airway stenosis. Disease induction will be achieved through initiating senescence in airway stem cells through Mdm2 deletion. We have Mdm2fl/fl mice and preliminary data show induction of airway epithelial-specific recombination using oropharyngeal delivery of Ad-Cre. This project will characterise the model in detail, including qPCR and Western blot confirmation of the loss of Mdm2 in the airway epithelium (but not stroma), assessment of senescence marker proteins, fibroblast activation and collagen deposition, and longitudinal monitoring of airway stenosis using both histological endpoints and microCT imaging (Centre for Advanced Biomedical Imaging, UCL Cancer Institute). These longitudinal analyses will allow timepoint selection for further experimental readouts, including flow cytometry to determine the immune composition of airway granulation tissue formation and high-performance liquid chromatography quantitation of hydroxyproline to quantify collagen deposition.
Aim 3: Investigate senolytic drugs as anti-stenotic therapies.
The combination of the senolytic drugs dasatinib and quercetin has been shown to inhibit epithelial cell senescence in the lung. The candidate will investigate whether dosing of senolytics by oral gavage reverses airway stenosis in the mouse model. Mice will be monitored for weight and survival, and the efficacy of senolytic treatment will be assessed by longitudinal micro-CT imaging. At the endpoint, tracheas will be assessed for histological evidence of fibrosis according to the phenotypes observed above. This represents the first investigation of the potential utility of senolytic drugs in the context of airway stenosis, and this approach would be primed for clinical translation given that intermittently dosed D+Q is well-tolerated in patients with pulmonary fibrosis.
Timeline
The mouse model is established so in vivo work is expected to commence from year one. The student will obtain a Home Office license, and receive full training and post-doctoral supervision. Model characterisation is expected during the first 12-18 months, with senolytic treatments tested during year two and human cell work in year three.
References
1) Dorris et al. Post-intubation subglottic stenosis: aetiology at the cellular and molecular level. European Respiratory Journal (2021; https://doi.org/10.1183/16000617.0218-2020)
2) Yao et al. Senescence of Alveolar Type 2 Cells Drives Progressive Pulmonary Fibrosis. Am J Respir Crit Care (2021; https://doi.org/10.1164/rccm.202004-1274oc)
Contact Information:
Rob Hynds