Hypoxia Induced Compromise Of Urothelium In Neuropathic Bladders: Recovery By Epigenetic Modifying Agent
Anna Radford, FRCS, PhD1, Alex Turner, FRCS, PhD2, Carl Fishwick, PhD3, Jennifer Southgate, PhD4, Ramnath Subramaniam, FRCS, FEAPU, PhD1.
1Leeds Teaching hospitals NHS trust, Leeds, United Kingdom, 2Alex Turner, Leeds, United Kingdom, 3Jack Birch Unit, Biology Dept, York University, United Kingdom, 4Jack Birch Unit, Biology Dept, University of York, United Kingdom.
Tissue Engineering and Regenerative Medicine (TERM) provides opportunities to introduce new techniques into paediatric urology practice. In this work, improvements to the concept of composite cystoplasty: where high pressure in end-stage disease bladders is alleviated by augmentation using bowel smooth muscle lined by an autologous in vitro-grown bladder epithelium, were considered.
Immunohistochemical characterisation of neuropathic bladders was performed. A hypoxia model was created in order to mimic the disease phenotype using propagated normal human urothelial cells and tissue in vitro. Cell counts were used to assess proliferation. Trans-epithelial electrical resistance (TEER) was used to measure barrier function in differentiated urothelium. Immunocytochemistry, immunoblotting and RTPCR were employed to identify mechanistic pathways leading to heritable changes in cell and tissue phenotype.
Assessment of human neuropathic bladder biopsies samples demonstrated significant expression of hypoxia-inducible factor (HIF-1 α) (median = 60.21, IQR = 42.37 – 95.05, n=15) compared to normal controls (median = 26.04, IQR = 11.66 – 33.98, n=9), (p=<0.001). Exposure to hypoxia reduced the proliferation and differentiation capacity of urothelial cells in vitro. Whereas proliferation recovered upon switching to normoxia, the compromised differentiation persisted as demonstrated by a significantly reduced TEER in previously hypoxic cells (1803.33 Ω.cm2 ± 235.02), compared to controls in normoxia (3203.33 Ω.cm2 ± 220.66), (p<0.001, n=11). Repressive epigenetic marks were found to alter in location and abundance in the compromised urothelium. These marks were targeted using an epigenetic-modifying agent, which significantly recovered the differentiated urothelial phenotype. This recovery was replicated in urothelium from diseased bladders.
It is proposed that hypoxia results in persistent compromise in differentiated urothelium. The urothelial phenotype is recoverable by the application of an epigenetic modifying agent. By using an integrated approach to regenerative medicine, epigenetic modification may provide strategies to improve the efficacy of autologous tissue engineering approaches in paediatric and adult urology.
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