The Developing Bladder is Hypoxic in Vivo and Bladder Angiogenesis is Controlled Through VEGF by Tissue Oxygenisation
Purpose During embryogenesis the fetus is subjected to a relative hypoxic condition. Tissue hypoxia in the neural tube and heart is considered to enhance embryonic angiogenesis, partly by upregulating VEGF expression. We hypothesized that the developing bladder is hypoxic in vivo, and that oxygen tensions modulate VEGF expression in theembryonic bladder. Material and methods Pimonidazole, a hypoxia-marker, was administered to pregnant mice at embryonic days (E)14,16 and 18, spanning bladder inception to a stage when the detrusor and lamina propria become well-defined and detected by immunohistochemistry (IHC) . Western blot and IHC were used to detect VEGF and CD31 (endothelial marker). Explanted E14 bladders were cultured in 20%O2 (representing normoxia for postnatal animals) or 3%O2 (representing a relatively hypoxic state, perhaps more equivalent to the embryo milieu). Percentages of CD31 expressing endothelia were measured by flowcytometry and capillary density was assessed using IHC Results At E14, both VEGF and hypoxic pimonidazole-adducts immunocolocalized to urothelia, whereas, at E16-18, both were more prominently detected in detrusor. At E14, endothelia (CD31) were visualized directly beneath the urothelium and, at later embryonic stages; capillaries were prominent in lamina propria and also detrusor layers. VEGF protein levels within explanted bladders themselves, and also in media conditioned by rudiments, was increased in 3%O2 versus 20%O2. Similarly, the amount and density of endothelia in explants increased after culture in hypoxic versus 20%O2 atmosphere Conclusions In vivo, the developing bladder contains hypoxic regions rich in VEGF and close to nascent capillary beds. Invitro, when oxygen tensions are experimentally altered, expression of VEGF protein positively correlates with endothelial mass and density.