Urothelial Inhibition of Transforming Growth Factor-β in a Bladder Tissue Recombination Model

Abstract

We examined the role of transforming growth factor-beta in urothelial and bladder development. Transforming growth factor-beta signaling was attenuated in the urothelial compartment and the subsequent effects were examined in a tissue recombination model. Urothelium was cultured from adult rat bladders and transfected with control vector C7Delta or mutant DNIIR (dominant negative transforming growth factor-beta receptor II). Grafts were created by recombining transfected urothelium plus embryonic day 18 bladder mesenchyma and placed beneath the renal capsule of athymic mouse hosts. Grafts were harvested at 21 and 42 days. Final tissues were evaluated with staining and immunohistochemistry using hematoxylin and eosin, Gomori's trichrome strain, broad-spectrum uroplakin, smooth muscle actin-alpha, phosphorylated SMAD2 and Ki67 antigen. Bladder structures were defined as having smooth muscle, suburothelial connective tissue and mature urothelium expressing uroplakin. Urothelial compartment diameters were measured and subcategorized as small--0.10 to 0.40, medium--0.41 to 1.0 and large--greater than 1.1 mm. At 21 days 14 C7Delta control and 15 DNIIR grafts were evaluated. No bladder tissue was seen in the C7Delta grafts vs 49 in DNIIR tissue, including 30 small, 9 medium and 10 large tissues. At 42 days 14 C7Delta and 12 DNIIR grafts were evaluated. Six bladder structures (5 small and 1 medium) were seen in the C7Delta cohort vs 27 (14 small, 7 medium and 6 large) in the DNIIR group. Immunohistochemical detection of phosphorylated-SMAD2 was significantly attenuated in DNIIR tissue. In addition, Ki67 proliferative indexes were 4.0-fold higher in the DNIIR cohort compared to those in C7Delta tissues. We successfully observed that primary urothelium cultures can be genetically manipulated and recombined with undifferentiated mesenchyma to grow bladder tissue. By attenuating transforming growth factor-beta signaling in the urothelium superior bladder tissue growth occurred, suggesting that transforming growth factor-beta is a growth inhibitor in this organ system.