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The present study tests the hypothesis that heterotypic stromal-epithelial interactions cause phenotypic changes in urothelium. The rational for the experimental design is to simulate heterotypic stromal-epithelial interactions that are created at the anastomotic site of intestinal-bladder augmentations and internal urinary diversions where the urothelium is in direct contact with the gastro-intestinal tract tissues. Tissue recombination experiments were performed by combining 14-day embryonic rat and mouse rectal mesenchyme with urothelium from embryonic, newborn, and adult mice or rats. All tissue recombinants were grown beneath the renal capsule of athymic mouse hosts for 6-16 weeks. Analyses were performed to detect expression of uroplakins, cytokeratin 7, 14, 19 and mucin secreting epithelial cells via Periodic Acid-Schiff (PAS). The phenotype of both mouse and rat urothelium was changed to a glandular morphology under the influence of rectal mesenchyme. Immunohistochemical staining revealed a loss of the urothelial specific uroplakins and cytokeratins 7, 14, and 19 (characteristic of urothelium). Histologic analysis revealed the presence of mucin secreting glandular structures which stained positive for PAS. The urothelial transdifferentiation into glandular epithelium was not a function of epithelial age and occurred in the embryonic, newborn and adult urothelium. Likewise, rectal mesenchyme from embryonic, neonatal, and adult animals was able to induce glandular differentiation in bladder epithelium. Urothelium exhibits the plasticity to change into an intestinal like epithelium as a result of mesenchymal/stromal stimulation from the gastro-intestinal tract. This experimental result is germane to heterotypic stromal-epithelial interactions that are created in patients with urinary tract reconstructions (intestinal augmentations, de-mucosalized urothelial lined bladder patches, and internal urinary diversion such as ureterosigmoidostomies). We propose that heterotypic stromal-epithelial interactions may play a role in determining histodifferentiation of urothelial cells at the anastomotic site between bowel and bladder tissue in patients with gastro-intestinal urothelial reconstructions.
PURPOSE - We previously showed that mesenchymal-epithelial interactions are necessary for the development of bladder smooth muscle. Specifically without bladder epithelium embryonic bladder mesenchyme does not differentiate into smooth muscle. We determine whether this process is specific to bladder epithelium or whether epithelial cells from other organ systems induce bladder mesenchyme to differentiate into smooth muscle, as well as whether epithelial age is an important variable.
MATERIALS AND METHODS - We recombined 14-day bladder mesenchyme before smooth muscle differentiation with rat epithelium from 14-day, 19-day, newborn and adult bladder, ureter, colon, ileum, stomach, cornea and epidermis. In addition, bladder epithelium was recombined with 14-day embryonic small intestinal, 14-day embryonic gastric and newborn seminal vesicle mesenchyme. All tissue recombinants were grafted under the renal capsule of an adult rat syngeneic host for 3 weeks.
RESULTS - Immunohistochemical analysis with antibodies directed against smooth muscle alpha-actin revealed that all epithelial types studied induced bladder mesenchyme to differentiate into smooth muscle, although to different degrees. Induction of smooth muscle was independent of urothelial age. In addition, bladder epithelium induced intestinal, gastric and seminal vesicle mesenchyme to differentiate into smooth muscle and express an overall morphological pattern indicative of the bladder fibromuscular wall.
CONCLUSIONS - The mechanism whereby urothelium induces bladder mesenchyme to differentiate into smooth muscle is not specific to embryonic urothelium. Older urothelium and heterotypic epithelium also induce smooth muscle differentiation. With the common use of bowel, stomach and ureteral segments for bladder augmentation it is important to understand the interaction of different types of epithelium with the native bladder.
During bladder development, undifferentiated mesenchymal and epithelial cells undergo an orderly sequence of differentiation defined by the expression of smooth-muscle (alpha-actin, myosin, vinculin, desmin, vimentin, and laminin) and epithelial (cytokeratins 5, 7, 8, 14, 18 and 19) protein markers. This process requires mesenchymal-epithelial interactions with bladder epithelium (urothelium) necessary for the differentiation of bladder smooth muscle. Peptide growth factors such as keratinocyte growth factor (KGF) and transforming growth factors (TGF) alpha and beta are likely candidates as mediators of these mesenchymal-epithelial interactions. Transcripts for KGF, TGF alpha, and TGF beta are regulated during bladder development and during smooth-muscle hypertrophy secondary to bladder-outlet obstruction. Finally, two experimental bladder models--(1) partial outlet obstruction and (2) regeneration of bladder smooth muscle into an acellular tissue matrix--are described in the context of mesenchymal-epithelial interactions in the bladder.
PURPOSE - To assess the role of cell-cell interactions in the development of bladder smooth muscle.
MATERIAL AND METHODS - Bladders from 14-day rat fetuses (prior to smooth muscle differentiation) were isolated and digested with trypsin to separate the mesenchyme and epithelium. Three types of specimens were prepared for grafting under the renal capsule of syngeneic adult hosts: a) intact bladder (BL) which had been isolated from fetuses of timed pregnant rats by surgical methods alone; b) bladder mesenchyme (BLM) alone (urothelium removed following trypsinization); and c) isolated BLM recombined with bladder urothelium (BLM + BLE). After 2 weeks of in vivo growth the grafts were assessed by immunocytochemical techniques for the expression of smooth muscle cells markers (actin, myosin, vinculin, laminin and desmin). The same experiments were repeated in vitro. In the final experiment, the induction of bladder smooth muscle was elicited in situ across species lines. Fourteen-day rat BLM was grafted onto the proximal ureter of an athymic nude mouse after ipsilateral nephrectomy.
RESULTS - Grafts of intact BL and BLM + BLE recombinants expressed smooth muscle differentiation. In contrast, grafts of BLM alone remained devoid of smooth muscle. This was also true for the in vitro studies in which, after 5 days of growth, BLM + BLE recombinants (n = 12) showed clear evidence of smooth muscle differentiation. In contrast, cultures of BLM alone (n = 12) exhibited poor growth without smooth muscle differentiation. In the final experiment testing the induction of smooth muscle across species, after 1 month in vivo growth the urothelium of the cut end of the ureter had invaded the grafted BLM. The BLM grafts (n = 3) had increased 30 times in size, and immunocytochemical staining showed clear expression of smooth muscle markers in the grafted BLM in proximity to the urothelium.
CONCLUSION - We have shown that the differentiation of smooth muscle in the rat bladder is dependent upon an inductive interaction with the epithelium.