We have studied the phenotypic changes in regenerating smooth muscle (SM) tissue of detrusor muscle after local application of a necrotizing, freeze-thaw injury to the serosal surface of rabbit bladder. Bromo-deoxyuridine (BrdU) incorporation and immunofluorescence studies were performed on bladder cryosections from day 2 up to day 15 after surgery with monoclonal antibodies specific for some cytoskeletal markers [desmin, vimentin, non-muscle (NM) myosin] and for SM-specific proteins (alpha-actin, myosin, and SM22). Four days after lesion, some clls incorporated in regenerating SM bundles are BrdU positive, but all display a phenotypic pattern identical to that of the interstitial, highly proliferating cells, i.e., expression of vimentin. By days 7-15 the differentiation profile of regenerating SM returns to that of uninjured SM tissue (appearance of desmin, SM-type alpha-actin, and SM myosin). A chemical denervation achieved by 6-hydroxydopamine treatment for 2 weeks induces the formation of vimentin/SM alpha-actin/NM myosin/SM22-containing myofibroblasts in the interstitial, fibroblast-like cells of uninjured bladder. In the bladder wall, alteration of reinnervation during the regenerating SM process produces: (1) in the outer region, the activation of vimentin/SM alpha-actin/desmin myofibroblasts in the de novo SM cell bundles; and (2) in the inner region of bladder, including the muscularis mucosae, the formation of proliferating, fully differentiated SM cells peripherally to newly formed SM cell bundles. These findings suggest that: (1) the de novo SM tissue formation in the bladder can occur via incorporation of interstitial cells into growing SM bundles; and (2) the alteration of reinnervation during the regenerating process induces a spatial-specific differentiation of interstitial myofibroblasts in SM cells before SM cell bundling.

Monoclonal antibodies specific for protein markers of smooth muscle and nonmuscle cell differentiation were applied to cryosections of normal, hyperplastic, and neoplastic human prostate specimens in order to determine whether differences in the distribution of target antigens could be detected among the various tissues. Immunofluorescence assays showed that vimentin, desmin, smooth-muscle-type alpha-actin, and both smooth muscle and nonmuscle myosin heavy chains do not change their patterns of labeling in the stromas of normal, BPH, and carcinomatous prostates. By contrast, cytokeratin 18, a differentiation marker of simple epithelia, and to a lesser extent cytokeratin 8, was consistently found in stromal tissue of the "transition zone", but only scarcely in the stroma of the "peripheral zone" from normal prostate, and was completely unexpressed in benign hyperplasia. Prostatic carcinoma from the "peripheral zone" expressed this cytoskeletal component only in trace amounts. Moreover, in prostate showing coexistence of hyperplasia and neoplasia (in the "peripheral zone"), the stroma of BPH closely resembled the stroma surrounding the carcinoma; that is, it was completely unreactive with the anti-cytokeratin 18 antibody. Expression of cytokeratins in extraepithelial tissues has been previously correlated with the achievement of a proliferative state, notably in embryogenesis, in tissue regeneration, and in various pathological forms of proliferation and growth, including some tumors of mesenchymal origin. Our results indicate the following: (1) cells in the stromal tissue of normal prostate are of smooth muscle type and are heterogeneous as concerns cytokeratin distribution; (2) we show, for the first time, the existence of a marker that is differentially distributed in the "transition" versus "peripheral" zone; (3) the expression of cytokeratins in the stroma is lost with the development of hyperplasia and only partially recovers with neoplasia; (4) the pattern of stromal tissue, concerning cytokeratin 18 expression, does not change with different BPH locations ("transition" versus "peripheral" zone); and (5) contrary to expectations, cytokeratin 18 expression disappears in conditions presumably involving stromal cell proliferation.

In an attempt to identify the growth factors or cytokines involved in the serosal thickening that occurs in rabbit bladder subjected to partial outflow obstruction, the following growth factors--transforming growth factor beta1, platelet-derived growth factor, epidermal growth factor, granulocyte colony-stimulating factor and granulocyte-monocyte colony-stimulating factor--were delivered separately onto the serosal surface of the intact bladder via osmotic minipumps. The proliferative/differentiative cellular response of the rabbit bladder wall was evaluated by bromodeoxyuridine incorporation and immunofluorescence staining with a panel of monoclonal antibodies to cytoskeletal proteins (desmin, vimentin, keratins 8 and 18 and non-muscle myosin) and to smooth muscle (alpha-actin, myosin and SM22) proteins. Administration of the transforming growth factor, but not of the other growth factors/cytokines, was effective in inducing serosal thickening. Accumulating cells in this tissue were identified as myofibroblasts, i.e. cells showing a mixed fibroblast-smooth muscle cell differentiation profile. The phenotypic pattern of myofibroblasts changed in a time-dependent manner: 21 days after the growth factor delivery, small bundles of smooth muscle cells were found admixed with myofibroblasts, as occurs in the obstructed bladder. These 'ectopic' muscle structures displayed a variable proliferating activity and expressed an immature smooth muscle cell phenotype. The complete cellular conversion to smooth muscle cells was not achieved if transforming growth factor beta1 was delivered to fibroblasts of subcutaneous tissue. These findings suggest a tissue-specific role for this growth factor in the cellular conversion from myofibroblast to smooth muscle cells.

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