Uroplakin gene expression in normal human tissues and locally advanced bladder cancer

The uroplakins are widely regarded as urothelium-specific markers of terminal urothelial cytodifferentiation. This study investigated the expression of the four uroplakin genes, UPIa, UPIb, UPII and UPIII, in a wide range of normal human tissues to determine tissue specificity and in advanced transitional cell carcinoma (TCC) to examine gene expression in primary and metastatic disease. In the urinary tract, all four uroplakins were expressed by urothelium and UPIII was also expressed by prostatic glandular epithelium. UPIa and UPII appeared to be urothelium-specific, but UPIb was detected in several non-urothelial tissues, including the respiratory tract, where it was associated with squamous metaplasia of tracheal and bronchial epithelia. The ten cases of primary TCC and corresponding lymph node metastases demonstrated that each uroplakin gene could be expressed at the mRNA level. No single uroplakin gene was expressed in all primary tumours or metastases, but 80% of the primary tumours and 70% of the lymph node metastases expressed at least one uroplakin gene. UPIII mRNA was often expressed in the absence of UPIII protein. These results confirm that in human tissues the expression of UPIa and UPII genes is highly specific to urothelium and suggest that the tight differentiation-restricted expression of uroplakin genes in normal urothelium is lost following malignant transformation.     

Iatrogenic changes in the urinary tract

A handful of therapeutic procedures are used to treat malignancies of the urinary tract, most frequently intravesical immunotherapy or chemotherapy, but also neoadjuvant systemic chemotherapy. These treatment modalities produce morphological changes in the urothelium that can be mistaken for carcinoma; in particular, these therapies frequently mimic urothelial carcinoma in situ (CIS) urothelial dysplasia or true invasive neoplasia. Drugs such as mitomycin C used after transurethral resection of bladder tumour to reduce recurrences, bacillus Calmette–Guérin (BCG) intravesical immunotherapy to treat high‐risk non‐muscle‐invasive bladder cancer and urothelial CIS and platin‐based systemic chemotherapy to improve postcystectomy disease‐specific survival are examples of therapy‐related atypia seen in the urinary tract. To complicate the pathologist's life, a number of systemic drugs in use to treat other diseases, such cyclophosphamide, used to treat some autoimmune disorders or certain haematological malignancies or, in the case of anaesthetics, ketamine, used increasingly as an illegal recreational drug, may produce similarly relevant atypical changes in the urothelium, and therefore need to be differentiated from intraepithelial neoplasia. Other less frequent procedures, such as photodynamic and laser therapy or the newer gene therapy to treat urothelial neoplasia, remain experimental. An immunohistochemical approach to reactive urothelium versus carcinoma in situ using p53, cytokeratin 20 and CD44 is also valid in the post‐therapy setting. The pathologist should be aware of these novelties, as he or she plays a crucial role in evaluating treatment efficacy, but at the same time needs to avoid misdiagnosing secondary atypia as intraepithelial neoplasia.     

Purinergic signalling in the lower urinary tract

The aim of this review is to describe the conceptual steps contributing to our current knowledge of purinergic signalling and to consider its involvement in the physiology and pathophysiology of the lower urinary tract. The voiding reflex involves ATP released as a cotransmitter with acetylcholine from parasympathetic nerves supplying the bladder and ATP released from urothelial cells during bladder distension to initiate the voiding reflex via P2X3 receptors on suburothelial low threshold sensory nerve fibres. This mechanosensory transduction pathway also participates, via high threshold sensory nerve fibres, in the initiation of pain in bladder and ureter. Treatment of prostate and bladder cancer with ATP is effective against the primary tumours in animal models and human cell lines, via P2X5 and P2X7 receptors, and also improves the systemic symptoms associated with advanced malignancy. Acupuncture is widely used for the treatment of urinary disorders, and a purinergic hypothesis is discussed for the underlying mechanism.     

Length-tension relations of smooth muscle from normal and denervated rat urinary bladders

Urinary bladders of rats were denervated by bilateral excision of the pelvic ganglion and removed 10 days after the operation. They were filled with 0.75 ml saline and a longitudinal muscle strip was marked out, measured and dissected out. Strips from normal bladders filled with the same volume were used as controls. Denervated bladders were 4–5 times heavier than control bladders. Muscle strips from denervated bladders showed, in contrast to controls, marked phasic spontaneous contractions which were unaffected by tetrodotoxin, indicating a myogenic origin. Active tension in response to AC stimulation was measured at different lengths. In relation to the in situ length (L_{in situ}) at 0.75 ml the denervated strips had to be stretched to much greater extent than controls in order to reach optimum length (L₀) for force development. Furthermore, the denervated strips shortened less in relation to L_{in situ} than the controls. If active length-tension relations were expressed in relation to Lo, the difference between denervated and control strips was abolished. Maximal active force was the same for denervated and control strips. Water content increased significantly in denervated bladders. The results suggest a remodelling of the smooth muscle structure in denervated bladders; the characteristics of the contractile machinery seem, however, to be unaltered.     

3-Dimensional morphometric analysis of murine bladder development and dysmorphogenesis

Background : Disorders of the urinary tract represent a major cause of morbidity and impaired quality of life. To better understand the morphological events responsible for normal urinary tract development, we performed 3‐D reconstructive analysis of developing mouse bladders in control, mgb?/?, and Fgfr2^Mes?/? mice. Results : Detrusor smooth muscle differentiation initiated in the bladder dome and progressed caudally with the leading edge extending down the right posterior surface of the bladder. Gender‐specific differences in detrusor smooth muscle development were observed during early embryonic development. Bladder trigone morphology transitioned from an isosceles to equilateral triangle during development due to the preferential lengthening of the urethra to ureter distance. The primary defect observed in mgb?/? bladders was a significant reduction in detrusor smooth muscle differentiation throughout development. Deviations from normal trigone morphology correlated best with VUR development in Fgfr2^Mes?/? mice, while alterations in intravesicular tunnel length did not. Conclusions : Multivariate morphometric analysis provides a powerful tool to quantify and assess urinary tract development. Developmental Dynamics 241:522–533, 2012. © 2012 Wiley Periodicals, Inc.     

Immunohistochemical analysis of thoracic endometriosis

Thoracic endometriosis is a rare disease responsible for catamenial pneumothorax. The immunohistochemical features of thoracic endometriosis are not well understood. An immunohistochemical examination of 84 diaphragmatic specimens of catamenial pneumothorax using antibodies against estrogen receptor (ER), progesterone receptor (PgR), CD10 and smooth muscle actin (SMA) was conducted. The endometrial tissue was small, and focally located around the chasm of the tendon on the side of the thoracic cavity. Endometrial stroma were detected in 84/84 (100%) of the specimens, endometrial glands were detected in 21/84 (25%) and smooth muscle was detected in 1/84 (1.2%). The endometrial stroma exhibited positive staining for ER in 74/84 (88.1%) of the specimens, PgR in 84/84 (100%), CD10 in 74/84 (88.1%) and SMA in 46/84 (54.8%). Because thoracic endometriosis is small in size, and only 25% of the resected tissue specimens were accompanied with the endometrial gland, an immunohistochemical analysis can be useful for their detection. The fact that over half of the thoracic endometrial stroma showed positive staining for SMA, and the existence of thoracic endometriosis accompanied by smooth muscle, indicated that some part of the thoracic endometriosis may have the ability to differentiate into smooth muscle, although further studies are needed to confirm this hypothesis.     

Smooth muscle homeostasis in human atherosclerotic plaques through interleukin 15 signalling

Interleukin (IL)-15 is a cytokine that has a broad tissue distribution and is important in maintaining homeostasis of cells and stability of tissues. When II-15 is also expressed by vascular smooth muscle cells (SMC), which are the dominant type of cells in most atherosclerotic plaques, it could be important in maintaining plaque tissue integrity and hence resistance of plaques towards development of clinically relevant complications such as plaque rupture and thrombosis. In this study, IL-15 and IL-15Rα in vitro expression by coronary artery SMC was investigated using RT -PCR and FACS analysis. Immunohistochemistry was used to study in situ expression of IL-15 and IL-15R by SMC of human carotid artery atherosclerotic plaques. Multiplex ligand-dependent probe amplification (MLPA) was used to investigate the mRNA expression of 40 pro- and anti inflammatory genes after stimulating coronary SMC with IL-15. We found that atherosclerotic SMC express both IL-15 and its receptor IL-15R, and TNF-γ and TNF-α enhance IL-15R expression in cultured SMC. MLPA studies on SMC revealed enhanced expression of PDGF beta mRNA after IL15 stimulation. In conclusion, our data suggest that IL-15 may contribute to atherosclerotic plaque integrity by stimulation of smooth muscle cells, probably in a PDGF dependent fashion.     

Cytoskeletal features of immature pulmonary vascular smooth muscle cells: the influence of pulmonary hypertension on normal development

Using an immunohistochemical technique, the development of the cytoskeletal proteins desmin, vimentin, and actin (using alpha isotype and non-isotype specific antibodies) was assessed using a semi-quantitative grading system in the pulmonary vascular smooth muscle of nine normal pigs and 19 normal humans at different ages, and in 13 children with pulmonary hypertensive congenital heart disease. In the normal of both species, immunostaining for vimentin decreased after birth and then increased gradually while immunostaining for desmin and alpha actin increased steadily with age. In pulmonary hypertension, immunostaining for alpha actin and vimentin showed an accelerated increase at between 2 and 8 months. Also, the media showed regional differences in immunostaining which preceded the development of intimal proliferation. The inner media showed less immunoreactivity for all cytoskeletal proteins studied than did the outer media. Within areas of intimal proliferation many cells were immunonegative. These results suggest that the cytoskeletal features of medial smooth muscle cells are remodelled in the normal infant; that this process is altered from at least 2 months in the pulmonary hypertensive infant; and that the smooth muscle cells immediately beneath the internal elastic lamina are remodelled before migrating to form intimal proliferation. Changes in cytoskeletal composition can be related to the previously described postnatal maturation of pulmonary vascular smooth muscle cells.     

Detection of neoplastic and preneoplastic urothelia by combined scanning and transmission electron microscopy of urinary surface of human and rat bladders

Scanning and transmission electron microscopy reveal distinct differences between the appearance of the luminal surface membrane of normal urothelium and those of transitional cell tumours of both human and rat bladder. The luminal surface membrane of the preneoplastic rat urothelium shows features similar to those seen in the fully developed tumour. In human tumour-bearing bladders the urothelium away from the transitional cell tumour is not recognizable as being preneoplastic by light microscopy. However, by scanning or transmission electron microscopy, the luminal membrane in some areas shows changes identical to those in the preneoplastic rat bladder. These observations are in accordance with in the clinical behaviour of the urothelium in patients with vesical transitional cell tumours which tend to recur in disparate sites. We suggest that scanning electron microscopy of the bladder urothelium away from the tumour may be of prognostic value for indicating the subsequent biological behaviour of the urothelium     

Dynamic expression pattern of Sonic hedgehog in developing cochlear spiral ganglion neurons

Sonic hedgehog (Shh) signaling plays important roles in the formation of the auditory epithelium. However, little is known about the detailed expression pattern of Shh and the cell sources from which Shh is secreted. By analyzing Shh^CreEGFP/+ mice, we found that Shh was first expressed in all cochlear spiral ganglion neurons by embryonic day 13.5, after which its expression gradually decreased from base to apex. By postnatal day 0, it was not detected in any spiral ganglion neurons. Genetic cell fate mapping results also confirmed that Shh was exclusively expressed in all spiral ganglion neurons and not in surrounding glia cells. The basal‐to‐apical wave of Shh decline strongly resembles that of hair cell differentiation, supporting the idea that Shh signaling inhibits hair cell differentiation. Furthermore, this Shh^CreEGFP/+ mouse is a useful Cre line in which to delete floxed genes specifically in spiral ganglion neurons of the developing cochlea. Developmental Dynamics 239:1674–1683, 2010. © 2010 Wiley‐Liss, Inc.     

Patterns of chromosomal aberrations in urinary bladder tumours and adjacent urothelium

Bladder cancer is often characterized by recurrent and multifocal growth, and tumours are frequently accompanied by precancerous alterations of the surrounding urothelium. These findings have led to the hypothesis that cells from areas of genetically aberrant but morphologically non-cancerous or even unremarkable mucosa may be the source of bladder carcinomas. Fluorescence in situ hybridization (FISH) was performed using ten probes targeting five different chromosomes that are known to be frequently altered in bladder cancer (centromere 1, 8, 9, 11, 17 and 1p36, 8p23, 9p21, 11q13, 17p13) on paraffin-embedded tissue sections of 11 superficial bladder cancers. Copy number changes of the tumours were compared to those in the urothelium adjacent to the tumour. Eleven of 11 (100%) tumours and eight of 11 (73%) samples of adjacent urothelium showed copy number changes of at least one chromosome. The occurrence of similar patterns of chromosomal aberrations in the tumours and their associated urothelium supports the hypothesis of a clonal relationship. It is concluded that FISH analysis targeting five different chromosomes is more sensitive than conventional histology for distinguishing between neoplastic and normal cells of the urothelium.     

Partial urethral obstruction of rabbit urinary bladder: stereological evidence that the increase in muscle content is mostly driven by changes in number, rather than size, of smooth muscle cells

The effects of partial urethral obstruction on the detrusor muscle of rabbit urinary bladder were investigated using stereological sampling and estimation tools. Twelve female Norfolk rabbits (2.5-3.0 kg body weight) were divided into four groups: 3, 7 and 12 weeks after surgical intervention to produce a standard partial obstruction and unobstructed controls. Following removal, bladder axes (craniocaudal, dorsoventral and laterolateral) and organ weights were recorded. Bladders were prepared for light microscopy by multistage random sampling procedures. Stereological methods were used to estimate the volume of muscle and the packing density and total number of myocyte nuclei in each bladder. We also estimated mean myocyte volume and the mean cross-sectional area and length of myocytes. Group comparisons were made by one-way analysis of variance. Changes in bladder axes were mainly laterolateral and craniocaudal. Mean bladder weight increased roughly six-fold by 3 weeks and 17-fold by 12 weeks and was accompanied, on average, by 12- and 33-fold increases in total muscle volume. These variables did not differ at 3 and 7 weeks post-obstruction. Increases in muscle content were not accompanied by changes in packing densities but were associated with increases in the total numbers of myocyte nuclei (13-fold by 3 weeks, 28-fold by 12 weeks). Mean myocyte volume did not vary significantly between groups but cells in obstructed groups were shorter and wider. These findings support the notion that partial outflow obstruction leads to an increase in the number, but not mean volume, of myocytes. If due solely to myocyte mitosis, the total of 43 x 10(8) cells found at 12 weeks could be generated by the original complement of 15 x 10(7) cells if an average of only 2.1 x 10(6) new cells was produced every hour. In reality, even this modest proliferation rate is unlikely to be achieved because myocyte proliferation rates are very low and it is possible that new myocytes can arise by differentiation of mesenchymal or other precursor cells.     

Towards defining roles and relationships for tenascin-C and TGFbeta-1 in the normal and neoplastic urinary bladder

Tenascin-C (TN-C) is an extracellular matrix glycoprotein expressed along epithelial/stromal boundaries during tissue remodelling events, such as those that occur during morphogenesis, wound healing, and tumour invasion. Using clinical specimens and a range of in vitro models that simulate homeostasis, wound healing, and malignant progression, this study sought to establish the patterns of TN-C expression in normal and neoplastic bladder and to determine the role of exogenous transforming growth factor beta-1 (TGFbeta-1), interleukin-4 (IL-4), basic fibroblast growth factor (bFGF), tumour necrosis factor alpha (TNFalpha), and interferon gamma (IFNgamma) in the induction of TN-C expression by bladder uro-epithelial cells. The findings indicate that normal urothelial cells may express TN-C, with both TGFbeta-1 and IL-4 able to induce expression. TN-C was not expressed in neoplastic urothelium, although both TN-C and TGFbeta-1 may be involved in tissue remodelling during papillary tumour formation and invasion. Furthermore, the urothelium of high-grade papillary tumours and carcinoma in situ specimens exhibited little TGFbeta-1 immunoreactivity, compared with the urothelium of low-grade tumours and normal specimens, suggesting an association between TGFbeta-1 expression and urothelial differentiation. A tumour invasion model, in which established bladder cancer cell lines were seeded onto a normal bladder stroma, corroborated the evidence from the clinical specimens and demonstrated that TN-C was strongly expressed around foci of stromal invasion. Thus, TN-C immunoreactivity may provide an additional tool in the assessment of early stromal invasion in bladder cancer.     

Non-epithelial basement membrane thickening in the urinary tract associated with phenacetin abuse

Summary In four cases of capillarosclerosis in the urinary tract associated with analgesic (phenacetin) abuse, the basement membrane (BM) thickening was not confined to the subepithelial capillaries, but was also found around the smooth muscle cells in the luminal part of the tunica muscularis. Electron microscopy confirmed that the changes in the BM around the smooth muscle cells were similar to those seen around capillaries. This non-vascular affection of BM in the urinary tract in patients with phenacetin abuse has not been reported previously. Thus, capillarosclerosis appears to be only part of a BM disorder, that clearly diminishes in intensity with increasing distance from the lumen. It is therefore suggested that the changes are caused by some agent (possibly a metabolite) in the urine diffusing from the lumen into the wall of the urinary tract.     

Immunohistochemical characterization of the intracellular pool of water channel aquaporin-2 in the rat kidney

Aquaporin-2 (AQP2) is a member of water channel proteins expressed in the kidney collecting duct cells, where it is stored in the intracellular compartment. Upon stimulation of antidiuretic hormone (ADH), AQP2 is recruited to the plasma membrane, and plays a critical role in urine concentration. We immunohistochemically characterized the intracellular compartment harboring AQP2 in the rat kidney using antibodies to the endoplasmic reticulum, Golgi apparatus, trans-Golgi network, lysosome, and endosome. Aquaporin-2 did not colocalize with calnexin, TGN38, Golgi 58K, cathepsin D or lgp-110. Small portions of AQP2-bearing vesicles were positive for early endosome antigen 1. These localization patterns were basically the same in water-loaded and ADH-treated animals. These results indicate that AQP2-bearing vesicles constitute a unique intracellular compartment distinct from the endoplasmic reticulum, Golgi apparatus, trans-Golgi network and lysosome. Partial colocalization of AQP2 with early endosomes suggests that the endosomal system might be involved in the trafficking of AQP2.     

Cellular morphological parameters of the human urinary bladder (malignant and normal)

The normal and malignant cellular morphological parameters (intra- and extracellular spaces of the human urinary bladder) were obtained from analysis of digital images of bladder histology sections. Then these cellular morphological parameters were compared with the same parameters obtained from the literature for the bladder tissue. However, the limited quantitative data about these parameters available in the literature for bladder cell sizes and other geometrical parameters such as extra-cellular space does not provide a scientific basis to construct accurate structural models of normal and malignant bladder tissue. Therefore, there is usually no quantitative discussion of cell sizes in literature but the measured data in this work can provide a reasonable estimation of expected morphological parameter changes of bladder tissue with pathology. To produce this quantitative information, and also, to build a suitable models in another study using electrical properties of the tissue, 10 digital images of histological sections of normal, and six sections from malignant areas of the human urinary bladder, were chosen randomly (ex vivo). Finally, the measured data showed that there is a significant difference between the cell dimensions (in basal and intermediate layers) of normal and malignant bladder tissues.     

Intracellular signalling pathways in the vasoconstrictor response of mouse afferent arterioles to adenosine

Aims: Adenosine causes vasoconstriction of afferent arterioles of the mouse kidney through activation of adenosine A₁ receptors and Gi‐mediated stimulation of phospholipase C. In the present study, we further explored the signalling pathways by which adenosine causes arteriolar vasoconstriction. Methods and results: Adenosine (10^?7 m ) significantly increased the intracellular calcium concentration in mouse isolated afferent arterioles measured by fura‐2 fluorescence. Pre‐treatment with thapsigargin (2 μm ) blocked the vasoconstrictor action of adenosine (10^?7 m ) indicating that release of calcium from the sarcoplasmic reticulum (SR), stimulated presumably by IP₃, is involved in the adenosine contraction mechanism of the afferent arteriole. In agreement with this notion is the observation that 2 aminoethoxydiphenyl borate (100 μm ) blocked the adenosine‐induced constriction whereas the protein kinase C inhibitor calphostin C had no effect. The calcium‐activated chloride channel inhibitor IAA‐94 (30 μm ) inhibited the adenosine‐mediated constriction. Patch clamp experiments showed that adenosine treatment induced a depolarizing current in preglomerular smooth muscle cells which was abolished by IAA‐94. Furthermore, the vasoconstriction caused by adenosine was significantly inhibited by 5 μm nifedipine (control 8.3 ± 0.2 μm, ado 3.6 ± 0.6 μm, ado + nifedipine 6.8 ± 0.2 μm) suggesting involvement of voltage‐dependent calcium channels. Conclusion: We conclude that adenosine mediates vasoconstriction of afferent arterioles through an increase in intracellular calcium concentration resulting from release of calcium from the SR followed by activation of Ca²⁺‐activated chloride channels leading to depolarization and influx of calcium through voltage‐dependent calcium channels.     

Immunohistochemical analysis of Fas ligand expression in normal human tissues

Cross-linking of Fas and Fas ligand (FasL) induces apoptosis in Fas-bearing cells and regulates apoptosis. Fas is widely expressed in normal human tissues, but FasL expression has been considered to be restricted to lymphoid tissues. Recent studies have demonstrated that FasL is also expressed in some nonlymphoid tissues. To screen the in situ expression of FasL in normal human tissues, immunohistochemistry was performed using paraffin-embedded human tissues. FasL immunostaining was easily detected in testis, neurons, trophoblasts, tonsil, lymph node, Paneth cells, hepatocytes, renal tubular epithelium and bronchial epithelium, consistent with previous reports. Surprisingly, FasL was also expressed in many other cell types, including thymic medulla, skeletal muscle, cardiac muscle, pituitary gland, parathyroid gland, prostate glands, oocytes, epithelium of fallopian tube, endometrial glands, and gastric parietal cells. These findings demonstrate that FasL is widely expressed in human tissues and suggest that wide but cell-type specific expression of FasL may not only be implicated in the regulation of immune homeostasis but also in the regulation of cell death and life in many cell types in vivo.