Expression of matrix metalloproteinase-2 (MMP-2) and of membrane-type-1-matrix metalloproteinase (MT1-MMP) in transitional cell carcinoma of the upper urinary tract.

Tumor cell invasion and metastasis are biologically dependent on the proteolytic destruction of surrounding matrix components. Matrix metalloproteinase-2 (MMP-2) is able to cleave type IV collagen, and membrane-type-1-matrix metalloproteinase (MT1-MMP) induces activation of proMMP-2. We investigated the expression of MMP-2 and MT1-MMP using in situ hybridization and immunohistochemistry in 102 cases of transitional cell carcinoma of the upper urinary tract (TCC-UUT). A positive expression of each metalloproteinase was recognized in all samples and was apparent within the cytoplasm of tumor epithelial cells and/or stromal cells situated at the interface between tumor and stroma. Our analysis of clinicopathologic findings showed a relationship between MMP-2 and MT1-MMP expression and stage. The correlation between the MMP-2 protein staining score for tumor epithelial cells and overall survival rate reached significance in the univariate analysis. However, only stage was associated with disease-free and overall survivals in the multivariate analysis. In conclusion, the detection of MMP-2 and MT1-MMP would appear to be of limited value in informing the prognosis of TCC-UUT.

Expression of bcl-2 oncoprotein in transitional cell carcinoma of the upper urinary tract.

We investigated the immunoreactivity for bcl-2 oncoprotein in 154 cases of transitional cell carcinoma of the upper urinary tract (TCC-UUT) and its relation with the immunoreactivity for p53 oncoprotein and proliferating cell nuclear antigen (PCNA) immunoreactivity. Immunohistochemically, bcl-2 oncoprotein was recognized as positive in 29.2% of the samples. The immunoreactivity for bcl-2 oncoprotein was significantly (P < 0.05) correlated only with stage, though there was a borderline correlation (P = 0.050) with PCNA immunoreactivity. Furthermore, in invasive TCC the immunoreactivity for bcl-2 oncoprotein was associated with PCNA immunoreactivity (P < 0.041). The 5-year disease-free and overall survival rates were 55.7% and 71.5%, respectively. A univariate analysis of survival revealed that stage, grade, pattern of growth, immunoreactivity for p53 oncoprotein, and PCNA immunoreactivity each had a significant effect on disease-free and overall survival rates, whereas the immunoreactivity for bcl-2 oncoprotein had no significant effect on either rate. In the final models of the multivariate analysis, stage was found to be the only prognostic factor for disease-free survival and for overall survival. Detection of immunoreactivity for bcl-2 oncoprotein appears to be of no real value in deciding the prognosis of TCC-UUT.

Tissue factor is associated with the nonbacterial thrombotic endocarditis induced by a hypobaric hypoxic environment in rats.

High-altitude hypoxia causes a hypercoagulable state. In our previous study on the blood coagulation system in rats, nonbacterial thrombotic endocarditis (NBTE) developed after 4-12 weeks' exposure to the equivalent of 5500 m in altitude. We hypothesized that TF (tissue factor)-producing cells in the cardiac valves might be induced by the hypobaric hypoxic environment (HHE) and then trigger NBTE. A total of 170 male Wistar rats were housed in a chamber at the equivalent of 5500 m altitude for 1-12 weeks. We measured TF activity in the plasma and studied morphological changes in the mitral valves using immunohistochemical and immunoelectrical methods for TF protein and in situ hybridization for TF mRNA. After 4 weeks or more of exposure to HHE, 28 of the 56 surviving rats had developed NBTE. After 4-8 weeks' exposure to HHE, the plasma TF activity level was significantly higher than in control rats. There was a significant correlation between plasma TF activity and the incidence of NBTE. After 1 weeks' exposure to HHE, immunoreactivity for TF protein was detected in foamy macrophages and stromal cells in the cardiac valves. In rats with NBTE, TF protein was present in foamy macrophages and spindle stromal cells and focally present in the extracellular matrix. TF mRNA was detected in some foamy macrophages within the thrombus, TF protein was localized to the rough endoplasmic reticulum and plasma membrane of many macrophages, some fibroblasts, and a few endocardial cells. TF is associated with the pathogenesis of the NBTE induced by exposure to HHE. The accumulation of TF-producing macrophages during exposure to HHE may be responsible for initiating thrombus formation.

Changes in atrial natriuretic peptide and brain natriuretic peptide associated with hypobaric hypoxia-induced pulmonary hypertension in rats.

Experimental pulmonary hypertension induced in a hypobaric hypoxic environment (HHE) is characterized by structural remodeling of the heart and pulmonary arteries. Atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) both have diuretic, natriuretic, and hypotensive effects, and both are involved in cardiovascular homeostasis as cardiac hormones. To study the effects of HHE on the natriuretic peptide synthesis system, 170 male Wistar rats were housed in a chamber at the equivalent of the 5500-m altitude level for 1-12 weeks. After 1 week of HHE, pulmonary arterial pressure was significantly raised, and the ratio of left ventricle plus septum over right ventricle of the heart showed a significant decrease (compared with those of ground-level control rats). In both ventricular tissues, the expression of ANP messenger (m)RNA and BNP mRNA increased after exposure to HHE. The amounts of ANP and BNP had decreased significantly in right atrial tissue at 12 weeks of HHE (compared with those of the controls), whereas in ventricular tissues at the same time point, both levels had increased significantly. In in situ hybridization and immunohistochemical studies, the staining of the mRNAs for ANP and BNP and of ANP and BNP themselves was more intense in both ventricular tissues after exposure to HHE than before (i.e., in the controls). The results suggest that, in response to HHE, the changes in ventricular synthesis are similar for ANP and BNP. These changes may play a role in modulating pulmonary hypertension in HHE. However, under our conditions, pulmonary hypertension increased progressively throughout the HHE period.

Mucin histochemistry in primary adenocarcinoma of the urinary bladder (of urachal or vesicular origin) and metastatic adenocarcinoma originating in the colorectum.

In order to evaluate the mucin histochemistry of primary adenocarcinomas (PA) of the urinary bladder and metastatic adenocarcinoma (MA) originating in the colorectum, 52 PA and nine MA were examined. It was determined that the percentage of cases in which more than 25% of the tumor was stained by each of the following: (i) Alcian blue pH 2.5 periodic acid-Schiff (AB-PAS); (ii) high iron diamine-AB (HID-AB); (iii) periodic acid-sodium borohydride-potassium hydroxide-PAS (PA-SB-PH-PAS); (iv) galactose oxidase- Schiff (GOS); and (v) paradoxical concanavalin A stain (PCS). For PA, the values obtained were: 75% of cases (blue, AB-PAS), 85% (magenta, AB-PAS), 71% (black, HID-AB), 75% (blue, HID-AB), 0% (PA-SB-PH-PAS), 19% (GOS), 8% (class II concanavalin A (Con A)-reactive mucin)), and 0% (class III Con A-reactive mucin). For MA, the corresponding values were 33, 22, 0, 11, 0, 0, 11, and 0%, respectively. A higher percentage of PA than MA cases showed staining in AB-PAS for acidic and neutral mucins, in HID-AB for sialo- and sulfomucins, and in GOS for terminal beta-galactose and beta-N-acetylgalactosamine. PA and MA were significantly different in terms of both frequency of staining with AB-PAS and frequency of staining with HID-AB. However, the overlap was such that in practice, it might be difficult, if not impossible, to use mucin histochemistry to inform a differential diagnosis. In view of the differences in AB-PAS and HID-AB positivity between PA and MA, we speculate that MA (originating in the colorectum) may have undergone structural distortion affecting the production and/or secretion of neutral mucins and acidic mucins (sialo- and sulfomucins) during metastasis or invasion.

Expression of telomerase catalytic subunit (hTERT) mRNA does not predict survival in patients with transitional cell carcinoma of the upper urinary tract.

Telomerase is a ribonucleoprotein enzyme that synthesizes telomeric repeats onto chromosomal ends using a segment of its RNA component as a template. Its activity has become an established indicator of the diagnosis, biological behavior, and prognosis of several tumors. However, few studies have investigated the diagnostic and prognostic importance of the expression of telomerase catalytic subunit (hTERT) mRNA in transitional cell carcinoma of the upper urinary tract (TCC-UUT). We investigated the expression of hTERT mRNA using in situ hybridization in 125 cases of TCC-UUT, and also its relation with the expression of telomerase RNA component (hTERC), proliferating cell nuclear antigen (PCNA) immunoreactivity, clinicopathologic parameters, and clinical outcome. A positive expression of hTERT mRNA was recognized in 93.6% of the samples and was apparent within the cytoplasm of tumor cells. In the normal urothelium examined in a few cases, its expression was barely detected. hTERT mRNA scores showed a significant association with hTERC score. However, no relationship was found between the expression of hTERT mRNA and clinicopathologic findings, PCNA index, or prognosis. These results suggest that the expression of hTERT mRNA does not predict prognosis in TCC-UUT.

Expression of p27(Kip1) protein in transitional cell carcinoma of the upper urinary tract.

The expression of p27(Kip1), a negative regulator of the cell cycle, has been reported to correlate with the biological behavior and prognosis of several tumors. However, its prognostic importance in transitional cell carcinoma of the upper urinary tract (TCC-UUT) has not previously been investigated. We investigated p27(Kip1) protein expression using immunohistochemistry in 132 cases of TCC-UUT and also its relation to proliferating cell nuclear antigen (PCNA) immunoreactivity, p53 oncoprotein immunoreactivity, clinicopathologic parameters, and clinical outcome. A positive expression of p27(Kip1) protein was recognized in 94.7% of the samples and was apparent within tumor nuclei. In the normal urothelium, its expression was identified in all cell layers. A positive expression of p53 oncoprotein was recognized in 27.2% of the patients. The PCNA index was 7.4 to 93.1% (mean, 66.4%). Examination of the relationships between the expression of p27(Kip1) protein and clinicopathologic findings, PCNA index, and the expression of p53 oncoprotein revealed that the expression of p27(Kip1) protein decreased significantly with stage and grade. In a univariate analysis of disease-free and overall survival rates, no correlation was found between the expression of p27(Kip1) protein and prognosis. The expression of p27(Kip1) protein appears to be of little or no value in informing the prognosis in TCC-UUT.

Urokinase-type plasminogen activator, its inhibitor, and its receptor in patients with upper urinary tract carcinoma.

BACKGROUND: Urokinase-type plasminogen activator (uPA) is a serine protease involved in tumor invasion and metastasis. Its activity during metastasis may be regulated by a plasminogen activator inhibitor (PAI). Furthermore, uPA exerts its action by binding to a membrane-bound receptor (uPAR). The authors attempted to examine the immunohistochemical expression of uPA, uPAR, and PAI-1 in patients with transitional cell carcinoma of the upper urinary tract (TCC-UUT). METHODS: Formalin fixed, paraffin embedded tumor tissues from 154 patients were analyzed using immunohistochemical staining. RESULTS: There was moderate to strong cytoplasmic staining for uPA, PAI-1, and uPAR in 57.8%, 96.1%, and 88.3%, respectively, of tumor epithelial cells, and in 22.7%, 53.9%, and 24.7%, respectively, of stromal cells at the tumor/stroma interface. Examination of the relationship between immunoreactive score and clinicopathologic findings revealed that the uPA score for stromal cells significantly correlated with the stage and pattern of growth of the tumors. The PAI-1 score for tumor epithelial cells and the uPAR score for stromal cells both correlated with stage, grade, and pattern of growth. The PAI-1-score for stromal cells correlated with stage and grade. The uPAR-score for tumor epithelial cells correlated with stage. When only the immunoreactive scores that were classified as "high" (if the score was > or = 5 or > or = 1, for tumor epithelial and stromal cells, respectively) were considered, univariate analysis revealed that a "high" PAI-1 score for tumor epithelial cells and a "high" uPAR score for stromal cells both were significantly associated with poor disease free and overall survivals, particularly early period survival. In the final models of the multivariate analysis, only stage (all periods, disease free survival and overall survival), and grade (12 months, overall survival) were found to be progressive or prognostic factors. CONCLUSIONS: Detection of immunoreactivity for plasminogen activator parameters appears to be of little or no value in determining the prognosis of patients with TCC-UUT, although some parameters were found to be associated with high stage or high grade of the tumors.

E-cadherin expression in upper-urinary-tract carcinoma.

Carcinoma of the upper urinary tract is a relatively rare neoplasm, and few studies have dealt with clinicopathological findings and prognosis in a large number of cases. The purpose of our investigation was to look for a possible relation between E-cadherin (E-CD) immunoreactivity and clinicopathologic findings or clinical outcome in transitional-cell carcinoma of the upper urinary tract (TCC-UUT). To this end, we investigated E-CD immunoreactivity in 154 cases of TCC-UUT. E-CD immunoreactivity was recognized as being of "normal" pattern in 29.2% of samples. The relationship between E-CD immunoreactivity and clinicopathologic findings was significant for stage, grade and pattern of growth. The 5-year disease-free rate for 147 cases and 5-year overall survival rate for 154 cases were 55.7% and 71.5%, respectively. A univariate analysis of survival revealed that stage, grade, pattern of growth and E-CD immunoreactivity all had a significant effect on disease-free and overall survival rates. In the final models of multivariate analysis, however, we found that, for disease-free survival and for overall survival, only stage was a factor for progression or prognosis. Detection of E-CD immunoreactivity appears to be of limited value in deciding the prognosis of TCC-UUT.

Expression of telomerase mRNA component (hTR) in transitional cell carcinoma of the upper urinary tract.

BACKGROUND: Telomerase is a ribonucleoprotein enzyme that synthesizes telomeric repeats onto chromosomal ends using a segment of its RNA component as a template. Its activity has become an established indicator of the diagnosis, biologic behavior, and prognosis of several tumors. However, to the authors' knowledge, no previous study has investigated the diagnostic and prognostic importance of the expression of telomerase RNA component (hTR) in transitional cell carcinoma of the upper urinary tract (TCC-UUT). METHODS: The authors investigated hTR expression using in situ hybridization in 130 cases of TCC-UUT, and also its relation to proliferating cell nuclear antigen (PCNA) immunoreactivity, immunoreactivity for p53 oncoprotein, clinicopathologic parameters, and clinical outcome. RESULTS: Positive hTR expression was recognized in 98.4% of the samples and was apparent within the cytoplasm of tumor cells. In normal urothelium, its expression was restricted to the basal cell layers, whereas in the dysplastic lesions of TCC-UUT it was detected with the same intensity and distribution as in the tumor itself. No correlation was found between hTR expression and clinicopathologic findings, PCNA index, or the expression of p53 oncoprotein, although hTR score did tend to increase with disease stage. In univariate and multivariate analyses of disease free and overall survival rates, high hTR expression was associated with significant decreases in both rates. CONCLUSIONS: The expression of hTR appears to be a useful indicator of prognosis for patients with TCC-UUT. In addition, evidence of up-regulation of hTR may also be useful in the diagnosis of this disease.

Expression of endothelin-1 in rats developing hypobaric hypoxia-induced pulmonary hypertension.

Experimental pulmonary hypertension induced in a hypobaric hypoxic environment (HHE) is characterized by structural remodeling of the heart and pulmonary arteries. Endothelin-1 (ET-1), a 21-amino acid peptide, is a novel and long-lasting vasoconstrictor that increases pulmonary arterial pressure in both in vivo and in vitro experiments. To study the effects of HHE on ET-1 activity in the lungs, 59 male rats were subjected to the equivalent of an altitude of 5500 m for 1 to 4 weeks. In rats exposed to HHE, the mean pulmonary arterial pressure increased significantly from 15.2+/-0.3 (ground level) to 30.6+/-1.5 mm Hg (5500-m level) at 4 weeks, whereas their mean systemic arterial pressure remained normal. The levels of ET-1 mRNA and protein, measured respectively by Northern blot analysis and enzyme immunoassay, increased rapidly in the lungs on exposure to HHE. By in situ hybridization and immunohistochemistry, respectively, ET-1 mRNA and protein were detected in control rats in nonciliated bronchiolar epithelial cells and alveolar epithelial cells, as well as in the endothelial cells of pulmonary arteries, but minimally in the smooth muscle cells of pulmonary arteries. ET-1 mRNA- and protein-reactive smooth muscle cells in pulmonary arteries and ET-1 mRNA-reactive airway epithelial cells were significantly more abundant in rats exposed to HHE than in ground level controls. These results suggest the possibility that in smooth muscle cells in pulmonary arteries and airway epithelial cells, ET-1 may play an autocrine or paracrine role in the remodeling of blood vessels during the development of the pulmonary hypertension that is induced by HHE.

Thrombopoietin expression in normal and hypobaric hypoxia-induced thrombocytopenic rats.

Thrombopoietin (TPO) is important as the physiologic regulator of platelet production. High-altitude hypoxia is a well-known cause of polycythemia and thrombocytopenia in animals. Fifty-two Wistar rats were housed for 0.5 to 21 days in a mechanical chamber in an environment equivalent to that found at 5500 m to determine (a) the cellular localization of TPO and (b) whether the decreased platelet and megakaryocyte counts in rats exposed to a hypobaric hypoxic environment (HHE) are associated with an altered TPO mRNA expression. In normal rats, there were high levels of TPO mRNA in the liver and kidney, intermediate levels in the brain and large intestine, and low levels in the skeletal muscle and small intestine. TPO mRNA and protein were expressed in Purkinje cells and neuronal cells in the brain, in proximal tubular cells and the mesangial cells of the glomeruli in the kidney, in hepatocytes and biliary duct epithelial cells, in absorptive epithelial cells in the large intestine, in the epidermis, and in the lung. The platelet count in the blood and megakaryocyte counts in the bone marrow and spleen were all decreased significantly after 5 or more days of exposure to HHE. In major producers such as the liver and kidney and in minor producers such as the brain, TPO mRNA levels, which tended to be decreased after 0.5 to 3 days of exposure to HHE, had returned to normal by about Day 5 or 7. Thus, during the HHE period with a decreased platelet count, no changes in TPO mRNA levels were detected in these three organs. In conclusion, we have demonstrated that TPO production occurs in various types of cells. In HHE, however, factors other than TPO may be involved in hypobaric hypoxia-induced thrombocytopenia in rats.

Expression of C-type natriuretic peptide during development of rat lung.

C-type natriuretic peptide (CNP), recently found to be secreted from vascular endothelial cells, is now viewed as a novel endothelium-derived relaxing peptide. However, the distribution and expression of CNP during cardiopulmonary development is unclear. To follow changes in the expression of CNP during lung development, we examined rat embryos and neonates using Northern blot analysis and in situ hybridization for CNP mRNA and radioimmunoassay and immunohistochemistry for CNP protein. A substantial expression of CNP mRNA was first detected on postnatal day 2, and it thereafter remained fairly steady. The level of CNP protein also increased rapidly after postnatal day 1, reaching a settled level on postnatal day 4. CNP protein and mRNA were detected in the endothelium and smooth muscle cells of blood vessels and in bronchial airway and alveolar epithelia. Immunoreactivity for CNP protein in the endothelium of blood vessels increased to an intense level after the saccular stage. These results suggest that the changes in CNP levels may be related to the occurrence of pulmonary vasodilation after birth.