Chemo-angiogenic profile of bovine urinary bladder tumors distinguishes urothelial carcinomas from hemangiosarcomas

Angiogenesis and inflammation are two processes regulated by numerous common molecular mechanisms. Inflammation can stimulate angiogenesis, and angiogenesis can facilitate inflammation; both mechanisms have been shown to be involved in carcinogenesis. With this study we sought to gain an understanding of the molecular mechanisms involved in tumor angiogenesis and inflammation in urinary bladder tumors. Tumor specimens were collected at slaughter from Friesian cows chronically exposed to bracken fern. Bracken chronic toxicity is characterized by the presence of multiple mixed tumors in the bladder, being reported throughout the world under the designation of bovine enzootic hematuria. We conducted molecular analyses of angiogenic factors and chemokine production by real-time RT-PCR, and also assessed microvessel density (MVD), microvessel pericyte coverage index (MPI) to reveal mature vessels, the extent of tumor-infiltrating leukocytes (TILk) and tumor cell apoptosis and proliferation in both epithelial and endothelial-derived bovine urinary bladder tumors. We defined a profile of chemokines/chemokine receptors (Mip1beta, CCR1) and angiogenesis-related factors (VEGF, VEGFR2) that allow distinguishing between urothelial carcinomas (epithelial origin) and hemangiosarcomas (endothelial origin). Taken together, our data reveals previously unrecognized paracrine and autocrine chemo-angiogenic loops in the context of bovine urinary bladder tumorigenesis.     

Correlation of vascular endothelial growth factor expression to overall survival in feline invasive mammary carcinomas

Samples from feline invasive mammary carcinomas (FMCs) were used to determine the prognostic significance of the immunohistochemical expression of vascular endothelial growth factor (VEGF) and microvessel density (MVD). Forty-eight queens bearing FMCs were included in a 2-year follow-up study. Mammary tumors were classified according to the World Health Organization system and graded on the basis of histologic criteria. Tumor sections were immunostained using anti-VEGF and anti-von Willebrand factor (vWf) antibodies. VEGF expression was quantified on the basis of the percentage of positive cells. MVD of vWf-positive microvessels was determined by both mean microvessel counts and highest microvessel counts. Normal mammary gland tissues showed an inconspicuous VEGF staining. In FMCs the proportion of VEGF-positive cells was significantly higher in papillary and solid carcinomas than in tubular and papillary cystic tumors. An increased number of cells expressing VEGF was also observed in poorly differentiated FMCS. Sixteen (33.3%) of the queens bearing invasive carcinomas were still alive at the end of the 2-year follow-up period, and 32 (66.7%) had died. The VEGF expression was significantly correlated with the clinical outcome, but no correlation was observed with the invasion of lymphatic vessels. A correlation between the higher percentage of VEGF-positive cells and the unfavorable prognosis was demonstrated by the estimation of curves for overall survival (P = 0.03). Univariate analysis showed that MVD did not correlate with the overall survival. The results of our study demonstrated that VEGF expression, although not associated with increased angiogenesis, is a prognostic indicator in feline mammary tumors. In contrast, there is no support for a role of neovascularization as an indicator of survivability.     

Expression of cyclooxygenase-2 in transitional cell carcinoma of the urinary bladder in dogs

OBJECTIVE: To evaluate expression of cyclooxygenase (COX)-1 and COX-2 in the urinary bladder epithelium of clinically normal dogs and in transitional cell carcinoma cells of dogs. ANIMALS: 21 dogs with transitional cell carcinoma of the urinary bladder and 8 dogs with clinically normal urinary bladders. PROCEDURE: COX-1 and COX-2 were evaluated by use of isoform-specific antibodies with standard immunohistochemical methods. RESULT: COX-1, but not COX-2, was constitutively expressed in normal urinary bladder epithelium; however, COX-2 was expressed in neoplastic epithelium in primary tumors and in metastatic lesions of all 21 dogs and in new proliferating blood vessels in 3 dogs. Also, COX-1 was expressed in the neoplastic cells. CONCLUSIONS AND CLINICAL RELEVANCE: Lack of expression of COX-2 in normal bladder epithelium and its substantial expression in transitional cell carcinoma cells suggest that this isoform may be involved in tumor cell growth. Inhibition of COX-2 is a likely mechanism of the antineoplastic effects of non steroidal antiinflammatory drugs.     

Evaluation of the Modifying Effect of Inhalation of Mainstream Cigarette Smoke on Mouse Bladder Carcinogenesis

Cigarette smoking is one of the major risk factors of bladder cancer in humans. To date, however, there is no experimental evidence for the effects of inhalation exposure to mainstream cigarette smoke on bladder carcinogenesis. The purpose of the present study was to evaluate the effect of inhalation of mainstream cigarette smoke on mouse bladder carcinogenesis using a cigarette smoke inhalation exposure system. Six-week-old male C57BL mice were administered 0.025% N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN) in their drinking water for 8 weeks and then divided into 2 groups and exposed to 0 or 300 mg/m³ wet total particulate matter mainstream cigarette smoke for 2 h per day, five times per week, for 22 weeks. The incidences of bladder tumors (papilloma and urothelial carcinoma) tended to increase in the cigarette smoke-exposed group (25.0%) compared with the controls (15.8%), albeit without a statistically significant difference. We also evaluated mRNA expression levels of cytochrome P450 (cyp) enzymes and proliferating cell nuclear antigen (PCNA) in the bladder epithelium. Expression of cyp1a1 was significantly increased in the cigarette smoke-exposed group. Cigarette smoke exposure did not have a significant effect on the expression of cyp1a2, cyp 1b1, cyp 2a4, cyp 2b10, cyp 2e1, or PCNA. In conclusion, limited exposure to mainstream cigarette smoke for 22 weeks, caused a significant increase in cyp1a1 expression. This increase coupled with the nonsignificant increase in bladder tumors suggests that a longer period of exposure is required to clarify the effects of cigarette smoke on bladder carcinogenesis.     

Involvements of Estrogen Receptor,Proliferating Cell Nuclear Antigen and p53 in Endometrial Adenocarcinoma Development in Donryu Rats

Involvements of estrogen receptor (ER)α, proliferating cell nuclear antigen (PCNA) and p53 in the uterine carcinogenesis process in Donryu rats, a high yield strain of the uterine cancer were investigated immunohistochemically. ERα was expressed in atypical endometrial hyperplasia, accepted as a precancerous lesion of the uterine tumors, as well as well- and in moderately-differentiated endometrial adenocarcinomas, and the intensities of expression were similar to those in the luminal epithelial cells of the atrophic uterus at 15 months of age. The expression, however, was negative in the tumor cells of poorly differentiated type. Good growth of implanted grafts of the poorly-differentiated adenocarcinomas in both sexes with or without gonadectomy supported the estrogen independency of tumor progression to malignancy. PCNA labeling indices were increased with tumor development from atypical hyperplasia to adenocarcinoma. The tumor cells in poorly-differentiated adenocarcinomas were positive for p53 positive but negative for p21 expression, suggesting accumulation of mutated p53. These results indicate that the consistent ERα expression is involved in initiation and promotion steps of uterine carcinogenesis, but not progression. In addition, PCNA is related to tumor development and the expression of mutated p53 might be a late event during endometrial carcinogenesis.     

Expression of NOS and VEGF in feline mammary tumours and their correlation with angiogenesis

In order to define the role of nitric oxide (NO) in feline mammary tumours, the expression of endothelial or inducible nitric oxide synthase (e/iNOS) and vascular endothelial growth factor (VEGF), and their relationship with angiogenesis, was investigated in 23 feline mammary tumours (two hyperplastic, 19 adenocarcinoma, one osteosarcoma and one squamous cell carcinoma) by immunohistochemistry. Tumour angiogenesis was assessed by CD31 immunostaining and was expressed as microvessel density (MVD). In general, iNOS immunoreactivity was localised in tumour cells and occasionally in stromal myofibroblasts, whereas eNOS and VEGF were localised in the cytoplasm of tumour epithelial cells and endothelium. In malignancy, expression of iNOS increased from well- to less-differentiated phenotypes (Grades 1-3) and was significantly higher in G3 and G2 when compared with G1 cases. However, increasing eNOS expression was limited only in hyperplastic lesions and showed no significant changes among the grades. In addition, expression of iNOS was positively correlated with VEGF and MVD in feline mammary tumours and both measures were significantly greater in less differentiated phenotypes (P<0.05). In conclusion, the expression of NOS isoforms in feline mammary tumours depended on tumour grade, and the positive correlations between iNOS and angiogenic markers suggests that iNOS synthesised by tumour cells promotes tumour growth. Thus, iNOS can be used as an important immunohistochemical marker to determine the degree of malignancy and prognosis of feline mammary carcinoma.     

Expressions of vascular endothelial growth factor and basic fibroblast growth factor in tumors induced by two different cloned cell lines established from transplantable rat malignant fibrous histiocytoma

In order to establish base-line data on angiogenic factors in development of mesenchymal tumors, expressions of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) in implanted MT-8 and MT-9 tumors, both derived from a transplantable malignant fibrous histiocytoma (MFH) in the F344 rat, were investigated by immunohistochemistry and Western blotting method. MT-8 and MT-9 tumors were developed in syngeneic rats by implant of a tumor tissue fragment. MT-8 tumors were examined on post-implantation (PI) days 3, 6, 9 and 17, and MT-9 tumors were on PI days 9, 14, 17 and 23. The growth of MT-8 tumors was faster than that of MT-9 tumors. Histologically, MT-8 tumors were features of undifferentiated sarcomas, whereas MT-9 tumors exhibited a typical storiform growth pattern of MFH. Immunohistochemically, all cells constituting MT-8 and MT-9 tumors reacted with antibodies to VEGF and bFGF, indicating production of these factors by mesenchymal neoplastic cells. However, there were no marked differences in these immunoreactions between tumors examined. Thus, the bands obtained in the Western blotting methods were densitometrically scanned. The expression levels of VEGF and bFGF gradually increased PI day 3 to 9 in MT-8 tumors and PI day 9 to 17 in MT-9 tumors. On last examination day, the levels of bFGF in both tumors and of VEGF in MT-9 tumors decreased, but the VEGF expression level in MT-8 tumors was still increased. These findings indicated that VEGF and bFGF may contribute cooperatively to angiogenesis in an early growth of mesenchymal tumor development.     

JSRV-Env重组质粒诱导转染TC-1、TC-1-Hyal2细胞后对TGF-α及VEGF因子表达的影响

通过脂质体法将JSRV-Env重组质粒瞬时转染小鼠肺上皮细胞（TC-1）和表达绵羊Hyal-2的小鼠肺上皮细胞（TC-1-Hyal2）,而后探讨两细胞中转化生长因子-α（TGF-α）及血管内皮生长因子（VEGF）mRNA和蛋白表达的变化,以此分析TGF-α、VEGF与绵羊肺腺瘤病（ovine pulmonary adenomatosis,OPA）发生的关联性及其在细胞癌变过程中的作用。体外培养TC-1和TC-1-Hyal2细胞,按照不同质粒可将TC-1和TC-1-Hyal2细胞分别设为pEGFP-C1-env转染组、pEGFP-C1转染组和未转染组。利用实时荧光定量PCR和ELISA方法对TGF-α、VEGF的表达水平进行检测。实时荧光定量PCR检测结果表明,相比对照组（pEGFP-C1转染组及未转染组）,在转染pEGFP-C1-env的两细胞组中VEGF mRNA的表达量均显著升高（P<0.05）;而两细胞组中TGF-α mRNA的表达量均极显著升高（P<0.01）。ELISA检测结果表明,同两对照组相比,转染pEGFP-C1-env的TC-1-Hyal2细胞上清液中VEGF、TGF-α蛋白浓度均极显著升高（P<0.01）;且VEGF在相同转染处理的TC-1细胞组中其蛋白浓度也极显著升高（P<0.01）,而TGF-α的蛋白浓度呈显著升高（P<0.05）。比较pEGFP-C1转染和未转染各细胞组中TGF-α、VEGF的mRNA和蛋白表达量,结果均无显著差异（P>0.05）。本研究发现,通过实时荧光定量PCR和ELISA方法检测经JSRV-Env诱导转染上述两细胞系后TGF-α、VEGF的mRNA和蛋白表达量均升高,结果提示JSRV-Env可能上调两细胞因子,进而可推测TGF-α、VEGF的表达变化与OPA发病存在一定的关联性,由此为衍生出的OPA针对二者进行基因靶向治疗方案提供重要的理论依据。     

Immunohistochemical expression of vascular endothelial growth factor and vascular endothelial growth factor receptor-2 in canine simple mammary gland adenocarcinomas

The expression of 5 markers associated with angiogenesis, proliferation, and apoptosis was studied in 26 canine simple mammary gland adenocarcinomas (SMGAs). The adenocarcinomas were graded histologically, and tissue sections were immunohistochemically stained for the expression of vascular endothelial growth factor (VEGF), VEGF receptor-2 (VEGFR-2), intra-tumor microvessel density, and tumor proliferation (PI) using antibodies against VEGF, VEGFR-2, von Willebrand factor, and Ki-67 antigen, respectively. Apoptotic indices (AI) were determined by an apoptosis assay. Markers VEGF and VEGFR-2 were detected in 96% and 100% of SMGAs, respectively. A high correlation between histologic grade and PI (r = 0.73), a moderate correlation between VEGF and histologic grade (r = 0.33), and between VEGF and PI (r = 0.42) were found. There was a significant difference in median PI among the 3 histologic grade groups (r < 0.05). Vascular endothelial growth factor may stimulate tumor cell proliferation through an autocrine loop, since VEGF and VEGFR-2 were expressed in most tumors.     

EP2受体激动剂Butaprost对奶牛子宫内膜上皮细胞中血管内皮生长因子基因表达的影响

[目的]观察EP2受体激动剂Butaprost对奶牛子宫内膜上皮细胞中血管内皮生长因子(vascular endothelial growth factor,VEGF)基因表达的影响,探讨前列腺素类化合物对奶牛子宫内膜组织修复的机制。[方法]分离培养奶牛子宫内膜上皮细胞,采用荧光定量PCR技术检测EP2受体激动剂Butaprost对奶牛子宫内膜上皮细胞中VEGFmRNA表达的影响。[结果]10-6mol/L的Butaprost作用于奶牛子宫内膜上皮细胞4、8、16、24h可显著(P<0.05)或极显著(P<0.01)地促进VEGFmRNA的表达。[结论]EP2受体激动剂Butaprost能够促进奶牛子宫内膜上皮细胞中VEGF基因的表达。     

Expression of epidermal growth factor receptor and vascular endothelial growth factor in malignant canine epithelial nasal tumours*

Epidermal growth factor (EGF) and vascular endothelial growth factor (VEGF) signalling pathways play a role in carcinogenesis. Inhibition of EGF receptor (EGFR) and of VEGF is effective in increasing the radiation responsiveness of neoplastic cells both in vitro and in human trials. In this study, immunohistochemical evaluation was employed to determine and characterize the potential protein expression levels and patterns of EGFR and VEGF in a variety of canine malignant epithelial nasal tumours. Of 24 malignant canine nasal tumours, 13 (54.2%) were positive for EGFR staining and 22 (91.7%) were positive for VEGF staining. The intensity and percentage of immunohistochemically positive neoplastic cells for EGFR varied. These findings indicate that EGFR and VEGF proteins were present in some malignant epithelial nasal tumours in the dogs, and therefore, it may be beneficial to treat canine patients with tumours that overexpress EGFR and VEGF with specific inhibitors in conjunction with radiation.     

High Urine Concentrations of Basic Fibroblast Growth Factor in Dogs With Bladder Cancer

Because dogs with bladder cancer often have advanced disease at the time of diagnosis, the identification and use of a tumor marker that could facilitate earlier diagnosis is a valid approach to improve prognosis. The objective of this study was to determine if urine concentrations of the proan-giogenic peptide, basic fibroblast growth factor (bFGF), are high in dogs with bladder cancer compared with normal dogs and dogs with urinary tract infection. We used a commercially available enzyme-linked immunosorbent assay test kit to quantitate bFGF in the urine of 17 normal dogs, 10 dogs with urinary tract infection, and 7 dogs with locally active transitional cell carcinoma of the urinary bladder. In normal dogs, the median urine bFGF concentration was 2.23 ng/g creatinine (quartile range, 1.53 to 5.12 ng/g creatinine). The median urine bFGF concentration in dogs with urinary tract infection did not differ significantly from normal dogs. Dogs with bladder cancer had significantly higher urine bFGF concentrations than normal dogs ( P < .002) and dogs with infection ( P < .02). The median urine bFGF concentration in dogs with transitional cell carcinoma was 9.86 ng/g creatinine (quartile range, 7.40 to 21.63 ng/g creatinine). Six of 7 dogs with bladder cancer had urine bFGF concentrations that were up to 7.4 times the 90th percentile value for normal dogs. Only 1 of 10 dogs with infection had a urine bFGF concentration that exceeded the 90th percentile of normal. These data suggest that canine bladder cancers export bFGF, and that urine bFGF may be useful as a diagnostic tumor marker or noninvasive indicator of treatment response. J Vet Intern Med 1996;10:231–234. Copyright © 1996 by the American College of Veterinary Internal Medicine .     

Steroid receptor expression and HER-2/neu (c-erbB-2) oncoprotein in the uterus of cats with cystic endometrial hyperplasia-pyometra complex

The presence of oestrogen-alpha receptor (ER), progesterone receptor (PR), and HER-2/neu (c-erbB-2) oncoprotein in the uterine walls of 10 healthy cats and 20 subjects with cystic endometrial hyperplasia-pyometra (CEH-P) were evaluated. Lesions were graded according to the severity of cystic dilation, hyperplasia and inflammation, and were classified as normal, mild uterine hyperplasia and severe uterine hyperplasia. The ER, PR and c-erbB-2 expression in the endometrium, glandular epithelium, stromal fibroblasts and myometrial smooth muscle cells was quantified by immunohistochemistry. The ER, PR and c-erbB-2 staining patterns differed between normal uteri and uteri with CEH-P. The ER expression was tended to be higher in the endometrial surface and glandular epithelium in the severe hyperplasia group (P > 0.05) and significantly lower in the mild hyperplasia cases compared with normal endometrium (P < 0.05), whereas the PR expression in both severe and mild hyperplasia cases tended to be higher in stromal cells and glandular epithelium than those in the normal uteri. C-erbB-2 immunoreactivity was observed only in the endometrial surface and glandular epithelium of the uterine wall and immunostaining was found to be highest in cases with severe hyperplasia. As a conclusion, we suggest that c-erbB-2 oncoprotein may play a role in the pathogenesis of the CEH together with the ER and PR in cats, and that ER does not have a role in the mechanism of pyometra, whereas PR plays a role in the pathogenesis of both CEH and pyometra.     

Detection of γ-H2AX,a Biomarker for DNA Double-strand Breaks,in Urinary Bladders of N -Butyl- N -(4-Hydroxybutyl)-Nitrosamine-Treated Rats

To evaluate the potential role of DNA repair in bladder carcinogenesis, we performed an immunohistochemical analysis of expression of various DNA repair enzymes and γ-H2AX, a high-sensitivity marker of DNA double-strand breaks, in the urothelium of male F344 rats treated with N-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN), a bladder-specific carcinogen. Our results clearly demonstrated that γ-H2AX aggregation was specifically generated in nuclei of bladder epithelial cells of BBN-treated rats, which was not found in untreated controls or mesenchymal cells. γ-H2AX-positive cells were detected not only in hyperplastic and neoplastic areas but also in the normal-like urothelium after BBN treatment. These data indicate that γ-H2AX has potential as a useful biomarker for early detection of genotoxicity in the rat urinary bladder. To the best of our knowledge, this is the first report demonstrating expression of γ-H2AX during bladder carcinogenesis.     

Expression of cell adhesion molecules in canine choroid plexus tumors

Choroid plexus tumor (CPT) is a primary intracranial neoplasm of the choroid plexus epithelium in the central nervous system. In the current World Health Organization classification, CPT is classified into two categories; choroid plexus papilloma (CPP) and carcinoma (CPC). In the present study, we investigated immunohistochemical expressions of N-cadherin, E-cadherin and β-catenin in 5 canine CPT cases (1 disseminated CPC, 2 CPCs and 2 CPPs). One CPP case was positive for N-cadherin and β-catenin, but negative for E-cadherin. The disseminated CPC case was positive for E-cadherin and β-catenin, but negative for N-cadherin. The other cases were positive for the three molecules examined. These results suggest that loss of the N-cadherin expression might associate with the spreading of CPC cells.     

The role of vascular endothelial growth factor and its receptor Flk-1/KDR in promoting tumour angiogenesis in feline and canine mammary carcinomas: a preliminary study of autocrine and paracrine loops

Vascular Endothelial Growth Factor (VEGF) and its receptor KDR are involved in the regulation of angiogenesis and are up-regulated in a number of tumours in humans and in particular, breast cancer. We therefore evaluated the prognostic potential of the angiogenetic process in feline and canine mammary carcinomas by the immunohistochemical assessment of VEGF expression and micro vessel density (MVD) quantification and examined the interplay between VEGF and KDR. These variables were related to some relevant clinicopathological parameters and to overall survival (OS). VEGF and KDR expression were evaluated in epithelial, stromal and endothelial compartments in order to identify autocrine and/or paracrine loops. In dogs an increased VEGF expression did not show any statistical correlation with the clinicopathological parameters examined and was not correlated to a poorer prognosis. MVD was found to be significantly correlated to the histologic type (P=0.04), tumour grading (P=0.02), and to the OS (P=0.01). In cats VEGF expression was significantly correlated to tumor grading (P=0.01) and OS (P=0.03), while no significant associations were found between MVD and the other parameters. VEGF and KDR were found to be detected on the epithelial, and/or endothelial and/or stromal cells of the carcinomas in both species, suggesting indications for some possible autocrine and paracrine loops. Our results encourage further studies on the possible prognostic role of VEGF and MVD in canine and feline mammary tumours and on the role of growth factors and their receptors in promoting tumour proliferation and an "angiogenetic shift". The VEGF/KDR system may play a role in malignant transformation and tumor progression.