Birgitt Wolfesberger

Expression of vascular endothelial growth factor and its receptors in canine lymphoma.

Vascular endothelial growth factor (VEGF) stimulates endothelial cell proliferation and has a pivotal role in tumour angiogenesis. The expression of VEGF and its receptors VEGFR-1 and VEGFR-2 was examined immunohistochemically in 43 specimens of canine lymphoma and in six normal lymph nodes. Western blotting and reverse transcriptase polymerase chain reaction (RT-PCR) were performed to detect VEGF protein and mRNA, respectively. VEGF protein was expressed by 60% of the tumours with diffuse cytoplasmic labelling of the neoplastic cells. Endothelial cells, macrophages and plasma cells were also immunolabelled. VEGFR-1 was expressed by variable numbers of neoplastic cells in 54% of lymphoma specimens. VEGFR-1 was also expressed by macrophages, plasma cells, reticulum cells, and vascular endothelial cells. Macrophages and lymphocytes in germinal centres of normal lymph nodes were also immunoreactive with anti-VEGF and VEGFR-1. Most tumours did not express VEGFR-2 but in 7% of sections there was focal labelling of neoplastic and endothelial cells, with a cytoplasmic and perinuclear pattern. The observed variability in expression of VEGF and its receptors probably relates to the fact that lymphoma is a heterogeneous lymphoproliferative tumour. Individual differences in VEGF and VEGFR expression must be taken into account when VEGF and VEGFR-targeted approaches for anti-angiogenic therapy are considered in dogs.

Ciprofloxacin causes cytoskeletal changes and detachment of human and rat chondrocytes in vitro

Abstract Quinolones cause damage of articular cartilage in different species by forming chelate complexes with divalent cations and inducing magnesium deficiency. Cations are important for regular function of integrins, a group of transmembrane proteins which connect extracellular matrix proteins with the intracellular cytoskeleton. We have shown that cultivation of rat chondrocytes in ciprofloxacin (CFX)-supplemented and Mg2+-free medium led to pronounced changes in the cytoskeleton and decreased adhesion of cells to the culture dish. In order to test whether or not these effects are species-specific, we extended our studies on human chondrocytes. Human chondrocytes cultivated in CFX-supplemented medium (10, 40, 80 and 160 μg/ml) or Mg2+-free medium showed decreased ability to adhere to growth support, cell shape changes, and alterations in actin and vimentin cytoskeleton in a concentration dependent manner. Attachment of human chondrocytes to collagen type II coated cover slips was reduced to 90% in CFX group and 75% in Mg2+-free group on day 1. This effect even increased after 4 days of culture in the respective medium (32% in CFX and 58% in Mg2+-free group). We concluded that Mg2+ deficiency is exerted via integrins, resulting in decreased ability to attach to extracellular matrix proteins and cytoskeletal changes. These effects are not species-specific. The attachment assay proves to be an easy to use experimental set-up to test ciprofloxacin and other quinolones for their chondrotoxic effects.

The tyrosine kinase inhibitor sorafenib decreases cell number and induces apoptosis in a canine osteosarcoma cell line

Canine osteosarcoma, an aggressive cancer with early distant metastasis, shows still despite good chemotherapy protocols poor long term survival. The aim of our study was to determine whether sorafenib, a novel multikinase inhibitor, has any effect on D-17 canine osteosarcoma cells. A cell proliferation kit was used for detecting surviving cells after treatment for 72 h with sorafenib or carboplatin or their combination. A significant decrease of neoplastic cells was observed after incubation with 0.5-16 microM sorafenib or with 80-640 microM carboplatin. Using immunocytochemistry for activated caspase 3 to evaluate apoptosis, we found significantly more positive cells in the sorafenib treated groups. Paradoxically, expression of the nuclear proliferation marker Ki-67 was also significantly higher in sorafenib treated cells. The drug sorafenib showed potent antitumour activity against D-17 canine osteosarcoma cells in vitro, suggesting a potential as a therapeutic tool in the treatment of bone cancer in dogs.

Angiogenic markers in canine lymphoma tissues do not predict survival times in chemotherapy treated dogs

Angiogenesis, which is essential for malignancies to progress, depends on various signalling proteins including vascular endothelial growth factor (VEGF) and vascular endothelial growth factor receptors 1 and 2 (VEGFR-1 and VEGFR-2). Microvessel density (MVD) is frequently used to evaluate angiogenesis. This study assessed the relationship between expression of VEGF, VEGFR-1 and VEGFR-2, MVD and the survival time in dogs with lymphoma. VEGF, VEGFR-1 and VEGFR-2 expression was evaluated immunohistochemically and microvessel profiles were counted in 34 lymphoma samples. Seventy-nine percent of the samples showed high VEGF expression and 62% were highly positive for VEGFR-1; VEGFR-2 immunoreactivity was mostly negative. Dogs treated with chemotherapy had a median survival time of 266days, but no significant relationships were found between overall survival time, MVD and expression of VEGF, VEGFR-1 or VEGFR-2. In this study, VEGF its receptors and the MVD were no prognostic factors in dogs with lymphoma.

Quantification of microvessels in canine lymph nodes

Quantification of microvessels in tumors is mostly based on counts of vessel profiles in tumor hot spots. Drawbacks of this method include low reproducibility and large interobserver variance, mainly as a result of individual differences in sampling of image fields for analysis. Our aim was to test an unbiased method for quantifying microvessels in healthy and tumorous lymph nodes of dogs. The endothelium of blood vessels was detected in paraffin sections by a combination of immunohistochemistry (von Willebrand factor) and lectin histochemistry (wheat germ agglutinin) in comparison with detection of basal laminae by laminin immunohistochemistry or silver impregnation. Systematic uniform random sampling of 50 image fields was performed during photo‐documentation. An unbiased counting frame (area 113,600 μm2) was applied to each micrograph. The total area sampled from each node was 5.68 mm2. Vessel profiles were counted according to stereological counting rules. Inter‐ and intraobserver variabilities were tested. The application of systematic uniform random sampling was compared with the counting of vessel profiles in hot spots. The unbiased estimate of the number of vessel profiles per unit area ranged from 100.5 ± 44.0/mm2 to 442.6 ± 102.5/mm2 in contrast to 264 ± 72.2/mm2 to 771.0 ± 108.2/mm2 in hot spots. The advantage of using systematic uniform random sampling is its reproducibility, with reasonable interobserver and low intraobserver variance. This method also allows for the possibility of using archival material, because staining quality is not limiting as it is for image analysis, and artifacts can easily be excluded. However, this method is comparatively time‐consuming. Microsc. Res. Tech., 2008.

Gene expression profile of vascular endothelial growth factor (VEGF) and its receptors in various cell types of the canine lymph node using laser capture microdissection (LCM).

The role of VEGF and its receptors has extensively been studied in tumours. In contrast, the presence and function of VEGF in normal tissues like the lymph node has not been given much attention until now. To study the expression of VEGF, VEGFR-1, VEGFR-2 and VEGFR-3 in the heterogenous cell population of the canine lymph node, laser capture microdissection was used to isolate pure cell fractions of macrophages, lymphocytes, endothelial cells, and capsule cells of the canine lymph node. To clarify if macrophages take up VEGF from the environment or express VEGF, VEGFR-1, VEGFR-2 or VEGFR-3 themselves, the mRNA expression was studied by real-time RT-PCR. After RNA isolation and subsequent analysis with the Agilent 2100 Bioanalyzer only RNA samples with appropriate RNA integrity were used for real-time PCR. For the accurate relative quantification of mRNA expression levels several reference genes were evaluated. It was shown that the reference genes HPRT1 and B2M serve as reliable reference genes for gene expression studies in the canine lymph node. Expression data analysis revealed no significant difference in VEGF expression levels between endothelial cells and the other investigated cells. VEGFR-1 expression was significantly lower in lymphocytes. Also macrophages showed a highly significant lower expression of VEGFR-1 compared to endothelial cells. In addition, the VEGFR-2 expression in lymphocytes and macrophages was significantly lower in comparison to endothelial cells. We were not able to detect VEGFR-3 mRNA in the lymphocyte cell population, in macrophages and cells of the lymph node capsule VEGFR-3 was expressed at very low levels. It was shown that laser capture microdissection in combination with quantitative real-time PCR is a valuable tool for studying the expression patterns of specific cells in their microenvironment. Our results support the hypothesis that VEGF and its receptors have other biological roles besides stimulating angiogenesis in the normal lymph node. These biological functions need to be clarified in further studies.

Human osteosarcoma cells respond to sorafenib chemotherapy by downregulation of the tumor progression factors S100A4, CXCR4 and the oncogene FOS

Osteosarcoma is a rare but aggressive bone neoplasm in humans, which is commonly treated with surgery, classical chemotherapy and radiation. Sorafenib, an inhibitor of a number of kinases targeting the Raf/MEK/ERK pathway, is a promising new chemotherapeutic agent in human medicine that has been approved since 2006 for the therapy of renal cell carcinoma and since 2007 for the treatment of hepatocellular carcinoma. Here, we studied the antimetastatic potential of 4 µM of this multikinase inhibitor in a human osteosarcoma cell line. DNA microarray-based gene expression profiling detected 297 and 232 genes upregulated or downregulated at a threshold of >2-fold expression alteration (P<0.05) in the sorafenib-treated cells. Three genes (CXCR4, FOS and S100A4) that are involved in tumor progression were chosen for validation by quantitative PCR (qPCR) and protein expression analysis. The decrease in RNA expression detected by microarray profiling was confirmed by qPCR for all three genes (P<0.01). On the protein level, sorafenib-induced reduction of S100A4 was verified both by western blotting and immunohistochemistry. For CXCR4 and c-Fos, a reduced protein expression was shown by immunohistochemistry, for c-Fos also by immunoblotting. We conclude that sorafenib could serve as a potent chemotherapeutical agent by which to inhibit the metastatic progression of osteosarcomas.

Sudden death in a dog with lymphoplasmacytic hypophysitis.

A 10-year-old dog with a history of progressive anorexia and weight loss died suddenly despite treatment. Histopathological examination revealed severe follicular lymphoplasmacytic adenohypophysitis and atrophy of the zona fasciculata and zona reticularis of the adrenal cortex. It is likely that lack of production of adrenocorticotropic hormone and cortisol was the cause of death of this dog.

Integrins mediate the effects of quinolones and magnesium deficiency on cultured rat chondrocytes

Chondrocyte-matrix interaction is mediated by a series of adhesion molecules. Both alpha and beta integrin subunits are involved and govern crucial functions of cell adhesion and signal transduction. These molecules modulate proliferation and differentiation, thus establishing cartilage integrity. We studied the influence of magnesium deficiency and quinolone antibiotics (which form chelate complexes with divalent cations) on chondrocytes in vitro in order to assess the role of Mg2+ ions in integrin function and to establish cellular changes mediated via integrin signal transduction. Mg2(+)-free medium and quinolone supplementation was found to decrease chondrocyte attachment to collagen type II-coated coverslips. Adhesion and growth of chondrocytes were reduced in the respective medium. Organisation of cytoskeletal fibers (vimentin) was changed and formation of stress fibers (f-actin) was disturbed. Additionally, rates of cell proliferation declined. These results indicate that quinolone-magnesium complex formation is important for chondrotoxicity of these substances. Cell-matrix detachment and morphological alterations described in vitro may explain the lesions observed in articular cartilage after quinolone administration in vivo. The attachment assay described could serve as a simple test to establish the susceptibility of chondrocytes of different species to different quinolones in use or new ones to be introduced.

Characterization of the T-cell receptor gamma chain gene rearrangements as an adjunct tool in the diagnosis of T-cell lymphomas in the gastrointestinal tract of cats.

Feline alimentary lymphoma is the most common hematopoietic neoplasia in cats. It affects mainly the small intestines and is most frequently of T-cell origin. Evaluation of a fine needle aspirate is often the first step in the diagnostic work-up. Differentiation between a resident mature lymphocyte population as encountered in inflammatory bowel disease and small cell lymphoma cannot be achieved by cytology alone. Even full thickness biopsies evaluated by histopathology can be inconclusive. These cases warrant the application of complementary tools like PCR-based T-cell receptor (TCR) clonality testing for confirmation. The aim of this study was to optimize the DNA extraction protocol for formalin fixed and paraffin embedded tissues (FFPE) and to establish a heteroduplex analysis to enhance resolution of the PCR fragments of the T-cell receptor gamma (TCRG) V-J gene. The new protocols resulted in improved quantity and quality of the extracted DNA. Heteroduplex analysis of the samples improved the resolution of the electrophoresis results so that rules for interpretation of the different patterns could be established. Application of this improved setup detected clonal rearrangements in at least one TCRG primer reaction in 31 of 36 of our feline intestinal lymphoma samples after DNA quality testing.