Atsuko Murai

Establishment of canine hemangiosarcoma xenograft models expressing endothelial growth factors, their receptors, and angiogenesis-associated homeobox genes

BackgroundHuman hemangiosarcoma (HSA) tends to have a poor prognosis; its tumorigenesis has not been elucidated, as there is a dearth of HSA clinical specimens and no experimental model for HSA. However, the incidence of spontaneous HSA is relatively high in canines; therefore, canine HSA has been useful in the study of human HSA. Recently, the production of angiogenic growth factors and their receptors in human and canine HSA has been reported. Moreover, the growth-factor environment of HSA is very similar to that of pathophysiological angiogenesis, which some homeobox genes regulate in the transcription of angiogenic molecules. In the present study, we established 6 xenograft canine HSA tumors and detected the expression of growth factors, their receptors, and angiogenic homeobox genes.MethodsSix primary canine HSAs were xenografted to nude mice subcutaneously and serially transplanted. Subsequently, the expressions of vascular endothelial growth factor (VEGF)-A, basic fibroblast growth factors (bFGF), flt-1 and flk-1 (receptors of VEGF-A), FGFR-1, and angiogenic homeobox genes HoxA9, HoxB3, HoxB7, HoxD3, Pbx1, and Meis1 were investigated in original and xenograft tumors by histopathology, immunostaining, and reverse transcription polymerase chain reaction (RT-PCR), using canine-specific primer sets.ResultsHistopathologically, xenograft tumors comprised a proliferation of neoplastic cells that were varied in shape, from spindle-shaped and polygonal to ovoid; some vascular-like structures and vascular clefts of channels were observed, similar to those in the original tumors. The expression of endothelial markers (CD31 and vWF) was detected in xenograft tumors by immunohistochemistry and RT-PCR. Moreover, the expression of VEGF-A, bFGF, flt-1, flk-1, FGFR-1, HoxA9, HoxB3, HoxB7, HoxD3, Pbx1, and Meis1 was detected in xenograft tumors. Interestingly, expressions of bFGF tended to be higher in 3 of the xenograft HSA tumors than in the other tumors.ConclusionWe established 6 xenograft canine HSA tumors in nude mice and found that the expressions of angiogenic growth factors and their receptors in xenograft HSAs were similar to those in spontaneous HSA. Furthermore, we detected the expression of angiogenic homeobox genes; therefore, xenograft models may be useful in analyzing malignant growth in HSA.

Immunohistochemical Analysis of the Akt/mTOR/4E-BP1 Signalling Pathway in Canine Haemangiomas and Haemangiosarcomas

The specific signalling pathways that are deregulated in canine endothelial tumours have not yet fully elucidated. Therefore, the aim of the present study was to examine activation of the Akt/mammalian target of rapamycin (mTOR)/eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) signalling pathway in spontaneously arising canine haemangiomas (HAs) and haemangiosarcomas (HSAs) in order to identify novel molecular targets for treatment. Surgically-resected samples of HA (n = 27), HSA (n = 37), granulation tissue (n = 4) and normal skin (n = 4) were investigated by immunohistochemistry. Approximately 80% of the HSA samples had moderate to intense expression of phosphorylated Akt at Ser473 (p-Akt Ser473), p-Akt Thr308, p-4E-BP1 Thr37/46 and eukaryotic initiation factor 4E, which was significantly higher than in the HAs and was similar to the expression in activated endothelial cells (ECs). Although p-mTOR complex1 (p-mTORC1) Ser2448 was expressed by most of the activated ECs, only 35% of the HSA samples had weak to moderate expression. Because mTORC2 and phosphorylates Akt Ser473 was activated in HSA samples, the present findings suggest that the mTORC2/Akt/4E-BP1 pathway, regulated independently of mTORC1, may be important for targeting therapy in canine HSAs.

Immunohistochemical Demonstration of Angiogenesis-Associated Homeobox Proteins in Canine Vascular Tumours

Angiogenic homeobox genes regulate the behaviour of endothelial cells (ECs) during angiogenesis, so the aim of this study was to determine whether expression of these genes may be a determinant of malignancy in canine haemangiosarcoma (HSA). Homeobox proteins were evaluated immunohistochemically in tissue samples from canine HSAs (n=78), haemangiomas (HAs; n=30) and samples of granulation tissue (n=8). Active ECs in granulation tissue were positively labelled by antisera specific for HoxA9, HoxB3, HoxD3, HoxB7, Pbx1 and Meis1. Quiescent ECs in granulation tissue did not express HoxD3 and Pbx1. There were significantly more neoplastic cells positively labelled for HoxA9, HoxB3, HoxD3 and Pbx1 in HSA compared with HA. Almost all tumours were positive for HoxB7 and Meis1. HoxB3, HoxD3, Pbx1 and Meis1 proteins were detected in 80-90% of the HSAs, but in <20% of the HAs. Overall, homeobox protein expression in HSA appears to have a phenotype similar to that of active ECs in angiogenesis. The expression of homeobox genes associated with angiogenesis might be associated with the malignant growth of HSA.

Squamous Cell Carcinoma in a Capybara (Hydrochoerus hydrochaeris)

ABSTRACT A 4-year and 2-month-old male capybara (Hydrochoerus hydrochaeris) was diagnosed with squamous cell carcinoma on the buttocks after chronic recurrent dermatosis. The capybara was euthanized, examined by computed tomography and necropsied; the tumor was examined histologically. Computed tomography showed a dense soft tissue mass with indistinct borders at the buttocks. Histological examination of the tumor revealed islands of invasive squamous epithelial tumor cells with a severe desmoplastic reaction. Based on the pathological findings, the mass was diagnosed as a squamous cell carcinoma. This is the first study to report squamous cell carcinoma in a capybara.

Glomerular Lipidosis Accompanied by Renal Tubular Oxalosis in Wild and Laboratory-Reared Japanese Rock Ptarmigans (Lagopus mutus japonicus)

SUMMARY. Glomerular lipidosis is a disease characterized by lipid accumulation in mesangial cells but that has not been fully investigated in avian species. We examined four wild and two laboratory-reared Japanese rock ptarmigans (Lagopus mutus japonicus)—an endangered avian species—presenting vacuolar deposits in the glomeruli. All cases had vacuolar deposits in the glomeruli. In the wild cases, fewer than 30% of all glomeruli were affected, compared with more than 90% in the laboratory-reared cases. In the wild cases, most deposits were mild and restricted to the mesangial areas of glomeruli. In the laboratory-reared cases, nearly all of the deposits covered entire glomeruli. Electron microscopy of mild deposits revealed vacuoles in the cytoplasm of mesangial cells. These vacuoles were positive for Sudan III, Sudan black B, oil red O, Nile blue, periodic acid-Schiff, Schultz test, and digitonin stain and were negative for performaric acid-Schiff stains. Based on these results, we diagnosed the glomerular lesion as glomerular lipidosis caused by uptake of low-density lipoprotein in mesangial cells. Except for one wild case, all cases exhibited renal tubular oxalosis. The severity of tubular oxalosis tended to be related to the severity of glomerular lipidosis: In cases of mild glomerular lipidosis, tubular oxalosis was also mild or absent. We therefore diagnosed the primary lesion as glomerular lipidosis accompanied by tubular oxalosis. The four wild cases came from different zones and therefore had no opportunities to interbreed and no common relatives. We believe these data support the hypothesis that glomerular lipidosis is a disease of the general population of Japanese rock ptarmigans. This is the first report of glomerular lipidosis accompanied by renal tubular oxalosis in an avian species.

OCCURRENCE OF THELAZIA CALLIPAEDA AND TOXOCARA CATI IN AN IMPORTED EUROPEAN LYNX (LYNX LYNX) IN JAPAN

A necropsy was performed on an adult European lynx, Lynx lynx (Linnaeus, 1758), held in captivity until its death, to determine level of parasitism. Examination of the eyes revealed the oriental eyeworm, Thelazia callipaeda, in the conjunctival sac and the third eyelid of both eyes. The species was confirmed by location and morphology. Intact worms were fixed, mounted, and identified. Examination of the alimentary tract revealed the common ascaroid nematode, Toxocara cati. Species was confirmed by the arrow-like anterior end. One hundred and forty-one adult worms were collected. The presence of these nematodes indicated the importance of eliminating the contact of zoo animals with Amiota spp. vectors and to prevent contamination with the infective T. cati eggs.