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A variety of VEGF inhibitors have been reported to treat cancers by suppressing tumor angiogenesis. Bevacizumab, a monoclonal VEGF antibody, was the first FDA approved anti-angiogenic agent for cancer treatments. However, bevacizumab shows modest therapeutic efficiency and often cause resistant problem in significant populations of cancer patients. To solve these problem, we investigated the therapeutic efficacy of siRNA drugs targeting VEGF and combination of the RNAi drug with bevacizumab for cancer treatments. For efficient VEGF siRNA delivery, chemically polymerized siRNAs were complexed with thiolated-glycol chitosan (psi(VEGF)/tGC). The poly-VEGF siRNA and thiolated-glycol chitosan formed stable nanoparticles via electrostatic interaction and chemical crosslinking, and showed high accumulation in tumor tissues resulting in efficient gene silencing. Both VEGF siRNA nanoparticles and bevacizumab had efficient therapeutic effects in tumor xenograft mouse models. Interestingly, most pronounced therapeutic efficacy was observed when the two distinct VEGF inhibitors were treated in combination revealing synergistic effects. The results showed that the psi(VEGF)/tGC nanoparticle mediated knockdown of VEGF exerts anti-tumor effects and the combination treatments with bevacizumab can extend the treatments options to conventional bevacizumab treatments for cancer therapy., (Copyright © 2017. Published by Elsevier Inc.)

Labeling of stem cells aims to distinguish transplanted cells from host cells, understand in vivo fate of transplanted cells, particularly important in stem cell therapy. Adipose-derived mesenchymal stem cells (ASCs) are considered as an emerging therapeutic option for tissue regeneration, but much remains to be understood regarding the in vivo evidence. In this study, a simple and efficient cell labeling method for labeling and tracking of stem cells was developed based on bio-orthogonal copper-free click chemistry, and it was applied in a mouse hindlimb ischemia model. The human ASCs were treated with tetra-acetylated N-azidoacetyl-d-mannosamine (Ac 4 ManNAz) to generate glycoprotein with unnatural azide groups on the cell surface, and the generated azide groups were fluorescently labeled by specific binding of dibenzylcyclooctyne-conjugated Cy5 (DBCO-Cy5). The safe and long-term labeling of the hASCs by this method was first investigated in vitro. Then the DBCO-Cy5-hASCs were transplanted into the hindlimb ischemia mice model, and we could monitor and track in vivo fate of the cells using optical imaging system. We could clearly observe the migration potent of the hASCs toward the ischemic lesion. This approach to design and tailor new method for labeling of stem cells may be useful to provide better understanding on the therapeutic effects of transplanted stem cells into the target diseases., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The aim of the present study was to determine the distribution and characteristics of microvessels in various histological types of canine renal cell carcinoma (RCC). The study compared microvessel density (MVD) and distribution of blood vessels according to histological type and evaluated the presence of angiogenesis-related proteins. Nine archival samples of canine RCC were studied. MVD was calculated as the mean number of blood vessels per mm(2). The diameter of blood vessels was calculated by determining either the length of the long axis of blood vessels (diameter(max)) or the mean distance from the centre of each blood vessel to the tunica adventia (diameter(mean)). A significant difference in MVD was evident between RCCs and normal kidneys (46.6 ± 28.0 versus 8.4 ± 2.2 microvessels/mm(2)). Diameter(max) in canine RCCs (34.1 ± 14.7 μm) was also significantly different from normal canine kidney (23.2 ± 3.4 μm). Vascular endothelial growth factor (VEGF) was expressed by tumour cells and vascular endothelial cells and tumour necrosis factor (TNF)-α expression was observed in vascular endothelial cells in both neoplastic and normal kidney. Although VEGF is involved in angiogenesis and correlates with tumour stage of development, no correlation was found between VEGF expression and MVD. Tumour-associated macrophages expressing TNF-α and hypoxia inducible factor 1α were identified in peritumoural tissue and may play an important role in angiogenesis., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Alzheimer's disease (AD) is the most common progressive form of dementia in aged people. Microscopical changes in the brains of AD patients include the formation of senile plaques (SPs), neurofibrillary tangles (NFTs) and granulovacuolar degeneration and the deposition of amyloid-beta (Aβ). Aged dogs are known to suffer from cognitive dysfunction and this state is associated with deposition of Aβ in the brain. The aim of the present study was to investigate tau phosphorylation of neurons and astrocytes in the brain of aged dogs with progressive cognitive impairment. Changes in the brain of aged dogs with cognitive dysfunction were compared with those in the brain of patients with AD of Braak stage V. Immunohistochemically, Aβ deposition, phosphorylated tau Ser396 (p-tau Ser396) and ubiquitin were observed in the parietal cortex and hippocampus of aged dogs with cognitive dysfunction. Astrocytes with expression of p-tau Ser396 and neurons with co-localization of p-tau Ser396 and ubiquitin were observed. Expression of p-tau Ser396 and accumulation of ubiquitin were significantly increased in the parietal cortex and dorsal part of the hippocampus of the brain of aged dogs when compared with expression of these molecules in human AD., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

CDX-2 is used as a specific cell marker for human intestinal adenocarcinoma. In human studies, HER-3 overexpression predicts poor survival for patients with various cancers including gastric cancer. Gastrointestinal adenocarcinoma is less common in dogs than in man and the expression of immunological markers by the canine tumours has not yet been extensively studied. CDX-2 and HER-3 expression was determined in 18 canine gastrointestinal adenocarcinomas: 13 were of colorectal origin and five were of gastric origin. CDX-2 expression was predominantly observed in the nuclei of normal colonic epithelium and in neoplastic epithelium and neoplastic gastric epithelial cells that which had metastasized to the gastric lymph node. CDX-2 was expressed in 11 of 13 (84.6%) colorectal adenocarcinomas and in all five (100%) gastric adenocarcinomas. HER-3 was consistently expressed in the cytoplasm of neoplastic epithelial cells. HER-3 expression was detected in 12 of 13 (92.3%) colorectal and in all five (100%) gastric adenocarcinomas. CDX-2 and HER-3 may be useful markers for canine gastrointestinal adenocarcinoma., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Angiogenesis plays a crucial role in tumour growth, invasion and metastasis. Mast cells (MCs) release angiogenic factors that promote endothelial proliferation and differentiation. Previous studies have suggested that MCs are involved in tumour angiogenesis due to the release of various pro-angiogenic factors. This study evaluated samples from 40 canine mammary carcinomas and eight healthy non-neoplastic canine mammary glands. Toluidine blue staining was performed to characterize the MCs. Immunohistochemical labelling was performed to detect the number of tryptase-positive MCs and microvessels. MCs accumulated in tumour tissue and were closely associated with blood or lymphatic vessels in the tumour microenvironment. Angiogenesis, as measured by microvessel density, increased in direct proportion to the number of MCs. The correlation coefficient was significantly higher for tryptase-positive MCs than for toluidine blue-stained MCs. These results suggest that MCs are involved in tumour angiogenesis, which in turn influences tumour growth, invasion and metastasis. In particular, MC tryptase may be influential in mediating this function of MCs., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Malignant tumours are often associated with a relatively high number of tumour-infiltrating lymphocytes (TILs) and associated local cytokine production and these factors are thought to play a role in tumour progression. These aspects of tumour microenvironment have not been studied in canine mammary gland tumours (MGTs). The present study investigates TILs and the presence of related cytokines, as well as the expression of breast cancer susceptibility gene-1 (BRCA1), in canine MGTs. Immunohistochemistry, immunoblotting and reverse transcriptase-polymerase chain reaction were performed to evaluate these parameters. Three times as many T lymphocytes as B cells infiltrated canine MGTs. A correlation was found between expression of interleukin (IL)-1 and IL-6 and metastasis. There was an association between the expression of TILs, cytokines and mutation of BRCA1, suggesting that all of these factors may play a role in tumour progression., (Copyright 2009 Elsevier Ltd. All rights reserved.)

Canine mast cell tumours (MCTs) may be graded microscopically for prognostic purposes. Grade I (well-differentiated) and grade II (intermediate differentiation) tumours have an abundance of metachromatic granules within the cytoplasm; however, grade III (poorly differentiated) MCTs may be difficult to diagnose as they frequently have fewer discernable granules. Herein we report that a cross-reactive anti-human CD1a monoclonal antibody (clone O10) may be used in immunohistochemistry to identify canine MCTs of all grades. The antibody was applied to tissue sections from 48 canine MCTs of different histological grades. Serial sections from each tumour were stained with toluidine blue and safranin O to compare diagnostic sensitivity. All MCTs were labelled positively by the CD1a antibody, but histochemical staining was often equivocal and identification of mast cells was extremely difficult in some cases. This antibody did not label neoplastic cells in cases of canine histiocytoma, plasmacytoma or amelanotic melanoma; therefore, the reagent may be a valuable marker for the diagnosis of canine MCTs, especially those tumours of histological grade III.