Aberrant expression of microRNAs and the miR-1/MET pathway in canine hepatocellular carcinoma.

Canine hepatocellular carcinoma (HCC) is the most common primary hepatic tumour in dogs. MicroRNA (miRNA) dysregulation has been reported in human HCC and shown to have diagnostic and prognostic value; however, there are no data on miRNA expression in canine HCC. The aim of the present study was to investigate differentially expressed miRNAs in canine HCC. Analysis of miRNA expression in canine HCC tissues and cell lines by quantitative reverse transcription PCR showed that miR-1, miR-122, let-7a, and let-7g were downregulated, whereas miR-10b and miR-21 were upregulated in canine HCC. MET is one of the target genes of miR-1. MET was upregulated in canine HCC at the gene and protein levels, and a significant correlation between the concomitant downregulation of miR-1 and upregulation of MET was observed. Fast/intermediate-proliferating canine HCC cell lines had higher MET gene and protein expression levels than the slow-proliferating cell line. These findings suggest that miRNAs are differentially expressed in canine HCC, and that the miR-1/MET pathway may be associated with canine HCC cell proliferation.

Disseminated Histiocytic Sarcoma in an African Hedgehog (Atelerix albiventris).

Disseminated histiocytic sarcoma (HS) was diagnosed on post-mortem examination of a 1.5-year-old African hedgehog (Atelerix albiventris) that was presented in poor physical condition and with diarrhoea. Leucocytosis and a hypoechoic abdominal mass were noted on haematological and ultrasonographical examinations. Gross pathological, histopathological, immunohistochemical and ultrastructural evaluation of the mass supported a diagnosis of disseminated HS. To our knowledge, this report represents the first documentation of disseminated HS in this species.

Radioresistance of cancer stem-like cell derived from canine tumours.

Cancer stem-like cells (CSCs)/cancer-initiating cells (CICs) are a small subpopulation of cancer cells that are responsible for the initiation, recurrence and metastasis of cancer. We previously demonstrated that, using the Hoechst 33342 dye-based side population technique, CSCs/CICs in canine lung adenocarcinoma cell line exist. In this study, as CSCs/CICs are known to form spheres in anchorage-independent environment in vitro, we evaluated the stemness of spheroid cells derived from canine lung adenocarcinoma and osteosarcoma cells by expression of stemness markers, and investigated radioresistance. Spheroid cells showed greater expression of stemness markers Oct-4 and CD133 gene than those of adherent-cultured cells. In nude mouse xenograft models, spheroid cells showed higher tumourigenic ability than adherent-cultured cells. In addition, spheroid cells showed significantly resistant against radioactivity as compared with adherent-cultured cells. These results suggest that spheroid cells could possess stemness and provide a CSCs/CICs research tool to investigate CSCs/CICs of canine tumour cells.

Development of podocyte injuries in Osborne-Mendel rats is accompanied by reduced expression of podocyte proteins.

Osborne-Mendel (OM) rats spontaneously develop glomerulopathy with progressive podocyte injury. Changes in protein expression levels in the foot processes of podocytes have been suggested to play an important role in the development of renal disease. The aim of this study was to investigate the temporal relationship between the expression of five podocyte proteins (nephrin, podocin, synaptopodin, α-actinin-4 and α3-integrin) and the development of podocyte injuries, proteinuria and glomerulosclerosis in OM rats. Male OM rats 5-20 weeks of age and age-matched Fischer 344 rats were used. Semiquantitative analysis of expression of the five podocyte proteins was performed by immunofluorescence labelling. Nephrin mRNA expression was determined by quantitative real-time reverse transcriptase polymerase chain reaction and nephrin protein expression was determined by mass spectrometry. Progressive reduction in expression of the podocyte proteins correlated with the progression of podocyte injuries, the development of proteinuria and the subsequent development of glomerulosclerosis. Nephrin mRNA expression and nephrin concentration also showed temporal decreases in OM rats. Altered expression of podocyte proteins preceded the development of proteinuria and glomerulosclerosis, suggesting that this event contributes to podocyte dysfunction and progression to glomerulosclerosis.

Expression of nephrin, podocin, α-actinin-4 and α3-integrin in canine renal glomeruli.

The biological features of podocytes that contribute to the pathogenesis of proteinuria have not been investigated in dogs. The aim of this study was to investigate the expression and localization of nephrin, podocin, α-actinin-4 and α3-integrin in canine renal glomeruli. Renal cortical tissue was collected from the kidneys of five normal adult beagles. Western blotting and immunofluorescence microscopy revealed specific expression and localization of the four proteins in canine glomeruli. Expression of genes encoding the four molecules in isolated glomeruli was detected by reverse transcriptase polymerase chain reaction. The results of this study will permit future exploration of podocyte injury and its involvement in protein leakage from the capillary wall in canine glomerular diseases.

Collagenofibrotic glomerulonephropathy with fibronectin deposition in a dog.

We report herein a case of collagenofibrotic glomerulonephropathy in a 3-year-old Shiba Inu with severe proteinuria. Histologically, renal glomeruli were enlarged with massive deposition of a homogeneous eosinophilic substance within the mesangium and capillary walls. The deposits reacted weakly with periodic acid-Schiff, stained deep blue with Masson's trichrome, and were positive by immunofluorescence for type III collagen and fibronectin. Ultrastructurally, the deposits consisted of fibrils and amorphous material in the mesangial matrix and beneath the glomerular capillary endothelium. The fibrils had transverse bands analogous to those of collagen fibrils. Electron microscopy also revealed focal detachment of podocytes and foot process effacement in glomerular tufts, which suggested that podocyte injury had contributed to the development of proteinuria in this dog. The current case resembles collagenofibrotic glomerulonephropathy (CFGN) in humans in histopathologic, immunofluorescence, and electron microscopic findings. This is the first report of CFGN in a nonhuman species with glomerular deposition of fibronectin and type III collagen.

Cloning and expression of the ecdysteroid receptor during ecdysis and reproduction in females of the soft tick, Ornithodoros moubata (Acari: Argasidae).

Molecular mechanisms of ecdysteroid regulation in development and reproduction have been thoroughly investigated in Diptera and Lepidoptera, but few studies report the molecular actions of ecdysteroids in hemimetabolous insects and more primitive arthropods. Ecdysteroids appear to be the main hormones regulating development and vitellogenesis in ticks. An ecdysteroid receptor that showed high homology with EcRs of other arthropods was isolated from Ornithodoros moubata (OmEcRA). OmEcR expression patterns coincided with ecdysteroid titres in the haemolymph during moulting and vitellogenesis and differed between mated and virgin females. Therefore, OmEcR appears to mediate the regulation of moulting and vitellogenesis by ecdysteroids in O. moubata females as seen in other arthropods.

Expression of cyclooxygenase-2, P-glycoprotein and multi-drug resistance-associated protein in canine transitional cell carcinoma.

Cyclooxygenase-2 (COX-2), P-glycoprotein (P-gp) and multi-drug resistance-associated protein (MRP) are considered important tumor-associated proteins in humans and dogs. In the present study, we immunohistochemically evaluated the expression of these proteins in canine patients with transitional cell carcinoma (TCC). Of 52 cases, 30 (57.7%) were positive for COX-2, 40 (76.9%) for P-gp, and only 10 (19.2%) for MRP. In addition, 27 samples (27/52, 51.9%) were positive for two markers, while 3 (5.7%) and 5 (9.6%) cases were positive and negative, respectively, for all three markers. No significant correlations were seen for COX-2 and P-gp on Fisher's exact test and Mann-Whitney's test, but a significance was seen on Spearman's rank correlation analysis using the IHC scoring system (P=0.043). These results suggest that P-gp expression is induced by overexpression of COX-2 in canine patients with TCC.

Spontaneous extraskeletal osteosarcomas of the subcutis in Djungarian hamsters (Phodopus sungorus): report of two cases.

Spontaneous subcutaneous extraskeletal osteosarcoma was diagnosed in the subcutaneous tissue of two Djungarian hamsters. Histologically, the tumour was characterized by multiple nests of osseous and cartilaginous components within a proliferation of pleomorphic cells. No abnormality was observed in any skeletal bones and no change suggesting tumorous growth was observed in any other sites. This is the first report of extraskeletal osteosarcomas in Djungarian hamsters.

Relation between clenching strength and occlusal force distribution in primary dentition.

The aim of this study was to investigate the relation between clenching strength and occlusal force distribution in primary dentition. Twenty healthy children with normal occlusions: 11 boys and 9 girls, ages 3.2-5.8 years (avg. 4.5 years) were selected. Setting the bilateral masseter muscular activity at maximum clenching in full intercuspation as 100%, the occluding forces at 20, 40, 60, 80 and 100% clenching were recorded with pressure-sensitive sheets (Dental Prescale 50H, type R, Fuji Photo Film Co.), and the force of each primary tooth was analysed by computer (Occluzer FPD703). Occlusal force distribution was expressed as a percentage of the total occlusal force of each tooth and was compared between each clenching. There were no significant differences between various clenching strengths in the occlusal force distribution in primary dentition [one-way repeated-measures analysis of variance (ANOVA)]. Thus, the results of the present study suggest that the distribution of occluding forces on a primary dental arch had its own pattern and that the clenching strength had no effect on that pattern. These patterns may be useful in determining occlusal function in children.

Oxyhemoglobin produces apoptosis and necrosis in cultured smooth muscle cells.

Confluent rat aortic smooth muscle cells were treated with OxyHb in a concentration- and time-dependent manner. A high concentration of OxyHb (100 microM) within 24 h decreased cell density. DNA analysis showed a smear pattern characteristic of cell necrosis. Transmission electron microscopy demonstrated disintegration of the cell membrane and destruction of cell organelles. Western blotting using PARP antibody revealed that 116 kDa PARP was not cleaved to 85 kDa, an apoptosis-related fragment. On the contrary, a low concentration of OxyHb (10 microM) produced apoptotic cell death at 72 h that was supported by DNA analysis and TUNEL staining. These results demonstrated that a high level of OxyHb induced necrosis within 24 h and a low concentration of OxyHb produced apoptosis after 72 h in cultured smooth muscle cells. Morphological alterations induced by OxyHb might contribute to the vascular wall changes in the cerebral arteries following subarachnoid hemorrhage (SAH).

Microvasculature of hydronephrotic kidneys in KK-A(Y) mice.

Spontaneous hydronephrosis in KK-A(Y) mice was studied using light and electron microscopy and scanning electron microscopy of resin casts to evaluate micro vascular changes in the kidney. The renal parenchyma was extremely thin as a result of tubular atrophy. Histologically, varying degrees of glomerulosclerosis were observed. Ultrastructurally, marked thickenings of the glomerular basal lamina, an increase in mesangial cells and matrix, and marked effacement of foot processes were observed. In resin casts, a marked reduction in number of glomeruli was evident. The capillaries were thin, strangulated and tom-off to varying degrees in severely affected glomeruli. In the medulla, the three-dimensional capillary network running along the tubules was lost and changed to a two-dimensional vascular bed. Despite severe hydronephrosis, the glomerular capillary network was relatively well preserved, being either slightly or moderately injured in approximately 60% of surviving glomeruli.

Apoptosis of endothelial cells in vessels affected by cerebral vasospasm.

BACKGROUND: Cerebral vasospasm after subarachnoid hemorrhage is a prolonged contraction that leads to cerebral ischemia or infarction. Morphological studies of cerebral arteries during vasospasm have shown extensive necrosis of smooth-muscle cells and desquamation and dystrophy of endothelial cells. The mechanism of cellular death is unknown. METHODS: We report an observation of apoptotic changes in the cerebral arteries of a patient who died after suffering severe cerebral vasospasm caused by aneurysmal rupture. Subarachnoid hemorrhage and cerebral vasospasm were confirmed by computed tomography scanning and angiogram. Histological and immunohistological examinations for apoptosis were performed in cerebral arteries. For control, the arteries from another patient, who died of trauma without head injury, were used. RESULTS: Corrugation of the internal elastic lamina and increased amounts of connective tissue was demonstrated by light microscopy. Apoptotic changes, characterized by condensation of chromatin of the nucleus and detachment from the basal membrane, were found on transmission electron microscopy in endothelial cells. Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling reaction revealed positive staining of the nuclei of the endothelial cells. CONCLUSIONS: This study demonstrates that apoptosis occurred in the cerebral arteries in a patient who died of cerebral vasospasm. The possible role of apoptosis in cerebral vasospasm is discussed.

Effect of endothelin receptor antagonists on non-muscle matrix compaction in a cell culture vasospasm model.

Endothelin-1 (ET-1), a potent vascular smooth muscle constrictor, is one of the possible spasmogens in cerebral vasospasm. However, the role of ET-1 in non-muscle compaction (another aspect of the pathogenesis of cerebral vasospasm) has not been reported. This study was undertaken to demonstrate the effect of ET-1, as well as erythrocyte lysate and bloody cerebrospinal fluid (CSF), on fibroblast populated collagen lattice (FPCL) compaction. Human dermal fibroblasts were used to form FPCL. The concentration-dependent effect of ET-1 was examined in the absence and presence of an ETA receptor antagonist (BQ-485), or an ETB receptor antagonist (BQ-788), or both. FPCL compaction was determined by measuring reduction of areas over five days following treatment. To compare the effect of ET-1 on lattice compaction, erythrocyte lysate and bloody CSF obtained from a cerebral vasospasm patient were also tested. We found that ET-1 increased FPCL compaction in a concentration-dependent (but not time-dependent) manner. Erythrocyte lysate produced the strongest compaction, however, without time-dependence. Bloody CSF promoted FPCL compaction in a time-dependent fashion. Compaction induced by ET-1 was inhibited by BQ-485 but not by BQ-788. We concluded that ET-1 promotes FPCL compaction by activation of ETA receptors. Other components in bloody CSF or erythrocytes may also contribute to FPCL compaction.

Mitogen-activated protein kinase plays an important role in hemolysate-induced contraction in rabbit basilar artery.

OBJECT: Mitogen-activated protein kinase (MAPK) is an important signaling factor in the vascular proliferation and contraction, the two features of cerebral vasospasm following subarachnoid hemorrhage. We studied the possible involvement of MAPK in hemolysate-induced signal transduction and contraction in rabbit basilar artery. METHODS: Isometric tension was used to record the contractile response of rabbit basilar artery to hemolysate. Western blots using antibodies for MAPK were conducted. 1) Hemolysate produced a concentration-dependent contraction of rabbit basilar artery. Pre-incubation of arteries with MAPK kinase inhibitor PD-98059 markedly reduced the contraction induced by hemolysate. PD-98059 also relaxed, in a concentration-dependent fashion, the sustained contraction induced by hemolysate (10%). 2) Hemolysate produced a time-dependent elevation of MAPK immunoreactivity in Western blot in rabbit basilar artery. MAPK was enhanced 3 min after hemolysate exposure and the effect reached maximum at 5 min. The immunoreactivity of MAPK decayed slowly with time, but the level of MAPK was still higher than the basal level even at two hours after exposure to hemolysate. 3) Pre-incubation of arteries with MAPK kinase inhibitor PD-98059 abolished the effect of hemolysate on MAPK immunoreactivity. CONCLUSION: Hemolysate produced contraction of rabbit basilar artery possibly by activation of MAPK. MAPK inhibitors may be useful in the treatment of cerebral vasospasm.

Role of tyrosine kinase in fibroblast compaction and cerebral vasospasm.

Hemolysate, a proposed causative agent for cerebral vasospasm following subarachnoid hemorrhage, produces contraction of cerebral arteries by activation of tyrosine kinases. In addition, hemolysate accelerates fibroblast collagen compaction that could play a role in cerebral vasospasm. We studied the effect of hemolysate on tyrosine phosphorylation and fibroblast collagen compaction in cultured dog cerebral and human dermal fibroblasts using tyrosine kinase inhibitors and tyrosine antibodies (Western blot). 1) Hemolysate was found to enhance tyrosine phosphorylation of two proteins approximately 64 and 120 kDa. The effect of hemolysate was time- and concentration-dependent. 2) Two main components in hemolysate, oxyhemoglobin and adenosine triphosphate (ATP), produced similar results to that of hemolysate. 3) Tyrosine kinase inhibitor genistein and tyrphostin A51 (30 microM) markedly reduced the effect of hemolysate on tyrosine phosphorylation. 4) In another study, hemolysate increased fibroblast collagen compaction and the effect of hemolysate was reduced by genistein and tyrphostin A51. We conclude that hemolysate activates tyrosine kinase that may lead to acceleration of fibroblast compaction. This effect of hemolysate may contribute to cerebral vasospasm.

Oxyhemoglobin produces necrosis in cultured smooth muscle cells.

OBJECT: Myonecrosis in the tunica media, which is defined morphologically, is one of the most striking alterations in the cerebral arterial wall following subarachnoid hemorrhage (SAH). In this study, oxyhemoglobin (OxyHb) was added to cultured rat aortic smooth muscle cells to determine the pattern of cell death by morphological and biochemical techniques. METHODS: Confluent rat aortic smooth muscle cells were treated with OxyHb in a concentration- and time-dependent manner. Cell density was assayed by counting the number of cells that attached to the culture dishes after exposed to OxyHb. To identify cell death pattern, DNA analysis, electron microscopy, and Western blotting using poly (ADP-ribose) polymerase (PARP) antibody were performed. CONCLUSIONS: OxyHb decreased cell density in a concentration- and time-dependent manner. DNA analysis showed a smear pattern characteristic of cell necrosis. Transmission electron microscopy demonstrated disintegration of cell membrane and destruction of cell organelles. No apoptotic changes, such as condensation of chromatin or apoptotic bodies were observed. Western blotting using PARP antibody revealed that 116 kDa PARP was not cleaved to 85 kDa, an apoptosis-related fragment. These results demonstrated morphologically and biochemically that OxyHb induced necrosis, not apoptosis, in cultured smooth muscle cells.

Overexpression and genetic abnormality of p53 in parathyroid adenomas.

To study the significance of p53 abnormality in parathyroid tumors, 32 parathyroid adenomas and 22 hyperplastic glands from 14 cases of secondary hyperparathyroidism were analysed using immunohistochemistry, polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), single-strand conformation polymorphism (SSCP) and DNA sequencing. Immunohistochemical study revealed p53 overexpression in four parathyroid adenomas, of which two showed diffuse and one showed focal nuclear pleomorphism. Genetic analysis disclosed allelic loss in one, and a point mutation (R290H) and a polymorphism (L257 L) in another of the two other adenomas with diffuse nuclear pleomorphism. No abnormalities were discovered in the other two adenomas, although one had a R72P polymorphism in exon 4. There was no evidence of malignancy of the four tumors in either clinical or pathological terms. None of the 22 hyperplastic glands showed p53 overexpression. These results demonstrate that p53 abnormality can occur in benign parathyroid adenomas and is more prevalent in those with nuclear pleomorphism than in those without.

Hemolysate induces tyrosine phosphorylation and collagen-lattice compaction in cultured fibroblasts.

Hemolysate, a proposed causative agent for cerebral vasospasm after subarachnoid hemorrhage, produces contraction of cerebral arteries by activation of tyrosine kinases. In addition, hemolysate increases fibroblast-collagen compaction that could play a role in cerebral vasospasm. We studied the effect of hemolysate on tyrosine phosphorylation and fibroblast-collagen compaction in cultured canine basilar and human dermal fibroblasts using tyrosine kinase inhibitors and tyrosine antibodies. Hemolysate enhanced tyrosine phosphorylation of two proteins, 64 and 120 kDa, in cultured canine basilar artery and human dermal fibroblast cells. The effect of hemolysate was time-dependent and concentration-dependent. Oxyhemoglobin and ATP, the two major components of hemolysate, produced similar tyrosine phosphorylation, however, with a different time course. Tyrosine kinase inhibitors genistein and tyrphostin A51 abolished the effect of hemolysate in both cerebral and dermal fibroblasts. Hemolysate increased fibroblast-populated collagen-lattice compaction and tyrosine kinase inhibitors genistein and tyrphostin A51 attenuated the effect of hemolysate. We conclude that hemolysate activates tyrosine kinase that leads to the increase of fibroblast compaction. This effect of hemolysate may contribute to cerebral vasospasm.