Teruaki Oka

Increased expression of vascular endothelial growth factor in human hepatocellular carcinoma

BACKGROUND/AIMS Angiogenesis is critical for the development and progression of solid tumors. The purpose of this study was to evaluate the possible role of vascular endothelial growth factor (which is considered to be one of the most important factors involved in tumor-associated angiogenesis), in human hepatocellular carcinoma. METHODS Vascular endothelial growth factor gene and protein expression were analyzed by means of Northern hybridization and immunohistochemical methods in 5 hepatocellular carcinoma cell lines. Secretion of vascular endothelial growth factor was evaluated by immunoblotting of conditioned medium of these hepatocellular carcinoma cells. Further, we compared the level of vascular endothelial growth factor expression in hepatocellular carcinoma tissues along with that in surrounding tumor-free tissues obtained from 20 patients. We also analyzed mRNA expression of Flt-1, one of the vascular endothelial growth factor specific high-affinity receptors, in hepatocellular carcinoma cell lines. RESULTS Northern hybridization analysis and immunohistochemistry revealed that all cultured hepatocellular carcinoma cells exhibited a high level of vascular endothelial growth factor mRNA. In addition, vascular endothelial growth factor secretion by Hep G2, one of the hepatocellular carcinoma cell lines, was shown by Western blot. In vivo, we observed vascular endothelial growth factor expression in both hepatocellular carcinoma and non-hepatocellular carcinoma tissues. However, in 12 of 20 cases, vascular endothelial growth factor mRNA levels were significantly up-regulated in hepatocellular carcinoma tissues. In the majority of cases (10 out of 12 cases) with abundant tumor vascularity, vascular endothelial growth factor mRNA up-regulation in hepatocellular carcinoma tissues was observed. We failed to detect Flt-1 mRNA expression in hepatocellular carcinoma cells. CONCLUSIONS This study suggests that the possibility that hepatocellular carcinoma cells overexpress the vascular endothelial growth factor gene and protein. These findings support the hypothesis that vascular endothelial growth factor is one of the important factors involved in the angiogenesis of hepatocellular carcinoma, and may even be involved in the development and/or progression of hepatocellular carcinoma itself.

Embryonic Lethality and Defective Neural Tube Closure in Mice Lacking Squalene Synthase

Squalene synthase (SS) catalyzes the reductive head-to-head condensation of two molecules of farnesyl diphosphate to form squalene, the first specific intermediate in the cholesterol biosynthetic pathway. We used gene targeting to knock out the mouse SS gene. The mice heterozygous for the mutation (SS+/−) were apparently normal. SS+/− mice showed 60% reduction in the hepatic mRNA levels of SS compared with SS+/+ mice. Consistently, the SS enzymatic activities were reduced by 50% in the liver and testis. Nevertheless, the hepatic cholesterol synthesis was not different between SS+/− and SS+/+ mice, and plasma lipoprotein profiles were not different irrespective of the presence of the low density lipoprotein receptor, indicating that SS is not a rate-limiting enzyme in the cholesterol biosynthetic pathway. The mice homozygous for the disrupted SS gene (SS−/−) were embryonic lethal around midgestation. E9.5–10.5 SS−/−embryos exhibited severe growth retardation and defective neural tube closure. The lethal phenotype was not rescued by supplementing the dams either with dietary squalene or cholesterol. We speculate that cholesterol is required for the development, particularly of the nervous system, and that the chorioallantoic circulatory system is not mature enough to supply the rapidly growing embryos with maternal cholesterol at this developmental stage.

Macrophage colony stimulating factor prevents the progression of atherosclerosis in Watanabe heritable hyperlipidemic rabbits

The early atherosclerotic lesion is characterized by the presence of macrophage-derived foam cells. Macrophage colony stimulating factor (M-CSF) specifically stimulates the functions of the monocyte-macrophages. To elucidate the effects of M-CSF in the atherogenic process in vivo, we administered human recombinant M-CSF into Watanabe heritable hyperlipidemic (WHHL) rabbits, an animal model for familial hypercholesterolemia. Three hundred micrograms of M-CSF were intravenously injected into WHHL rabbits aged 2.5 months, three times a week for 8.5 months. After the M-CSF treatment, we found very retarded progression of atherosclerosis. The accumulation of cholesterol ester was remarkably decreased in the aortae of M-CSF-treated animals (0.60 +/- 0.32 mg/g tissue), as compared to those of controls (4.32 +/- 0.61 mg/g tissue). Furthermore, the percentage of the surface area of the aorta with macroscopic plaque in animals treated with M-CSF was 14.3 +/- 6.2%, much less than that in controls receiving saline injection (38.8 +/- 8.0%). Thus, M-CSF definitely prevented the progression of atherosclerosis in WHHL rabbits by influencing macrophage functions.

Advanced glycation end products in human optic nerve head

AIMS To localise advanced glycation end products (AGEs) in human optic nerve head. METHODS Optic nerve samples from 13 elderly individuals (seven diabetics and six non-diabetics) were obtained at necropsy. Pyrraline, an advanced glycation end product, was immunohistochemically localised in the optic nerve heads. RESULTS In the diabetic subjects, moderate to intense immunoreactivity for pyrraline was detected in sclera, pia mater, cribriform plates, connective tissues in the optic nerve, and around vessels in the optic nerve and pia mater. Immunoreactivity for pyrraline was also detected around retinal vessels. In the non-diabetic subjects, slight or no immunoreactivity for pyrraline was found in cribriform plates and around the optic nerve vessels. CONCLUSION Accumulation of AGEs in cribriform plates and around vessels in the optic nerve may contribute to the development of optic neuropathy in diabetic patients.

Bladder tumor producing granulocyte colony-stimulating factor and parathyroid hormone related protein.

A 51-year-old man with gross hematuria and fever was admitted to our hospital for evaluation. Computerized tomography of the pelvis demonstrated a 6.8 cm. heterogeneous enhancing mass in the bladder that was suggestive of tumor. Laboratory testing showed marked leukocytosis (30,200/mm., normal 4,000 to 7,000) and hypercalcemia (11.2 mg./dl., normal 8.4 to 9.7). However, no evidence of foci of infection was detected. Two sessions of transurethral resection of bladder tumor were performed. Following resection leukocytosis and hypercalcemia resolved completely. Pathological findings for specimens obtained from resection revealed transitional cell carcinoma, G3, mixed with squamous cell carcinoma. After intra-arterial injection of cisplatin and pirarubicin we performed total cystectomy and ileal neobladder. Serum levels of G-CSF and PTH related protein were elevated preoperatively (G-CSF 98.3 pg./ml., normal 3.7 to 32.3; PTH related protein 2.0 pmol./l., normal less than 1.1). Immunohistochemical testing for anti-G-CSF and anti-PTH related protein revealed positive staining for both substances in paraffin embedded specimens from the bladder tumor (see figure). Serum levels of G-CSF and PTH related protein returned to normal postoperatively. No evidence of tumor recurrence was seen during 40-month followup. DISCUSSION

Churg-Strauss syndrome (allergic granulomatous antiitis) with multiple perforating ulcers of the small intestine, multiple ulcers of the colon, and mononeuritis multiplex

A case of Churg-Strauss syndrome with multiple perforations of the small intestine is described. A 31-year-old woman was admitted with a complaint of epigastric pain. She had a history of bronchial asthma. One week before admission, white blood cell count was 20 800/mm3 with 59% eosinophils. Neurological examination on admission disclosed mononeuritis multiplex with paresthesia in both the lower and upper extremities. At colonoscopy, there were scattered aphthous ulcers in the colon. Ophthalmological examination revealed allergic conjunctivitis. After admission, hypereosinophilia increased to as high as 36 000/mm3. Oral administration of prednisolone (60 mg/day) was begun. On the 3rd day of the treatment, the eosinophil count decreased dramatically, to 400/mm3, while severe abdominal pain developed. Since abdominal X-ray film revealed free air in the abdominal cavity, emergency laparotomy was performed and multiple intestinal ulcers with perforations were found. Partial ileectomy was performed. Pathological findings of the resected specimen were interpreted as a necrotizing angiitis with extravascular granuloma. Since the operation, the patient has been asymptomatic, except for neurological symptoms. Hypereosinophilia has decreased without treatment to counts averaging 270/mm3, within 3 months. On the basis of the clinical features and histopathological findings, a diagnosis of Churg-Strauss syndrome was established.

Multiple small intestinal stromal tumors with skeinoid fibers in association with neurofibromatosis 1 (von Recklinghausen's disease)

A case of multiple small Intestinal stromal tumors (SIST) with skeinoid fibers of the jejunum arising in a 50 year old male with neurofibromatosis 1 (NF‐1) is reported. Seven small tumors of the jejunal wall were incidentally found and excised during an operation for abdominal and retroperitoneal neuroflbromas. Histologlcally, the tumors were composed of uniform spindle‐shaped cells with fascicular pattern, almost indistinguishable from the histology in leiomyoma. Periodic acid Schiff stain‐positive hyaline globules were observed among the tumor cells. Ultrastructurally, these globules were stromal tangles of curvilinear, fluffy fibrils, consistent with skeinoid fibers. The electron‐dense granules, possibly neurosecretory granules, were found In the cytoplasm of the tumor cells. Immunohistochemically, the tumor cells were positive for vimentin, neuron specific enolase and CD34, but negative for muscle markers and S100 protein. The association of NF‐1 and multiple SIST with skeinoid fibers may have clinical implications. The multiple occurrence of SIST with skeinoid fibers seems to be often cited as one of the gastrointestinal manifestations of NF‐1. The possible site of origin of SIST with skeinoid fibers in NF‐1 may be the enteric autonomlc nerve plexus in the small intestinal wall.

Serial Histopathological Examination of the Lungs of Mice Infected with Influenza A Virus PR8 Strain

Avian influenza H5N1 and pandemic (H1N1) 2009 viruses are known to induce viral pneumonia and subsequent acute respiratory distress syndrome (ARDS) with diffuse alveolar damage (DAD). The mortality rate of ARDS/DAD is extremely high, at approximately 60%, and no effective treatment for ARDS/DAD has been established. We examined serial pathological changes in the lungs of mice infected with influenza virus to determine the progress from viral pneumonia to ARDS/DAD. Mice were intranasally infected with influenza A/Puerto Rico/8/34 (PR8) virus, and their lungs were examined both macro- and micro-pathologically every 2 days. We also evaluated general condition, survival rate, body weight, viral loads in lung, and surfactant proteins in serum. As a result, all infected mice died within 9 days postinfection. At 2 days postinfection, inflammation in alveolar septa, i.e., interstitial pneumonia, was observed around bronchioles. From 4 to 6 days postinfection, interstitial pneumonia with alveolar collapse expanded throughout the lungs. From 6 to 9 days postinfection, DAD with severe alveolar collapse was observed in the lungs of all of dying and dead mice. In contrast, DAD was not observed in the live infected-mice from 2 to 6 days postinfection, despite their poor general condition. In addition, histopathological analysis was performed in mice infected with a dose of PR8 virus which was 50% of the lethal dose for mice in the 20-day observation period. DAD with alveolar collapse was observed in all dead mice. However, in the surviving mice, instead of DAD, glandular metaplasia was broadly observed in their lungs. The present study indicates that DAD with severe alveolar collapse is associated with death in this mouse infection model of influenza virus. Inhibition of the development of DAD with alveolar collapse may decrease the mortality rate in severe viral pneumonia caused by influenza virus infection.

Interleukin 6B cell stimulatory factor-II is expressed and released by normal and transformed human bronchial epithelial cells

Airway epithelial cells have a potential to participate in local immune and inflammatory responses via releasing biologically active compounds. We studied the expression and release of interleukin 6 (IL-6), a multifunctional cytokine possibly involved in tissue immune responses. Primary culture of normal human bronchial epithelial cells and its transformed cell line BEAS-2B released significant amount of biologically and immunologically intact IL-6 into media. A protein synthesis inhibitor cycloheximide abolished the IL-6 release, suggesting a de novo synthesis. Northern blot analysis demonstrated the expression of the specific IL-6 mRNA. Human bronchial epithelial cells can produce IL-6 and contribute to the local activity of IL-6, suggesting that these cells may play a role in the regulation of airway immune responses.

Parachordoma : an ultrastructural and immunohistochemical study

A case of parachordoma of the left calf in a 19-year-old Chinese female is reported. The tumour showed multinodular growth pattern and consisted of round or oval tumour cells with abundant eosinophilic cytoplasm and myxoid matrix. Tumour cells formed small nests and sometimes showed concentric arrangement. Physaliferous-like cells and undifferentiated spindle cells were occasionally observed among the cell nests. The myxoid matrix was positive for high-iron diamine stain, indicating the presence of chondroitin 4- and 6- sulphates and keratan sulphate. Ultrastructurally, well-developed rough endoplasmic reticulum, abundant intermediate filaments, microvillous cytoplasmic processes, pinocytic vesicles, and desmosome-like junctional structures were found. Tumour cells were positive for S-100 protein and vimentin, but negative for cytokeratin, epithelial membrane antigen, carcinoembryonic antigen, and desmin. These results are consistent with the definition of parachordoma as a soft tissue neoplasm consisting of cells with histology and ultrastructure similar to those of chordoma cells but with immunohistochemistry similar to that of chondroid tumour cells.