A clinical study of Japanese patients with ulcer induced by low-dose aspirin and other non-steroidal anti-inflammatory drugs.

BACKGROUND: The incidence and severity of non-steroidal anti-inflammatory drugs (NSAIDs)-induced gastro-duodenal ulcer have not been extensively studied in Japan. AIM: We performed a prospective study to clarify NSAIDs-induced gastro-duodenal injury, focusing especially on low-dose aspirin (L-A). METHODS: Two hundred and thirty-eight patients with bleeding peptic ulcers admitted to our hospital. History of taking NSAIDs and anti-ulcer drugs was obtained from all patients who underwent endoscopic examinations. The lesion scores of patients taking L-A were classified numerically from zero (no lesion) to five (ulcer). RESULTS: The NSAIDs were associated with 28.2% of hemorrhagic ulcers. The rates of patients using L-A, loxoprofen, diclofenac, and combination of two of these drugs were 27, 16, 10 and 9%, respectively. Co-administered anti-ulcer drugs were cytoprotective anti-ulcer drugs (27%), H2 receptor antagonists (16%), PPI (4%), and none (53%). In patients taking L-A, H2 receptor antagonists were used most frequently. The HP was positive in 63% of L-A-induced ulcer cases and in 69% of NSAIDs other than low-dose aspirin-induced ulcer cases. The lesion scores of patients taking L-A with H2 receptor antagonists or PPI were significantly lower than those of patients who were taking only L-A (P < 0.05). CONCLUSIONS: Approximately one-third of hospitalized patients with NSAIDs-induced hemorrhagic ulcer showed an association with L-A. Prospective randomized controlled trials including H2 receptor antagonists are required to establish preventive efforts aimed at L-A-induced gastro-duodenal injury.

Hepatic stellate cells express Ca2+ pump-ATPase and Ca2+-Mg2+-ATPase in plasma membrane of caveolae.

Intracytoplasmic free calcium ions (Ca2+) are maintained at a very low concentration in mammalian tissue by the extrusion of Ca2+ across a steep extracellular Ca2+ gradient, mainly through the activity of plasma membrane Ca2+ pump-ATPase. The present study aimed to identify, by electron cytochemical and electron immunogold methods, the ultrastructural localizations of two types of plasma membrane Ca2+-ATPase; Ca2+-Mg2+-ATPase and Ca2+ pump-ATPase, in hepatic stellate cells. Liver tissues and isolated hepatic stellate cells (HSCs) were studied. The ultrastructural localization of Ca2+-Mg2+-ATPase activity was examined by the electron cytochemical method of Ando. The localization of Ca2+ pump-ATPase was identified by immunofluorescence. The ultrastructural localization of Ca2+ pump-ATPase was identified by the electron immunogold method. The cytochemical reaction products of Ca2+-Mg2+-ATPase activity were localized on the outer (cavity) side of the plasma membrane of caveolae. Immunofluorescence of Ca2+ pump-ATPase was seen as small dots along the cell edge in HSCs. Immunogold particles indicating the presence of Ca2+ pump-ATPase were identified on the inner (cytoplasmic) side of the plasma membrane of caveolae. We localized Ca2+ pump-ATPase on the inner side of the plasma membrane caveolae and Ca2+-Mg2+-ATPase on the outer leaflet of the caveolar plasma membrane in stellate cells, suggesting that Ca2+ pump-ATPase may play a key role in the Ca2+ reflux.

Increased expression of osteopontin in activated Kupffer cells and hepatic macrophages during macrophage migration in Propionibacterium acnes-treated rat liver.

Osteopontin is an extracellular matrix component that can act as a chemokine to induce macrophage migration. The significance of osteopontin in macrophage infiltration into the liver was examined in rats given heat-killed Propionibacterium acnes. In normal rats, osteopontin mRNA expression in the liver was minimal, determined by quantitative-competitive reverse transcription-polymerase chain reaction (RT-PCR) assay. Northern blot analysis revealed that osteopontin mRNA was not expressed in Kupffer cells isolated from normal rats. When rats received heat-killed P. acnes intravenously, marked macrophage accumulation, forming granulomas, developed in the liver later than 3 days after the injection and its extent became maximal between 5 and 7 days. In these rats, osteopontin mRNA expression was increased in the liver later than 1 day (with its peak at 3 days after the injection), and the mRNA expression was increased markedly in Kupffer cells and hepatic macrophages isolated at 7 days. The mRNA expression of monocyte chemotactic protein-1 (MCP-1) and macrophage inflammatory protein-1alpha (MIP-1alpha), chemokines for monocytes and macrophages, was also increased in the liver of P. acnes-treated rats, with peak expression at 3 days. We conclude that osteopontin derived from Kupffer cells and hepatic macrophages may contribute to the infiltration of monocytes and macrophages into the liver cooperatively with the actions of MCP-1 and MIP-1alpha in P. acnes-treated rats.

Effects of neurotropic pyrimidine heterocyclic compound, MS-430, on cultured hepatic parenchymal and stellate cells.

MS-430 is a novel synthetic pyrimidine derivative that stimulates regeneration of the nerve as a promoter for various growth factors such as epidermal growth factor (EGF) and nerve growth factor, and differentiation of astrocytes. The effects of MS-430 on the liver were tested using hepatocytes and stellate cells in primary culture isolated from rats. MS-430 enhanced EGF-induced DNA synthesis in hepatocytes while it alone failed to increase the basal DNA synthesis. Albumin mRNA expression in the cells and its amount in the medium were not changed by addition of EGF or MS-430 alone or both. Basic fibroblast growth factor (bFGF) increased DNA and but not collagen synthesis by hepatic stellate cells. Addition of MS-430 inhibited DNA synthesis by hepatic stellate cells at either presence or absence of bFGF, and collagen synthesis at the presence of bFGF. However, MS-430 had no effects on basal or bFGF-stimulated TGFbeta mRNA expression in the cells. These results suggest that MS-430 stimulated proliferation of hepatocytes as a comitogen for EGF without affecting albumin synthesis, and suppressed proliferation of activated hepatic stellate cells and their collagen synthesis without affecting TGFbeta expression.

Inhibitory effect of tranilast on activation and transforming growth factor beta 1 expression in cultured rat stellate cells.

Stellate cells, the primary extracellular matrix-producing cells in the liver, undergo activation characterized by fibrogenesis, proliferation and smooth muscle alpha-actin expression, in hepatic fibrosis or when cultured on plastic. TGF beta 1 is known to have a pivotal role in fibrogenesis. Tranilast, a drug used for allergic diseases with anti-inflammatory effects, is known to inhibit collagen synthesis by cultured fibroblasts. Thus, effects of tranilast on activation and TGF beta 1 expression in stellate cells was investigated in vitro. Tranilast reduced collagen synthesis in a dose-related manner up to 50.8% of the control. This effect was reversible after tranilast withdrawal. The mobility of procollagen on gel electrophoresis and the ratio of intracellular procollagen to extracellular collagen concentrations were not affected by tranilast. Tranilast decreased DNA synthesis and increased smooth muscle alpha-actin expression. mRNA expressions of procollagen and TGF beta 1 were reduced by tranilast. Tranilast with anti-fibrogenic and anti-inflammatory actions merits consideration as a candidate for therapeutic agent of hepatic fibrosis.

Increased expressions of vascular endothelial growth factor and its receptors, flt-1 and KDR/flk-1, in regenerating rat liver.

Sinusoidal endothelial cells proliferate following hepatocyte regeneration in the liver after partial resection. The expressions of vascular endothelial growth factor (VEGF) and its receptors, flt-1 and KDR/flk-1, were studied by Northern blotting in isolated rat liver cells and 70% resected rat liver. VEGF was expressed in hepatocytes immediately after isolation, and both flt-1 and KDR/flk-1 were expressed in non-parenchymal cells including sinusoidal endothelial cells. The VEGF expression in hepatocytes decreased during primary culture for 48 hr. This expression was maintained at 48 hr of culture by addition of EGF to the medium at 24 hr, increasing thereafter. VEGF, flt-1, and KDR/flk-1 were also expressed in normal rat liver. In 70% resected rat liver, VEGF expression increased with a peak at 72 hr after the operation, followed by expressions of flt-1 and KDR/flk-1 increasing between 72 and 168 hr. These results suggest that VEGF expression increases in regenerating hepatocytes, which may contribute to proliferation of sinusoidal endothelial cells of rat liver following partial resection, probably through flt-1 and KDR/flk-1 receptors upregulated on sinusoidal endothelial cells.

VEGF can act as vascular permeability factor in the hepatic sinusoids through upregulation of porosity of endothelial cells.

VEGF is shown to be a vascular permeability factor (VPF) as well as a growth stimulatory factor on endothelial cells. In the hepatic sinusoids, endothelial cells express flt-1 and KDR/flk-1, receptors for VEGF. These cells, in primary culture, proliferate in response to VEGF stimulation. However, the role of VEGF as VPF in the hepatic sinusoids is to be elucidated. The effect of VEGF on the porosity of sinusoidal endothelial cells was studied. Sinusoidal endothelial cells were isolated from rats and cultured in DMEM containing 10% FCS on plastic dishes coated with type I collagen for 16 and 48 h for morphological examination and cell-number measurement, respectively. When the cells were cultured without VEGF addition, their number was decreased at 48 h compared to that at 16 h. However, the number was unchanged in the cells cultured with VEGF at 10 ng/mL and increased with addition of VEGF at 100 ng/mL. Scanning electron microscopic examination revealed that sieve-plate appearance of the cells was impaired in culture with no VEGF addition, but the appearance was maintained in culture with VEGF at 10 ng/mL or more. The cells cultured with VEGF at 100 ng/mL showed significantly increased number and size of pores compared to the cells cultured with VEGF at 10 ng/mL, suggesting that sinusoidal endothelial cells proliferating in response to VEGF may increase their porosity. It is concluded that VEGF can act as VPF in the hepatic sinusoids through regulation of endothelial cell porosity.

Apoptosis in dissociation between DNA synthesis and cellular functions of activated hepatic stellate cells--a study with carbon tetrachloride-induced rat liver injury.

It is widely believed that DNA synthesis and expressions of smooth muscle alpha actin and TGF-beta are all together increased in activated hepatic stellate cells both in vitro and in vivo. Our previous reports disclosed that these increases did not always coexist under experimental conditions. Liver necrosis was induced in rats by oral administration of carbon tetrachloride. Hepatic stellate cells were isolated from these rats 2 days later. When these cells were cultured on plastic dishes for 3 days, they showed marked DNA synthesis and smooth muscle alpha actin and TGF-beta mRNA expressions assessed by (3)H-thymidine incorporation and Northern blotting, respectively. In the cells further cultured for 7 days, the DNA synthesis was decreased, whereas both smooth muscle alpha actin and TGF-beta mRNA expressions were increased, compared to the cells cultured for 3 days. The cells cultured for 10 days showed apoptotic nuclei positive for nick-end labeling, and DNA extracted from the cells revealed laddering patterns on agarose gels by electrophoresis. Apoptotic nuclei were also immunohistochemically found in stellate cells in the liver of rats 4 days after the intoxication. We conclude that apoptosis developed in activated hepatic stellate cells both in vitro and in vivo, and this may contribute to the discrepancy between DNA synthesis and cellular functions of the cells.

The mechanisms of hepatic sinusoidal endothelial cell regeneration: a possible communication system associated with vascular endothelial growth factor in liver cells.

Vascular endothelial growth factor (VEGF) has been shown to induce proliferation of sinusoidal endothelial cells in primary culture. To elucidate the mechanisms of sinusoidal endothelial cell regeneration in vivo, mRNA expression of VEGF and its receptors, flt-1 and KDR/flk-1, were studied in rat livers. Northern blot analysis revealed that VEGF-mRNA was expressed in hepatocytes immediately after isolation from normal rats. In contrast, non-parenchymal cells, including sinusoidal endothelial cells, expressed VEGF receptor-mRNA. Vascular endothelial growth factor-mRNA expression in hepatocytes was decreased during primary culture, but increased following a peak of DNA synthesis, induced by addition of epidermal growth factor or hepatocyte growth factor to the culture medium at 24 h of plating. In a 70% resected rat liver, VEGF-mRNA expression increased with a peak at 72 h after the operation, and mRNA expression of VEGF receptors between 72 and 168 h. In such a liver, mitosis was maximal in hepatocytes at 36 h and in sinusoidal endothelial cells at 96 h. Also, mRNA expression of both VEGF and its receptors was significantly increased in carbon tetrachloride-intoxicated rat liver compared with normal rat liver. Vascular endothelial growth factor expression was minimal in Kupffer cells isolated from normal rats, but marked in activated Kupffer cells and hepatic macrophages from the intoxicated rats. Vascular endothelial growth factor-mRNA expression was also increased in activated stellate cells from these rats and in the cells activated during primary culture compared with quiescent cells. We conclude that increased levels of VEGF expression in regenerating hepatocytes may contribute to the proliferation of sinusoidal endothelial cells in partially resected rat liver, probably through VEGF receptors up-regulated on the cells. Also, VEGF derived from activated Kupffer cells, hepatic macrophages and stellate cells may be involved in this proliferation in injured rat liver.

Expressions of vascular endothelial growth factor in nonparenchymal as well as parenchymal cells in rat liver after necrosis.

Vascular endothelial growth factor (VEGF) can induce proliferation of sinusoidal endothelial cells. Its mRNA expression was increased in proliferating rat hepatocytes in primary culture. To clarify a role of VEGF in liver after necrosis, expressions of VEGF and its receptors were measured in the liver or liver cells isolated from rats after carbon tetrachloride intoxication. Hepatic VEGF mRNA expression increased later than 24 h after the intoxication and became prominent at 168 h when liver necrosis disappeared, while hepatic mRNA expressions of its receptors increased between 24 and 72 h. VEGF mRNA expression was increased in Kupffer cells, hepatic macrophages and stellate cells isolated from rats between 24 and 72 h after the intoxication and in hepatocytes at 168 h compared to those cells from normal rats. Immunohistochemical VEGF stains were comparable to such results. Vascular endothelial cells existed abundantly in the necrotic areas, and sinusoidal endothelial cells appeared following disappearance of the necrotic areas. VEGF mRNA expression in hepatocytes isolated from 70% resected liver was increased at 12 h after the operation and became marked between 72 and 168 h. Similar increase of hepatic VEGF expression was immunohistochemically seen. In conclusion, VEGF derives from nonparenchymal as well as parenchymal cells in rat liver after necrosis. The former might contribute to vascular endothelial cell proliferation and the latter to sinusoidal endothelial cell regeneration.

Inhibition of hepatic stellate cell contraction during activation in vitro by vascular endothelial growth factor in association with upregulation of FLT tyrosine kinase receptor family, FLT-1.

Activated hepatic stellate cells produce vascular endothelial growth factor (VEGF). VEGF has been shown to act on mesenchymal cells as well. If hepatic stellate cells can express FLT tyrosine receptor family, flt-1 and KDR/flk-1, their function might be regulated by VEGF in an autocrine manner. This hypothesis was tested using hepatic stellate cells isolated from normal rats. Northern blot analysis and immunocytochemical study revealed that hepatic stellate cells cultured for 3 days on plastic dishes expressed both flt-1 and KDR/flk-1. When the culture was prolonged to 10 days, the flt-1 mRNA expression was increased, whereas both KDR/flk-1 mRNA and protein expressions diminished. DNA and collagen syntheses were minimal in the cells cultured for 3 days, but marked in those cultured for 10 days. Addition of recombinant human VEGF to the culture medium did not change both syntheses but attenuated an increase of smooth muscle alpha-actin expression in the cells during culture on plastic dishes and also contraction of collagen gels on which the cells were cultured. We conclude that VEGF may inhibit contraction of hepatic stellate cells appearing during activation by culture, probably through attenuation of smooth muscle alpha-actin expression via upregulated VEGF receptor, flt-1.

Expression of osteopontin in Kupffer cells and hepatic macrophages and Stellate cells in rat liver after carbon tetrachloride intoxication: a possible factor for macrophage migration into hepatic necrotic areas.

Activated Kupffer cells and macrophages accumulate in necrotic areas in the liver. Osteopontin, an extracellular matrix with RGD sequence, has been shown to act as a chemokine that can induce monocyte migration. The possibility that osteopontin can play a role in infiltration of both cells into hepatic necrotic areas was investigated in rats. Northern blot analysis revealed that osteopontin mRNA expression was minimal in Kupffer cells and hepatocytes immediately after isolation from normal rats, but slight in hepatic stellate cells assumed nearly quiescent in function after 3 days of culture on plastic dishes. When rat received carbon tetrachloride, liver necrosis developed between 1 and 3 days following the intoxication. In these rats, osteopontin mRNA expression assessed by quantitative competitive RT-PCR was increased in the liver later than 1 day with its peak at 2 days following the intoxication. Kupffer cells and hepatic macrophages and hepatic stellate cells isolated from such liver showed marked expression of osteopontin mRNA on Northern blotting. Immunohistochemical examination disclosed that osteopontin was stained in macrophages including Kupffer cells and stellate cells in the necrotic areas. On electron microscopy, osteopontin stains were present in the Golgi apparatus in these cells. Recombinant human osteopontin promoted migration of Kupffer cells isolated from normal rats and cultured in a Transwell cell culture chamber in a dose-related manner. We conclude that activated Kupffer cells and hepatic macrophages and stellate cells express osteopontin. These cells might contribute to the infiltration of Kupffer cells and macrophages into hepatic necrotic areas by expressing osteopontin.