Increased ADAMTS-13 proteolytic activity in rat hepatic stellate cells upon activation in vitro and in vivo.

INTRODUCTION: ADAMTS-13 is a member of A Disintegrin And Metalloprotease with ThromboSpondin type 1 repeats (ADAMTS) family, primarily synthesized in hepatic stellate cells (HSCs), one of the major cell types transdifferentiating into myofibroblasts during liver fibrosis. However, the association between ADAMTS-13 expression and HSC activation or liver fibrosis is not known. METHODS: In this study, we determined the ADAMTS-13 mRNA, protein, and activity in isolated primary HSCs upon activation on a plastic dish and in liver after administration of carbon tetrachloride (CCl(4)) in rats. RESULTS: We showed that ADAMTS-13 antigen and proteolytic activity in the activated rat HSCs were dramatically increased, whereas ADAMTS-13 mRNA in these cells was only minimally altered. Similarly, the ADAMTS-13 antigen and proteolytic activity in rat liver after CCl(4) injury were also significantly increased, whereas the ADAMTS-13 mRNAs in these liver tissues were only slightly increased compared with normal. Surprisingly, despite the dramatic up-regulation of ADAMTS-13 protein synthesis in the activated HSCs after CCl(4) administration, the plasma levels of ADAMTS-13 protease in rats did not increase concordantly. CONCLUSION: We conclude that the up-regulation of ADAMTS-13 protein expression in rat HSCs during activation in vitro and in vivo suggests the possibility of ADAMTS-13 proteolysis, an important part of function of the activated HSCs, perhaps through modulation of liver regeneration or formation of liver fibrosis after various injuries. The data also suggest the minimal contribution of the activated HSCs in regulation of plasma levels of ADAMTS-13 protease.

Acquired type 3-like von Willebrand syndrome preceded full-blown systemic lupus erythematosus.

We report a quite rare case of acquired type 3-like von Willebrand syndrome (vWS) that preceded full-blown systemic lupus erythematosus (SLE). A 16-year-old woman with no previous disease history and no family history of hemorrhagic diathesis was referred to our hospital because of recurrent epistaxis and gingival bleeding. She was diagnosed as having atypical type 3 von Willebrand disease because of prolonged bleeding time with normal platelet count and prolonged activated partial thromboplastin time (aPTT), and an almost complete absence of von Willebrand factor (vWF) antigen, ristocetin cofactor activity (vWF:RCo) and ristocetin-induced platelet agglutination (RIPA). Furthermore, electrophoretic analysis of plasma vWF revealed a trace amount of vWF and an absence of the multimeric form of vWF. Infusions of either vasopressin or factor VIII/vWF concentrates improved bleeding symptoms and corrected the aPTT and RIPA. However, she complained of low-grade fever, general fatigue and polyarthralgia 5 months later, and leukocytepenia and hypo-complementemia developed. Anti-double-stranded DNA antibodies and lupus erythematosus cells became positive. These findings were compatible with SLE. Mixing the patient's platelet-poor plasma (PPP) with normal platelet-rich plasma (PRP) (PPP/PRP = 2/1) resulted in a complete inhibition of RIPA, suggesting the presence of vWF inhibitor in her plasma. Treatment with prednisolone (40 mg/day) started and the bleeding tendency gradually improved. One month later, all of the laboratory data including aPTT, bleeding time, RIPA and vWF:RCo became normal. These findings indicate that she has an acquired type 3-like vWS associated with SLE.

[Transition of branched-chain amino acids and tyrosine ratio (BTR) in the blood of acute hepatitis patients].

The molar ratio of branched-chain amino acids to tyrosine (BTR) correlates well with the Fischer ratio, and can be measured in a short period of time. It is regarded as the method of analysis that will eventually replace the Fischer ratio. But clinical significance of BTR in terms of acute liver disorders has not been examined thoroughly as of yet. In this study, we measured BTR of 34 patients with acute hepatitis, and examined the transition of the acute period of acute hepatitis and its recovery process. Thirty-four patients diagnosed with acute viral hepatitis became subjects of examination (16 patients of A type, 15 patients of B type, 1 patient of C type, 2 patients of non-A, non-B, non-C type). Out of the 34 patients, 11 were in serious stages (HPT under 40%), including 3 in fulminant condition. By using preserved serum obtained during the acute period (within 1 week of the highest transaminase value), recovery period (within 4 weeks), and treatment period (3 months and later), measurements were conducted with Diacolor:BTR (enzymatic analysis, ONO Pharmaceutical Co., Ltd.), and the results were compared with those of 50 healthy subjects (25 men, 25 women). BTR correlated well with the Fischer ratio for chronic hepatic patients, and with albumin (Alb), PT, and ICGR15 as well, proving that it is useful as an indicator of hepatic reserve ability. But BTR has not been thoroughly examined as it relates to acute liver disorders. In this study, BTR fell in the acute period, correlating with the serious period, proving that it is a useful indicator. For acute liver damage, BTR supports conventional indicators (Alb, Ch-E, HGF, etc.) for assessing serious damage. Also, it has been suggested that measuring the passage of BTR could be the indicator of true recovery, including amino acid metabolism for liver disorders.

Massive culture of human liver cancer cells in a newly developed radial flow bioreactor system: ultrafine structure of functionally enhanced hepatocarcinoma cell lines.

With a view to initiating clinical trials, cell morphology and function for a newly developed artificial liver support system employing highly functional human liver cell line, FLC-7, cultured in a radial flow bioreactor were compared to cells grown in a conventional monolayer culture. The radial flow bioreactor consists of a vertically extended cylindrical matrix comprised of porous glass bead microcarriers through which liquid medium flows from the periphery in toward the central axis generating a beneficial concentration gradient of oxygen and nutrients, while preventing excessive shear stresses or buildup of waste products. The three-dimensional culture system supports high-density (1.1 x 10(8) cells/ml-matrix), large scale cultures (4.4 x 10(10) cells/400 ml-bioreactor) with long-term viability. Scanning and transmission electron microscopy (SEM and TEM) revealed that cells cultured in a monolayer system were flattened and extended with numerous cytoplasmic projections. Cells in the three-dimensional culture were spherical and covered with microvillilike processes resembling liver cells in vivo. The cells were solidly attached on the surfaces and within the pores of the microcarriers in highly dense colonies. The spherical cells remained in close contact with adjacent cells, while circulation of liquid medium flowed freely through spaces between cells. FLC-7 cells produced albumin at a rate of 6.41 micrograms/24 h/10(6) cells. Alpha-fetoprotein (AFP) production dropped nearly threefold in comparison to monolayer cultures. Results demonstrated that the new artificial liver support systems (ALSS) provides a superior three-dimensional culture environment that allows cells to perform at naturally functioning levels.

Hepatitis B virus variants in patients with acute hepatitis in whom various clinical forms develop.

Ten patients who suffered from acute hepatitis with various clinical forms due to hepatitis B virus (HBV) were studied. HBV variants with a mutation in the precore region were dominant in two patients with fulminant hepatitis and in a patient with the most severe acute hepatitis. However, these mutant viruses were not detected in a patient who had the fulminant form of acute HBV infection on chronic liver damage or in most patients who had severe acute hepatitis. Furthermore, mutant viruses were also not detected in a patient with complicating myopathy and in one who had an atypical clinical course with three transaminase peaks. These results suggest that precore mutants may be involved in the pathogenesis of some cases of severe acute hepatitis, the same as for fulminant hepatitis, but not in other clinical forms of acute hepatitis.

[Clinical significance of serum levels of human hepatocyte growth factor in patients with acute viral hepatitis].

We studied the relationship of serum levels of human hepatocyte growth factor (hHGF) to causative viruses and clinical features in 63 patients at our hospital with serologically diagnosed acute viral hepatitis. Serum levels of hHGF were not correlated with the type of hepatitis virus (A, B, and C) during the acute phase (p < 0.60) but were correlated with results of the hepaplastin test (p < 0.01). Furthermore, the difference in serum levels of hHGF between severe (levels on hepaplastin test < 40%) and nonsevere cases of hepatitis was significant (p < 0.001), and serum levels of hHGF became normal as levels of alanine aminotransferase decreased. However, serum levels of hHGF in prolonged cases of hepatitis (time until normalization of alanine aminotransferase > 13 weeks) tended to be slightly lower than in nonprolonged cases (p < 0.47). These results suggest that serum levels of hHGF are useful to determine the prognoses of patients with severe hepatitis and to estimate the time until liver damage heals.

[A new liver support system composed of functional human cells and a radial-flow bioreactor].

An artificial liver will be useful for the treatment of acute hepatic failure and a bridge of liver transplantation. The current reports suggest that the hybrid type of artificial liver composed of functional human liver cells and a bioreactor is practical for clinical use. In the present study, we succeeded high density culture on a large-scale of human functional hepatoma (JHH-7) using a newly developed radial flow packed-bed bioreactor. Since the shear stress of this bioreactor is lower than the other type, high density culture without cell damage is possible. JHH-7 cells produced large amounts of human albumin and other liver specific proteins, and then have the function of ammonia metabolism in the system. This study suggests that a radial flow bioreactor will be developed as a new type of artificial liver.

Regulation of vitamin A transport into cultured stellate cells of rat liver: studies by anchored cell analysis and sorting system.

Stellate cells (SC) in the liver store the most retinoid in the body, but the mechanisms of specific retinoid transport into SC remain to be elucidated. In this study, to analyze the retinoid content of cultured SC, we employed an anchored cell analysis and sorting system (ACAS), which provides fluorescence analysis of single cultured cells under the phase-contrast microscope by utilizing a laser. First, we examined the effect of retinol binding protein (RBP) on retinol transport into cultured SC of rat liver. Rat holo-RBP added to the medium inhibited retinol uptake into SC. We also prepared RBP-free human serum by affinity chromatography using conjugated anti-human RBP IgG and compared retinoid fluorescence of SC cultured in human serum with or without RBP. No significant difference in retinoid fluorescence intensity was observed between SC cultured with and without holo-RBP. Second, the removal of cellular retinol by esterification may be important for the continued uptake of retinol. Retinyl esters are stored in lipid droplets of SC. Therefore we examined the relationship between the lipid droplet number and the retinoid fluorescence intensity in SC which were cultured in medium containing retinol for 1-3 days. The increases in lipid droplet number and in retinoid fluorescence in SC were almost parallel. Progesterone, previously shown to increase the esterification of retinol by lecithin:retinol acyltransferase (LRAT) in vitro, was added to the SC medium; progesterone facilitated retinol uptake in cultured SC. In conclusion, RBP did not facilitate specific retinol transport into SC. However, the specific transport of retinol is likely to be dependent on the intracellular esterification of retinol by LRAT in SC.

Retinol transport in cultured stellate cells of rat liver: studies by light and electron microscope autoradiography.

The mechanisms of specific transport of retinoids into stellate cells (SC) of liver remain to be elucidated. In the present study, we have conducted experiments to observe the intracellular retinoid metabolism of cultured SC of rat liver using light and electron microscope autoradiography (LMARG and EMARG). After 72 h of culture, the cells were incubated in the medium containing 1.06 x 10(-6) or 6.36 x 10(-8) M of [3H]retinol for either 5 or 30 min. In some cases, incubation with the labeled retinol was followed by a medium containing nonlabeled 1 x 10(-6) M retinol for 90 min. First, the incorporation of labeled retinol (1.06 x 10(-6) M) into SC and hepatocytes was compared by LMARG. After 5 min, silver grains were already present on both cells. After 30 min, label was concentrated on the lipid droplets of SC. After the chase, the number of grains on hepatocytes decreased. On the other hand, grains on the lipid droplets of SC remained. Second, we studied the fine morphology and intracellular retinoid metabolism in SC using EMARG. The SC, which contained abundant multivesicular bodies (MVB) and lamellar bodies, were found to have a heavy accumulation of grains. Even in the medium containing a lower concentration of retinol (6.36 x 10(-8) M), SC also took up retinol. After 90 min of chase, many grains moved on the lipid droplets in SC. The labeled MVB were often accompanied by lamellar bodies and found near the lipid droplets. Sometimes we noticed small labeled lipid droplets bound by membranes in MVB. From the results of this study, we concluded that the MVB and lamellar bodies might be important organelles for retinyl ester formation and the initial storage of retinoid in the lipid droplets in SC.

[Consideration on the mechanism of liver regeneration on experimental results].

Liver has enough functional capacity and regeneration ability. Liver parenchymal cells are usually stable, and it is thought being at stage of Go in cell cycle. But liver cells are easily into progressive stage and the liver recovers its functions and volume after partial hepatectomy or liver injury. Usually the studies of liver regeneration are done by studying control of growth of isolated hepatocytes in primary culture in vitro, and by pathological considerations of experimental injured models of liver in vivo. In this paper, we considered known several hepatotrophic factors including our experimental results, and regeneration mechanisms by noticing appearance of albumin positive hepatocytes in injured Nagase analbuminemic rat (NAR).

Characteristics and significance of albumin-positive hepatocytes in analbuminemic rats.

Analbuminemic rats (NAR) are a mutant strain in which splicing of the albumin mRNA is blocked due to a seven-base-pair deletion in an intron of the albumin gene. NAR liver contains a few hepatocytes that react with anti-rat albumin antibody (Alb+ hepatocytes), and these cells increase in number during aging and on treatment with hepatocarcinogens. To characterize these Alb+ hepatocytes, we examine their albumin mRNA, the biochemical specificity of their albumin, and its intracellular distribution. Signals of albumin mRNA were observed in a few hepatocytes by in situ hybridization. Moreover, a small amount of cytoplasmic albumin mRNA was detected by RNA blot analysis in the liver of aged NAR and NAR treated with 3'-methyl-4-diaminoazobenzene (DAB). Immunoelectron microscopic examination revealed the cisternae of the rough and smooth endoplasmic reticula, Golgi complexes, and secretory vesicles of the Alb+ hepatocyte of NAR being filled with material that reacted with anti-rat albumin antibody. These facts suggested that albumin was gradually synthesized in Alb+ hepatocytes but that its secretion was disturbed. The albumin-like proteins of NAR were shown by Western blot analysis to consist of three species of 68 kDa, 50 kDa, and 25 kDa proteins. The 50 kDa albumin was thought to be formed by exon-skipping splicing of the albumin mRNA precursor, which was recently reported by Shalaby and Shafritz (Proc. Natl. Acad. Sci. USA 87, 2652-2656 (1990)). The 25 kDa protein was suspected to be formed by fragmentation of the 50 kDa protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Integration of hepatitis B virus DNA into cells of six established human hepatocellular carcinoma cell lines.

The authors successfully established 5 hepatocellular carcinoma cell lines, JHH-1, 2, 4, 5 and 6, derived from hepatitis B surface antigen seronegative patients and one line. JHH-7, from a patient considered to be a hepatitis B virus carrier. In the culture media of any of the JHH cell lines, including JHH-7, hepatitis B surface antigen was not detected by radioimmunoassay. However, in JHH-7, integration of hepatitis B virus DNA was confirmed at two sites on the chromosomes of this line by Southern blot hybridization. In contrast, in other JHH cell lines derived from hepatitis B surface antigen seronegative patients, integration of hepatitis B virus DNA into the chromosomes of cells was not detected. These cell lines will be useful in the investigation of hepatocellular carcinoma development, especially for research into non-A/non-B hepatitis viruses.

[Analysis for the integrated hepatitis B virus genome in cells of established human hepatocellular carcinoma cell line JHH-7].

Authors have successfully established 7 human hepatocellular carcinoma (HCC) cell lines. In one of these cell lines, JHH-7, derived from HBs antigen positive HCC, hepatitis B virus (HBV) genome was analyzed by Southern blot hybridization. Digesting with Hind III restriction endonuclease, two bands were obtained at 6.0 and 2.5 kb, that is the integration of HBV genome was confirmed at the two sites of chromosomes in this cell line. And analyzing with 32P labeled HBV-DNA fragments for probes, it was indicated that integrated HBV genome was incomplete one containing from X to C region.