Koji Miyanishi

Resolution of liver cirrhosis using vitamin A-coupled liposomes to deliver siRNA against a collagen-specific chaperone

There are currently no approved antifibrotic therapies for liver cirrhosis. We used vitamin A–coupled liposomes to deliver small interfering RNA (siRNA) against gp46, the rat homolog of human heat shock protein 47, to hepatic stellate cells. Our approach exploits the key roles of these cells in both fibrogenesis as well as uptake and storage of vitamin A. Five treatments with the siRNA-bearing vitamin A–coupled liposomes almost completely resolved liver fibrosis and prolonged survival in rats with otherwise lethal dimethylnitrosamine-induced liver cirrhosis in a dose- and duration-dependent manner. Rescue was not related to off-target effects or associated with recruitment of innate immunity. Receptor-specific siRNA delivery was similarly effective in suppressing collagen secretion and treating fibrosis induced by CCl4 or bile duct ligation. The efficacy of the approach using both acute and chronic models of liver fibrosis suggests its therapeutic potential for reversing human liver cirrhosis.

Colorectal cancer: genetics of development and metastasis.

It has been well documented that there are two major pathways in colorectal carcinogenesis. One is the chromosomal instability pathway (adenoma–carcinoma sequence), which is characterized by allelic losses on chromosome 5q (APC), 17p (p53), and 18q (DCC/SMAD4), and the other is a pathway that involves microsatellite instability. Recent progress in molecular biology, however, has shown that colorectal carcinogenesis is not necessarily clearly divided into these two pathways, but is in fact more complicated. Other routes, including the transforming growth factor-β/SMAD pathway, the serrated pathway, and the epigenetic pathway, have been reported. Cross talk among these pathways has also been reported. In the invasion and metastasis steps of colorectal cancers, many more genes have now been identified as being involved in proteolysis, adhesion, angiogenesis, and cell growth. Recently accumulated evidence indicates that colorectal cancer is a genetically heterogeneous and complicated disease.

Long-term phlebotomy with low-iron diet therapy lowers risk of development of hepatocellular carcinoma from chronic hepatitis C.

BackgroundWe have previously demonstrated that in patients with chronic hepatitis C (CHC), iron depletion improves serum alanine aminotransferase (ALT) levels as well as hepatic oxidative DNA damage. However, it has not been determined whether continuation of iron depletion therapy for CHC favorably influences its progression to hepatocellular carcinoma (HCC).MethodsWe conducted a cohort study on biopsy-proven CHC patients with moderate or severe liver fibrosis who failed to respond to previous interferon (IFN) therapy or had conditions for which IFN is contradicted. Patients were divided into two groups: subjects in group A (n = 35) underwent weekly phlebotomy (200 g) until they reached a state of mild iron deficiency, followed by monthly maintenance phlebotomy for 44–144 months (median, 107 months), and they were advised to consume a low-iron diet (5–7 mg iron/day); group B (n = 40) comprised CHC patients who declined to receive iron depletion therapy.ResultsIn group A, during the maintenance phase, serum ALT levels decreased to less than 60 IU/l in all patients and normalized (<40 IU/l) in 24 patients (69%), whereas in group B no spontaneous decrease in serum ALT occurred. Hepatocarcinogenesis rates in groups A and B were 5.7% and 17.5% at the end of the fifth year, and 8.6% and 39% in the tenth year, respectively. Multivariate analysis revealed that iron depletion therapy significantly lowered the risk of HCC (odds ratio, 0.57) compared with that of untreated patients (P = 0.0337).ConclusionsLong-term iron depletion for CHC patients is a promising modality for lowering the risk of progression to HCC.

Ex vivo large-scale generation of human red blood cells from cord blood CD34+ cells by co-culturing with macrophages

We generated red blood cells (RBC) from cord blood (CB) CD34+ cells using a four-phase culture system. We first cultured CB CD34+ cells on telomerase gene-transduced human stromal cells in serum-free medium containing stem cell factor (SCF), Flt-3/Flk-2 ligand, and thrombopoietin to expand CD34+ cells (980-fold) and the total cells (10,400-fold) (first phase). Expanded cells from the first phase were liquid-cultured with SCF, interleukin-3 (IL-3), and erythropoietin (EPO) to expand (113-fold) and differentiate them into erythroblasts (second phase). To obtain macrophages for the next phase, we expanded CD34+ cells from a different donor using the same co-culture system. Expanded cells from the first phase were liquid-cultured with granulocyte-macrophage colony stimulating factor, macrophage-colony stimulating factor (M-CSF), IL-3, and SCF to generate monocytes/macrophages (75-fold), which were incubated with type AB serum and M-CSF to fully differentiate them into macrophages. Erythroblasts were then co-cultured with macrophages in the presence of EPO to expand (threefold) and fully differentiate them (61% orthochromatic erythroblasts plus 39% RBC) (third phase). RBC were purified from erythroblasts and debris through a deleukocyting filter to generate 6.0 × 1012 RBC from 1.0 unit of CB (3.0 transfusable units). Qualitatively, these RBC showed a hemoglobin content, oxygenation of hemoglobin, and in vivo clearance similar to those of adult peripheral RBC. Finally, an almost complete enucleation of orthochromatic erythroblasts (99.4%) was achieved by the cultivation method recently described by Miharada et al. in the absence of macrophages and cytokines (fourth phase). RBC were purified from remnant erythroblasts and debris by passage through a deleukocyting filter to generate 1.76 × 1013 RBC from 1.0 unit of CB (8.8 transfusable units), the highest yield ever reported. Thus, this method may be useful for generating an alternative RBC supply for transfusions, investigating infectious agents that target erythroid cells, and as a general in vitro hematopoietic model system.

Hepatitis C virus core protein promotes proliferation of human hepatoma cells through enhancement of transforming growth factor α expression via activation of nuclear factor-κB

Background: Hepatitis C virus (HCV) infection is a major cause of human hepatocellular carcinoma (HCC). The precise mechanism of hepatocarcinogenesis in humans by HCV is currently unclear. It was recently shown, however, that transgenic mice with the HCV core gene often develop HCC, suggesting tumorigenic activity of the HCV core protein. Further, the HCV core protein expressed in HepG2 cells transfected with the core gene was shown to stimulate proliferation of transfectants through activation of nuclear factor-κB (NF-κB). The downstream target molecule(s) of NF-κB activated by the HCV core protein to evoke cell proliferation is not yet identified. Transforming growth factor (TGF) α, which is often overexpressed in various tumour tissues such as HCC, has been shown to stimulate hepatocyte proliferation through activation of the mitogen-activated protein kinase or extracellular signal-related protein kinase (MAPK/ERK) cascade. Aims: To explore the possibility that TGFα might be a target molecule for NF-κB activated by the HCV core, and that TGFα participates in the growth promotion of the core transfectants in an autocrine manner, activating the MAPK/ERK pathway. Methods: A HCV core expression vector was transfected into human hepatoma Huh-7, HepG2 and Hep3B cells. NF-κB activity was examined by an electrophoretic mobility shift assay. TGFα transcription was assessed by a luciferase reporter assay. TGFα protein was determined by immunoblot and ELISA. MAPK/ERK activity was examined by an in vitro kinase assay. Cell proliferation was assessed by a water-soluble tetrazolium salt-1 assay. Results: In the HCV core transfectants, NF-κB bound to the κB site in the TGFα proximal promoter region, resulting in an increase in TGFα transcription. Immunoblot as well as ELISA showed increased TGFα expression in the HCV core transfectants. SN50, a specific inhibitory peptide for NF-κB, cancelled HCV core-induced TGFα expression. HCV core protein increased cell proliferation as well as ERK activity of the HCV core transfectants as compared with the mock transfectants. The growth-promoting activity and activation of ERK by the HCV core protein were negated by treatment with anti-TGFα antibodies. Conclusions: These results suggest that the HCV core protein promotes proliferation of human hepatoma cells by activation of the MAPK/ERK pathway through up regulation of TGFα transcription via activation of NF-κB. Our finding provides a new insight into the mechanism of hepatocarcinogenesis by HCV infection.

Effect of transjugular intrahepatic portosystemic shunt formation on portal hypertensive gastropathy and gastric circulation

OBJECTIVES:The aim of this study was to investigate the effect of a transjugular intrahepatic portosystemic shunt (TIPS) on portal hypertensive gastropathy (PHG) and gastric hemodynamics.METHODS:A total of 16 patients with cirrhosis and portal hypertensive gastropathy were prospectively studied. Of these, 12 patients underwent TIPS for esophageal varices and four for refractory ascites. Gastric mucosal blood flow (GMBF) was assessed by laser Doppler flowmeter, and total blood flow (TBF) in submucosa and mucosa by near-infrared endoscopy. Portal venous pressure was obtained by a transducer during the TIPS procedure. The severity of portal hypertensive gastropathy was classified as none, mild, or severe. The examinations were performed before and 2 wk after the procedure.RESULTS:TIPS significantly reduced portal venous pressure. PHG improved in all four patients with severe PHG and in five of 12 patients with mild PHG after treatment. Gastric mucosal blood flow increased from 49.0 to 55.6 ml/min/100 g after TIPS. In contrast, TBF decreased from 0.35/s to 0.27/s after treatment. Liver function tests showed no significant changes before and after the procedure.CONCLUSIONS:It is considered that TIPS may have a beneficial effect on PHG at least for a short time. The mechanism by which PHG improves may be closely related to the improvement of the injured gastric perfusion in cirrhotic patients with PHG.

Aberrant Crypt Foci as Precursors of the Dysplasia-Carcinoma Sequence in Patients with Ulcerative Colitis

Purpose: Long-standing ulcerative colitis (UC) predisposes patients to the development of colorectal cancer, but surveillance of colitis-associated cancer by detecting the precancerous lesion dysplasia is often difficult because of its rare occurrence and normal-looking appearance. In sporadic colorectal cancer, aberrant crypt foci (ACF) have been reported by many investigators to be precursor lesions of the adenoma-carcinoma sequence. In the present study, we analyzed the genetic background of ACF to determine whether they could be precursors for dysplasia, and we examined the usefulness of endoscopic examination of ACF as a surrogate marker for surveillance of colitis-associated cancer. Experimental Design: ACF were examined in 28 UC patients (19 patients with UC alone and 9 patients with UC and dysplasia; 2 of those patients with dysplasia also had cancer) using magnifying endoscopy. K-ras, APC, and p53 mutations were analyzed by two-step PCR RFLP, in vitro–synthesized protein assay, and single-strand conformation polymorphism, respectively. Methylation of p16 was analyzed by methylation-specific PCR. Results: ACF that appeared distinct endoscopically and histologically were identified in 27 out of 28 UC patients. They were negative for K-ras, APC, and p53 mutations but were frequently positive for p16 methylation (8 of 11; 73%). In dysplasia, K-ras and APC mutations were negative but p53 mutation (3 of 5; 60%) and p16 methylation (3 of 5; 60%) were positive. There was a significant stepwise increase in the number of ACF from patients with UC alone to patients with dysplasia and to patients with cancer. Univariate and multivariate analyses showed significant correlations between ACF and dysplasia. Conclusions: We have disclosed an ACF-dysplasia-cancer sequence in colitis-associated carcinogenesis similar to the ACF-adenoma-carcinoma sequence in sporadic colon carcinogenesis. This study suggests the use of ACF instead of dysplasia for the surveillance of colitis cancer and warrants further evaluation of ACF as a surveillance marker in large-scale studies.

Aberrant Crypt Foci: Detection, Gene Abnormalities, and Clinical Usefulness

Human aberrant crypt foci (ACF) were first identified as lesions consisting of large thick crypts in colonic mucosa of surgical specimens after staining with methylene blue. Previously we succeeded in identifying ACF by using magnifying endoscopy and analyzed the number, size, and dysplastic features of ACF in normal controls and patients with adenoma or cancer patients. On the basis of these analyses, we strongly suggested that ACF, particularly dysplastic ACF, are precursor lesions of the adenoma-carcinoma sequence in humans. In most sporadic ACF, K-ras mutations were positive, but APC mutations were negative irrespective of nondysplastic or dysplastic features. Conversely, in most ACF from familial adenomatous polyposis patients, APC mutations were positive but K-ras mutations were negative. These results may suggest that the molecular mechanism of sporadic colon carcinogenesis is not necessarily the same as that of familial adenomatous polyposis. It was shown that ACF acquired resistance to apoptosis induced by bile salts, whereas normal colonic epithelial cells are turning over consistently by apoptosis. This apoptosis resistance was closely associated with glutathione S-transferase P1-1 expression. One of the most important clinical applications of ACF observation with magnifying endoscopy is its use as a target lesion for chemoprevention. Because ACF are tiny lesions, they should be eradicated during a short time by administration of chemopreventive agents. In fact, we performed an open chemopreventive trial of sulindac and found that the number of ACF was reduced markedly in 2 months. We currently are proceeding with a randomized double-blind trial targeting ACF.

Rapid on-site evaluation by endosonographer during endoscopic ultrasound-guided fine needle aspiration for pancreatic solid masses.

Endoscopic ultrasound‐guided fine needle aspiration (EUS‐FNA) is an established diagnostic method for patients with suspected pancreatic ductal carcinoma. Rapid on‐site evaluation (ROSE) has been reported to improve the accuracy. However, an on‐site cytopathologist is not routinely available in many institutions. One of the solutions may be ROSE by endosonographer. The aim was to examine whether diagnostic accuracy increases through ROSE by endosonographer using our cytological criteria.

Treatment of pancreatic fibrosis with siRNA against a collagen-specific chaperone in vitamin A-coupled liposomes

Background and objective Fibrosis associated with chronic pancreatitis is an irreversible lesion that can disrupt pancreatic exocrine and endocrine function. Currently, there are no approved treatments for this disease. We previously showed that siRNA against collagen-specific chaperone protein gp46, encapsulated in vitamin A-coupled liposomes (VA-lip-siRNAgp46), resolved fibrosis in a model of liver cirrhosis. This treatment was investigated for pancreatic fibrosis induced by dibutyltin dichloride (DBTC) and cerulein in rats. Methods Specific uptake of VA-lip-siRNAgp46, conjugated with 6′-carboxyfluorescein (FAM) by activated pancreatic stellate cells (aPSCs), was analysed by fluorescence activated cell sorting (FACS). Intracellular distribution of VA-lip-siRNAgp46-FAM was examined by fluorescent microscopy. Suppression of gp46 expression by VA-lip-siRNAgp46 was assessed by immunoblotting. Collagen synthesis in aPSCs was assayed by dye-binding. Specific delivery of VA-lip-siRNAgp46 to aPSCs in DBTC rats was verified following intravenous VA-lip-siRNA-FAM and 3H-VA-lip-siRNAgp46. The effect of VA-lip-siRNA on pancreatic histology in DBTC- and cerulein-treated rats was determined by Azan-Mallory staining and hydroxyproline content. Results FACS analysis revealed specific uptake of VA-lip-siRNAgp46-FAM through the retinol binding protein receptor by aPSCs in vitro. Immunoblotting and collagen assay verified knockdown of gp46 and suppression of collagen secretion, respectively, by aPSCs after transduction of VA-lip-siRNAgp46. Specific delivery of VA-lip-siRNAgp46 to aPSCs in fibrotic areas in DBTC rats was confirmed by fluorescence and radioactivity 24 h after the final injection. 10 systemic VA-lip-siRNAgp46 treatments resolved pancreatic fibrosis, and suppressed tissue hydroxyproline levels in DBTC- and cerulein-treated rats. Conclusion These data suggest the therapeutic potential of the present approach for reversing pancreatic fibrosis.