Quantitative trait loci for cellular defects in glucose and fatty acid metabolism in hypertensive rats.

Coronary heart disease, hypertension, non-insulin-dependent diabetes and obesity are major causes of ill health in industrial societies. Disturbances of carbohydrate and lipid metabolism are a common feature of these disorders. The bases for these disturbances and their roles in disease pathogenesis are poorly understood. The spontaneously hypertensive rat (SHR), a widely used animal model of essential hypertension, has a global defect in insulin action on glucose metabolism and shows reduced catecholamine action on lipolysis in fat cells. In our study we used cellular defects in carbohydrate and lipid metabolism to dissect the genetics of defective insulin and catecholamine action in the SHR strain. In a genome screen for loci linked to insulin and catecholamine action, we identified two quantitative trait loci (QTLs) for defective insulin action, on chromosome 4 and 12. We found that the major (and perhaps only) genetic determinant of defective control of lipolysis in SHR maps to the same region of chromosome 4. These linkage results were ascertained in at least two independent crosses. As the SHR strain manifests many of the defining features of human metabolic Syndrome X, in which hypertension associates with insulin resistance, dyslipidaemia and abdominal obesity, the identification of genes for defective insulin and catecholamine action in SHR may facilitate gene identification in this syndrome and in related human conditions, such as type-2 diabetes and familial combined hyperlipidaemia.

A multicenter validation of an endoscopic classification with narrow band imaging for gastric precancerous and cancerous lesions.

BACKGROUND AND STUDY AIM: The reliability and external validity of narrow band imaging (NBI) in the stomach have not been described consistently. The aim of the current study was to describe and estimate the accuracy and reliability of a simplified classification system for NBI in the diagnosis of gastric lesions. METHODS: Consecutive patients undergoing NBI endoscopy at two reference centers (n=85, 33% with dysplasia) were included in two studies. In total, 224 different areas were biopsied and recorded onto video. In the derivation study, previously described NBI features were analyzed in order to develop a simplified classification. In the validation study the accuracy and reliability of this classification were estimated among three groups of endoscopists with different levels of expertise in NBI. RESULTS: The reliability/accuracy results from the derivation study allowed the creation of a simplified NBI classification. In the validation study, "regular vessels with circular mucosa" (pattern A) was associated with normal histology (accuracy 83%; 95% confidence interval [CI] 75 %-90%); "tubulo-villous mucosa" (pattern B) was associated with intestinal metaplasia (accuracy 84%; 95CI 77%-91%; positive likelihood ratio [LR+]=4.75); and "irregular vessels and mucosa" (pattern C) was associated with dysplasia (accuracy 95%; 95CI 90%-99%; LR+=44.33). The reproducibility of these patterns was high (k=0.62). "Light-blue crest" was moderately reliable (k=0.49) but specific (87%) for intestinal metaplasia. A variable vascular density (additional pattern+) was the best feature for Helicobacter pylori gastritis (accuracy 70%; 95CI 59%-80%) but showed only fair reliability (k=0.38). Non-experienced endoscopists presented lower agreement (k=0.6 vs. k=0.75) and accuracy (74% vs. 86%) than international experts/experienced endoscopists. CONCLUSION: A simplified NBI classification is accurate and reliable for the diagnosis of intestinal metaplasia and dysplasia. The classification should be further assessed and validated on a per-patient assessment of NBI, and by comparing NBI with other imaging technologies.

The indolent course and high incidence of t(14;18) in primary duodenal follicular lymphoma.

BACKGROUND: Information on the clinical behavior of the recently proposed primary duodenal follicular lymphoma (DFL) is limited. PATIENTS AND METHODS: Demographic data, signs, symptoms, disease stage, and treatment of the patients diagnosed in National Cancer Center Hospital from 1999 to 2007 were collected and analyzed. RESULTS: Twenty-seven patients were studied. Nineteen patients were asymptomatic at the time of diagnosis. Twenty patients had stage I disease. The histological grade was 1 or 2 in 26 patients. IgH/BCL2 fusion was shown in 20 of the examined 24 cases (83%). Fourteen patients received therapy upon diagnosis (local radiotherapy in 2 patients and chemotherapy in 12 including rituximab therapy), their response rate was 85%, and the estimated progression-free survival (PFS) rate at 3 years was 70%. One patient developed histological transformation. The other 13 patients were followed up; their estimated PFS rate at 3 years was 74%. Five among six cases responded to treatment even after progressive disease. All 27 patients have survived with a median follow-up time of 47.9 months. CONCLUSIONS: The majority of primary DFL patients have a localized tumor of low-grade histology and are positive for t(14;18). Watchful waiting might be an alternative approach for its indolent course; however, further studies are warranted.

Endoscopic resection of early gastric cancer treated by guideline and expanded National Cancer Centre criteria.

BACKGROUND: Criteria for endoscopic resection in patients with early gastric cancer (EGC) have been expanded recently by the National Cancer Centre (NCC). This study compared long-term outcomes in patients with EGC who underwent endoscopic treatment according to guideline criteria with those treated according to expanded criteria. METHODS: Baseline and outcome data from patients undergoing curative endoscopic resection for EGC between January 1999 and December 2005 were collected from electronic medical records. Survival time hazard ratios and 95 per cent confidence intervals were calculated using the Cox proportional hazards model. RESULTS: Of 1485 patients who had a curative resection, 635 (42.8 per cent) underwent resection according to traditional criteria and 625 (42.1 per cent) according to expanded criteria. There was no significant difference in overall survival between the groups. CONCLUSION: Patients who have treatment following the expanded criteria have similar long-term survival and outcomes to those treated according to guideline criteria.

Use of Gascon and Pronase either as a pre-endoscopic drink or as targeted endoscopic flushes to improve visibility during gastroscopy: a prospective, randomized, controlled, blinded trial.

OBJECTIVE: To assess whether endoscopic flushes of the bubble-bursting agent Gascon and the mucolytic agent Pronase are as effective in terms of improving endoscopic mucosal visibility as a pre-endoscopic drink of the same agents. MATERIAL AND METHODS: A total of 112 patients attending a Japanese tertiary referral centre for upper gastrointestinal endoscopy were randomized to receive either the standard Japanese procedure of a pre-endoscopic drink of water containing Gascon and Pronase with endoscopic flushes of 20-ml aliquots of water, or no pre-endoscopic therapy but endoscopic flushes of 20-ml aliquots of water containing Gascon, with or without Pronase as necessary. RESULTS: Visibility scores were significantly better in the pre-endoscopic drink group than in either of the endoscopic flush groups. The group receiving a pre-endoscopic drink required fewer flushes during the procedure and there was no difference in the endoscopic time between the three groups. CONCLUSIONS: Our results suggest that endoscopic spraying of these bubble-bursting and mucolytic agents is not able to offer equivalent improvements in endoscopic mucosal visibility when compared with the standard Japanese therapy of a pre-endoscopic drink of these agents. The addition of Pronase to the spray solution had no measurable benefit over Gascon alone. We therefore cannot recommend endoscopic spraying of mucous clearing agents over their use as a pre-endoscopic drink.

Our perspective on endoscopic resection for colorectal neoplasms.

Endoscopic mucosal resection (EMR) is a minimally invasive technique for effective treatment of early stage colorectal lesions with no invasive potential. However, the high frequency of local recurrence after piecemeal EMR for large lesions is considered a serious problem. In contrast, endoscopic submucosal dissection (ESD) allows en-bloc resection, irrespective of the lesion's size. ESD has been established as a standard method for the endoscopic removal of early cancers in the upper gastrointestinal tract in Japan. Although the use of ESD for colorectal lesions has been studied clinically, ESD is not yet established as a standard therapeutic method. We define the indications for en-bloc resection, based on extensive clinicopathological analyses, as a laterally spreading tumor (LST) non-granular type (LST-NG) lesion greater than 20 mm and an LST granular (LST-G) type lesion greater than 40 mm. Both of these lesions had a high submucosal invasion rate. Especially, LST-NG type lesions greater than 20 mm are technically difficult to remove completely even by piecemeal EMR and are considered a "definite indication for en-bloc resection". The ESD procedure is undoubtedly an ideal method to achieve en-bloc resection, however, the prevalences of suitable lesions among all neoplastic lesions and among all early cancers were not high (1.0% and 5.0%, respectively). Therefore, it is crucial to master more fundamental therapeutic techniques and have knowledge of surveillance strategy after endoscopic treatment.

Risk factors for cardiac and pyloric stenosis after endoscopic submucosal dissection, and efficacy of endoscopic balloon dilation treatment.

BACKGROUND AND STUDY AIMS: Bleeding and perforation are major complications of endoscopic submucosal dissection (ESD) for early gastric cancer (EGC), but post-ESD stenosis represents a severe delayed complication that can result in clinical symptoms such as dysphagia and nausea. The aims of this study were to determine the risk factors and evaluate the clinical treatment for post-ESD stenosis. METHODS: A total of 2011 EGCs resected by ESD at our institution between 2000 and 2005 were reviewed retrospectively. Resection was defined as cardiac when any mucosal defect was located in the squamocolumnar junction, and as pyloric when any mucosal defect was located < 1 cm from the pylorus ring. Post-ESD stenosis was defined when a standard endoscope could not be passed through the stenosis. We examined the incidence of post-ESD stenosis, its relationship with relevant factors, and the clinical course of post-ESD stenosis patients. RESULTS: Post-ESD stenosis occurred with seven of 41 cardiac resections (17 %) and eight of 115 pyloric resections (7 %). Circumferential extent of the mucosal defect of > 3/4 and longitudinal extent > 5 cm were each significantly related to occurrence of post-ESD stenosis with both cardiac and pyloric resections. All 15 affected patients were successfully treated by endoscopic balloon dilation. CONCLUSIONS: A circumferential extent of the mucosal defect of > 3/4 or longitudinal extent of > 5 cm in length were both demonstrated to be risk factors for post-ESD stenosis, in both cardiac and pyloric resections, and endoscopic balloon dilation was shown to be effective in treating post-ESD stenosis.

Treatment strategy after non-curative endoscopic resection of early gastric cancer.

BACKGROUND: Endoscopic resection (ER) is indicated for patients with early gastric cancer who have a negligible risk of lymph node metastasis (LNM). Histological examination of the resected specimen may indicate a possible risk of LNM or a positive resection margin. These patients are considered to have undergone non-curative ER. The aim of this study was to determine the appropriate treatment strategy for such patients. METHODS: A total of 298 patients who had non-curative ER were classified into those with a positive lateral margin only (group 1; 72 patients) and those with a possible risk of LNM (group 2; 226 patients). RESULTS: Surgery was performed within 6 months of non-curative ER in 19 patients in group 1 and 144 in group 2. In group 1, nine patients were found to have local residual tumours, all limited to the mucosal layer without LNM. In Group 2, 13 patients had residual disease, including four local tumours without LNM, two local tumours with LNM and seven cases of LNM alone. The rate of LNM after surgery was 6.3 per cent in group 2. CONCLUSION: Surgery remains the standard treatment after non-curative ER in patients with a possible risk of LNM.

Total endoscopic resection of Barrett esophagus.

Barrett's esophagus with high grade intraepithelial neoplasia is associated with disease progression at rates of greater than 10% per year. Endoscopic resection is a lower risk alternative to surgery for the management of high grade intraepithelial neoplasia and intramucosal cancer. Two endoscopic approaches have been used, namely localized resection of the lesion and total endoscopic resection of all Barrett's mucosa. The latter strategy removes all at-risk mucosa. Currently it is performed mainly using piecemeal endoscopic mucosal resection techniques. In recent years endoscopic submucosal dissection has been attempted to obtain en bloc resection. This review will describe the techniques of total endoscopic resection, and summarize the key published data.

Routine coagulation of visible vessels may prevent delayed bleeding after endoscopic submucosal dissection--an analysis of risk factors.

BACKGROUND AND STUDY AIM: Endoscopic submucosal dissection (ESD) has been reported to be associated with a higher complication rate than standard endoscopic mucosal resection. We aimed to clarify the risk factors for delayed bleeding after ESD for early gastric cancer (EGC). METHODS: 1083 EGCs in 968 consecutive patients undergoing ESD during a 4-year period were reviewed. Post-ESD coagulation (PEC) preventive therapy of visible vessels in the resection area, using a coagulation forceps, was introduced and mostly performed during the later 2 years. Various factors related to patients, tumors, and treatment including PEC were investigated using univariate and multivariate analysis with regard to delayed post-ESD bleeding, evidenced by hematemesis or melena, that required endoscopic treatment. RESULTS: Delayed bleeding occurred after ESD of 63 lesions (5.8 % of all lesions and 6.5 % of patients), controlled in all cases by endoscopic hemostasis; blood transfusion was required in only one case. Tumor location in the upper third of the stomach and PEC were independent factors indicating a lower rate of delayed bleeding according to both univariate and multivariate analysis. CONCLUSIONS: This retrospective study suggested that preventive coagulation of visible vessels in the resection area after ESD may lead to a lower bleeding rate.

Macrophage colony-stimulating factor regulates both activities of neutral and acidic cholesteryl ester hydrolases in human monocyte-derived macrophages.

Macrophage colony-stimulating factor (M-CSF) regulates cholesterol metabolism in vivo and in vitro. We studied the effects of M-CSF on enzyme activities of acidic cholesteryl ester (CE) hydrolase, neutral CE hydrolase, and acyl-coenzyme A:cholesterol acyltransferase (ACAT), all of which are involved in cellular cholesterol metabolism in macrophages. During the differentiation of monocytes to macrophages, these enzyme activities were induced and further enhanced in response to M-CSF. M-CSF (100 ng/ml) enhanced acidic and neutral CE hydrolase and ACAT activities by 3.2-, 4-, and 2.3-fold, respectively, in the presence of acetyl LDL. The presence of acetyl LDL influenced these enzyme activities. ACAT and acidic CE hydrolase activities were increased and neutral CE hydrolase activity was decreased, indicating that these enzymes are regulated by intracellular cholesterol enrichment. M-CSF increased the ratios of acidic CE hydrolase to ACAT activity and of neutral CE hydrolase to ACAT activity. The results suggest that M-CSF enhances net hydrolysis of CE by stimulating the two CE hydrolases to a greater extent than ACAT, and M-CSF may reduce the rate of atherogenesis.

Induction of sustained expression of proto-oncogene c-fms by platelet-derived growth factor, epidermal growth factor, and basic fibroblast growth factor, and its suppression by interferon-gamma and macrophage colony-stimulating factor in human aortic medial smooth muscle cells.

Vascular medial smooth muscle cells migrate, proliferate and transform to foam cells in the process of atherosclerosis. We have reported that the intimal smooth muscle cells express proto-oncogene c-fms, a characteristic gene of monocyte-macrophages, which is not normally expressed in medial smooth muscle cells. In the present study, we demonstrated that combinations of platelet-derived growth factor (PDGF)-BB and either epidermal growth factor (EGF) or fibroblast growth factor (FGF) induced high expression of c-fms in normal human medial smooth muscle cells to the level of intimal smooth muscle cells or monocyte-derived macrophages, whereas c-fms expression by PDGF-BB alone was 1/10 and both EGF and FGF had no independent effect on c-fms expression. By contrast, interferon (IFN)-gamma and macrophage colony-stimulating factor (M-CSF) suppressed the induction of c-fms expression. These results indicate that multiple growth factors and cytokines may play a role in the phenotypic transformation of medial smooth muscle cells to intimal smooth muscle cells in atherosclerotic lesions by altering c-fms expression.

Inhibition of diet-induced atheroma formation in transgenic mice expressing apolipoprotein E in the arterial wall.

Apolipoprotein E (apoE) plays a crucial role in lipoprotein metabolism both in plasma and in peripheral tissues. To test whether apoE in the vascular wall has a direct and local effect on atherogenesis, we established transgenic mice expressing human apoE under control of H2 Ld promoter. Studies on mRNA levels and immunohistochemistry demonstrated that this line was characterized by high expression of human apoE in the arterial wall while its expression was relatively low in other tissues as compared with the respective endogenous expression of mouse apoE. They showed no difference in plasma cholesterol levels and lipoprotein profile from controls when fed both normal and atherogenic diets. However, after 24 wk of an atherogenic diet, the formation of fatty streak lesions in proximal aorta was markedly inhibited in transgenic mice as compared with controls. Both lesion area and esterified cholesterol content were < 30% of those in controls. In a tissue cholesterol labeling study with 3H-cholesterol, the specific activity of aorta cholesterol was much less in transgenic mice, suggesting that apoE enhances cholesterol efflux from the aortic wall into plasma. Thus, apoE has anti-atherogenic action which is mediated via enhancing reverse cholesterol transport from arterial wall.

Plasma lipoprotein metabolism in transgenic mice overexpressing apolipoprotein E. Accelerated clearance of lipoproteins containing apolipoprotein B.

We have reported that transgenic mice overexpressing rat apo E shows marked reduction of plasma cholesterol and triglyceride levels due to the disappearance of VLDL and LDL. In this study, we investigated the metabolism of plasma lipoproteins in transgenic mice. After intravenous injection, the rates of clearance of 125I-VLDL and 125I-LDL were 3.0- and 2.4-fold greater in transgenic mice than in controls, respectively. Furthermore, clearance of chylomicron remnants estimated by oral retinyl palmitate-loading test was markedly enhanced in transgenic mice. The hepatic expression of LDL receptors by immunoblot analysis was similar in both groups. These data suggest that elimination of lipoproteins containing apo B was due to enhanced clearance of these lipoproteins enriched with apo E through hepatic LDL receptors. When fed a high cholesterol diet, controls showed twofold elevation of plasma cholesterol levels with marked increases in VLDL and LDL cholesterol on gel filtration chromatography. In contrast, cholesterol-fed transgenic mice showed resistance against these increases. High cholesterol feeding decreased the activity of hepatic LDL receptors and had no effect on enhancement of chylomicron remnant clearance in transgenic mice. Thus, overexpression of apo E facilitates metabolism of lipoproteins containing apo B presumably primarily via the LDL receptor pathway and possibly through an interaction with the chylomicron remnant receptor.

Heterogeneous mutations in the human lipoprotein lipase gene in patients with familial lipoprotein lipase deficiency.

The DNA sequences were determined for the lipoprotein lipase (LPL) gene from five unrelated Japanese patients with familial LPL deficiency. The results demonstrated that all five patients are homozygotes for distinct point mutations dispersed throughout the LPL gene. Patient 1 has a G-to-A transition at the first nucleotide of intron 2, which abolishes normal splicing. Patient 2 has a nonsense mutation in exon 3 (Tyr61----Stop) and patient 3 in exon 8 (Trp382----Stop). The latter mutation emphasizes the importance of the carboxy-terminal portion of the enzyme in the expression of LPL activity. Missense mutations were identified in patient 4 (Asp204----Glu) and patient 5 (Arg243----His) in the strictly conserved amino acids. Expression study of both mutant genes in COS-1 cells produced inactive enzymes, establishing the functional significance of the two mis-sense mutations. In these patients, postheparin plasma LPL mass was either virtually absent (patients 1 and 2) or significantly decreased (patients 3-5). To detect these mutations more easily, we developed a rapid diagnostic test for each mutation. We also determined the DNA haplotypes for patients and confirmed the occurrence of multiple mutations on the chromosomes with an identical haplotype. These results demonstrate that familial LPL deficiency is a heterogeneous genetic disease caused by a wide variety of gene mutations.

Secretion-recapture process of apolipoprotein E in hepatic uptake of chylomicron remnants in transgenic mice.

To investigate the role of apoE in hepatic uptake of chylomicron remnants, we studied chylomicron metabolism in transgenic mice overexpressing apoE in the liver. Plasma clearance of injected 125I-labeled human chylomicrons was fivefold faster in transgenic mice than in controls. Immunohistochemistry demonstrated that apoE was specifically localized at the basolateral surface of hepatocytes from fasted transgenic mice. After injection of a large amount of chylomicrons, the density of the cell surface apoE was markedly reduced and vesicular staining was observed in the cytoplasm, suggesting that the cell surface apoE was used for hepatic endocytosis of chylomicrons and remnants. Polyacrylamide gel analysis of chylomicrons and remnants that had been reisolated from plasma and from liver membrane after the injection of chylomicrons showed the particles to be enriched with apoE mainly after their influx into the liver rather than during their residence in plasma. These results provide strong evidence for the secretion-recapture process of apoE, whereby chylomicron remnants enter the sinusoidal space, acquire apoE molecules, and subsequently are endocytosed. Data from experiments with very low density lipoprotein and LDL showed that this system is specific for chylomicron remnants.

Apolipoprotein E prevents the progression of atherosclerosis in Watanabe heritable hyperlipidemic rabbits.

Apo E plays an important role in plasma lipoprotein metabolism through its high affinity binding to cell surface LDL receptor. In the present study, we studied the effects of apo E on the atherogenic process in Watanabe heritable hyperlipidemic rabbits which are deficient in LDL receptor and an animal model for familial hypercholesterolemia. We isolated apo E from plasma of 1% cholesterol-fed rabbits and administered 10 mg of purified apo E intravenously into five Watanabe heritable hyperlipidemic rabbits three times a week from their age of 2.5 months to 11 months for 8.5 months. After sustained administration to apo E, we found a significant reduction in the accumulation of cholesterol ester in aortae (1.55 +/- 0.07 mg/g tissue) as compared to control rabbits (4.32 +/- 0.61 mg/g tissue). Supporting this, the percentage of the surface area of the aorta with macroscopic plaque was remarkably decreased in apo E-treated animals (18.8 +/- 5.1% vs. 38.8 +/- 8.0% in control). Thus, apo E definitely prevented the progression of atherosclerosis in Watanabe heritable hyperlipidemic rabbits.

Transcription factor PU.1 mediates induction of c-fms in vascular smooth muscle cells: a mechanism for phenotypic change to phagocytic cells.

The macrophage colony-stimulating factor receptor encoded by the c-fms gene is expressed in vascular intimal smooth muscle cells isolated from atherosclerotic lesions. A combination of platelet-derived growth factor-BB and epidermal growth factor induces stable expression of c-fms in normal vascular medial smooth muscle cells. The mechanism by which these growth factors induce c-fms expression has now been investigated in an attempt to gain insight into the events that underlie the phenotypic conversion of vascular smooth muscle cells in atherosclerosis. Deletion analysis of the c-fms promoter revealed that the region including a binding site for transcription factor PU.1 was required for transcriptional activity in human aortic medial smooth muscle cells. Mutation in the PU.1 binding site markedly reduced promoter activity. Northern (RNA) blot analysis demonstrated that growth factors induced the expression of PU.1 mRNA in vascular medial smooth muscle cells and that PU.1 mRNA was expressed in vascular intimal smooth muscle cells. PU.1 antisense oligonucleotides inhibited growth factor-induced c-fms expression and foam cell formation. These results suggest that transcription factor PU.1 plays an essential role in the phenotypic conversion of vascular smooth muscle cells to macrophagelike cells by mediating the induction of c-fms.

Identification of liver X receptor-retinoid X receptor as an activator of the sterol regulatory element-binding protein 1c gene promoter.

In an attempt to identify transcription factors which activate sterol-regulatory element-binding protein 1c (SREBP-1c) transcription, we screened an expression cDNA library from adipose tissue of SREBP-1 knockout mice using a reporter gene containing the 2.6-kb mouse SREBP-1 gene promoter. We cloned and identified the oxysterol receptors liver X receptor (LXRalpha) and LXRbeta as strong activators of the mouse SREBP-1c promoter. In the transfection studies, expression of either LXRalpha or -beta activated the SREBP-1c promoter-luciferase gene in a dose-dependent manner. Deletion and mutation studies, as well as gel mobility shift assays, located an LXR response element complex consisting of two new LXR-binding motifs which showed high similarity to an LXR response element recently found in the ABC1 gene promoter, a reverse cholesterol transporter. Addition of an LXR ligand, 22(R)-hydroxycholesterol, increased the promoter activity. Coexpression of retinoid X receptor (RXR), a heterodimeric partner, and its ligand 9-cis-retinoic acid also synergistically activated the SREBP-1c promoter. In HepG2 cells, SREBP-1c mRNA and precursor protein levels were induced by treatment with 22(R)-hydroxycholesterol and 9-cis-retinoic acid, confirming that endogenous LXR-RXR activation can induce endogenous SREBP-1c expression. The activation of SREBP-1c by LXR is associated with a slight increase in nuclear SREBP-1c, resulting in activation of the gene for fatty acid synthase, one of its downstream genes, as measured by the luciferase assay. These data demonstrate that LXR-RXR can modify the expression of genes for lipogenic enzymes by regulating SREBP-1c expression, providing a novel link between fatty acid and cholesterol metabolism.