The combination of fatty liver and increased gamma-glutamyl transpeptidase levels as a risk factor for atherosclerotic plaque development in apparently healthy people

Background/aim: To elucidate how the combination of fatty liver and increased serum gamma-glutamyl transpeptidase (GGT) levels influences atherosclerotic plaque development in apparently healthy people. Materials and methods: The study population included people who had received an annual health checkup for more than 7 years and had no evidence of carotid plaque at baseline. We investigated the risk factors for carotid plaque occurrence using the Cox proportional hazards model. Results: A total of 107 people (76 men and 31 women; median age, 49 years) were enrolled. At baseline, fatty liver and a serum GGT level ≥50 U/L were observed in 13 and 38 people, respectively. During a median follow-up period of 13.3 years, carotid plaques appeared in 34 people. Multivariate analysis revealed that the combination of fatty liver and a serum GGT level ≥50 U/L was the only significant risk factor for carotid plaque occurrence (age- and sex-adjusted hazard ratio: 5.55; 95% confidence interval 1.70­18.14; P = 0.005). Conclusion: The combination of fatty liver and increased serum GGT levels raises the risk for atherosclerotic plaque development in apparently healthy people.

Relationship Between Short Term Variability (STV) and Onset of Cerebral Hemorrhage at Ischemia-Reperfusion Load in Fetal Growth Restricted (FGR) Mice.

Fetal growth restriction (FGR) is a risk factor exacerbating a poor neurological prognosis at birth. A disease exacerbating a poor neurological prognosis is cerebral palsy. One of the cause of this disease is cerebral hemorrhage including intraventricular hemorrhage. It is believed to be caused by an inability to autoregulate cerebral blood flow as well as immaturity of cerebral vessels. Therefore, if we can evaluate the function of autonomic nerve, cerebral hemorrhage risk can be predicted beforehand and appropriate delivery management may be possible. Here dysfunction of autonomic nerve in mouse FGR fetuses was evaluated and the relationship with cerebral hemorrhage incidence when applying hypoxic load to resemble the brain condition at the time of delivery was examined. Furthermore, FGR incidence on cerebral nerve development and differentiation was examined at the gene expression level. FGR model fetuses were prepared by ligating uterine arteries to reduce placental blood flow. To compare autonomic nerve function in FGR mice with that in control mice, fetal short term variability (STV) was measured from electrocardiograms. In the FGR group, a significant decrease in the STV was observed and dysfunction of cardiac autonomic control was confirmed. Among genes related to nerve development and differentiation, Ntrk and Neuregulin 1, which are necessary for neural differentiation and plasticity, were expressed at reduced levels in FGR fetuses. Under normal conditions, Neurogenin 1 and Neurogenin 2 are expressed mid-embryogenesis and are related to neural differentiation, but they are not expressed during late embryonic development. The expression of these two genes increased in FGR fetuses, suggesting that neural differentiation is delayed with FGR. Uterine and ovarian arteries were clipped and periodically opened to give a hypoxic load mimicking the time of labor, and the bleeding rate significantly increased in the FGR group. This suggests that FGR deteriorates cardiac autonomic control, which becomes a risk factor for cerebral hemorrhage onset at birth. This study demonstrated that cerebral hemorrhage risk may be evaluated before parturition for FGR management by evaluating the STV. Further, this study suggests that choosing an appropriate delivery timing and delivery method contributes to neurological prognosis improvement.

Reduced Uterine Perfusion Pressure (RUPP) Model of Preeclampsia in Mice.

Preeclampsia (PE) is a pregnancy-induced hypertension with proteinuria that typically develops after 20 weeks of gestation. A reduction in uterine blood flow causes placental ischemia and placental release of anti-angiogenic factors such as sFlt-1 followed by PE. Although the reduced uterine perfusion pressure (RUPP) model is widely used in rats, investigating the role of genes on PE using genetically engineered animals has been problematic because it has been difficult to make a useful RUPP model in mice. To establish a RUPP model of PE in mice, we bilaterally ligated ovarian vessels distal to ovarian branches, uterine vessels, or both in ICR-strain mice at 14.5 days post coitum (dpc). Consequently, these mice had elevated BP, increased urinary albumin excretion, severe endotheliosis, and mesangial expansion. They also had an increased incidence of miscarriage and premature delivery. Embryonic weight at 18.5 dpc was significantly lower than that in sham mice. The closer to the ligation site the embryos were, the higher the resorption rate and the lower the embryonic weight. The phenotype was more severe in the order of ligation at the ovarian vessels < uterine vessels < both. Unlike the RUPP models described in the literature, this model did not constrict the abdominal aorta, which allowed BP to be measured with a tail cuff. This novel RUPP model in mice should be useful for investigating the pathogenesis of PE in genetically engineered mice and for evaluating new therapies for PE.

Binge alcohol consumption aggravates oxidative stress and promotes pathogenesis of NASH from obesity-induced simple steatosis.

The pathogenesis of nonalcoholic steatohepatitis (NASH) is a two-stage process in which steatosis is the "first hit" and an unknown "second hit." We hypothesized that "a binge" could be a "second hit" to develop NASH from obesity-induced simple steatosis. Thirty-week-old male Otsuka Long-Evans Tokushima fatty (OLETF) rats were administered 10 mL of 10% ethanol orally for 5, 3, 2, and 1 d/wk for 3 consecutive weeks. As control, male Otsuka Long-Evans Tokushima (OLET) rats were administered the same amount of alcohol. Various biochemical parameters of obesity, steatosis and NASH were monitored in serum and liver specimens in untreated and ethanol-treated rats. The liver sections were evaluated for histopathological alterations of NASH and stained for cytochrome P-4502E1 (CYP2E1) and 4-hydroxy-nonenal (4-HNE). Simple steatosis, hyperinsulinemia, hyperglycemia, insulin resistance, hypertriglycemia and marked increases in hepatic CYP2E1 and 4-HNE were present in 30-wk-old untreated OLETF rats. Massive steatohepatitis with hepatocyte ballooning was observed in the livers of all OLETF rats treated with ethanol. Serum and hepatic triglyceride levels as well as tumor necrosis factor (TNF)-α mRNA were markedly increased in all ethanol-treated OLETF rats. Staining for CYP2E1 and 4-NHE demonstrated marked increases in the hepatic tissue of all the groups of OLETF rats treated with ethanol compared with OLET rats. Our data demonstrated that "a binge" serves as a "second hit" for development of NASH from obesity-induced simple steatosis through aggravation of oxidative stress. The enhanced levels of CYP2E1 and increased oxidative stress in obesity play a significant role in this process.

Serum endocan as a novel prognostic biomarker in patients with hepatocellular carcinoma.

Endocan is a vascular endothelium-derived factor regulated by angiogenic factors. The aim of this study was to determine whether serum endocan levels are prognostic for survival in patients with hepatocellular carcinoma (HCC). Serum endocan levels were measured in 64 HCC patients who were naïve to treatment, eight apparently healthy subjects, and 68 patients with liver cirrhosis; the latter two groups served as controls. Prognostic factors for the survival of HCC patients were examined using a Cox proportional hazards model. The median serum endocan levels were 1.145 ng/mL (range, 0.93-1.68 ng/mL) in healthy subjects, 1.93 ng/mL (range, 0.45-8.47 ng/mL) in liver cirrhosis patients, and 3.73 ng/mL (range, 0.74-10.95 ng/mL) in HCC patients (P = 0.0001). In HCC patients, elevated serum endocan levels were significantly associated with poor hepatic function (P = 0.015), a greater number of tumors (P = 0.034), and vascular invasion (P = 0.043). The median follow-up period was 23.0 months, and 33 HCC patients died during follow up. Multivariate analysis showed that serum endocan levels ≥ 2.20 ng/mL (hazard ratio 2.36, 95% confidence interval 1.22-5.36, P = 0.008) as well as elevated serum α-fetoprotein and des-γ-carboxy prothrombin levels were independent prognostic biomarkers for poor survival. The combination of serum endocan and these two additional markers was significantly predictive of worse survival (P < 0.0001). Thus, serum endocan may be a prognostic biomarker for survival in HCC patients, and the combination of serum endocan, α-fetoprotein, and des-γ-carboxy prothrombin levels can result in better prognostic stratification of these patients.

Advanced ultrasonography technologies to assess the effects of radiofrequency ablation on hepatocellular carcinoma.

BACKGROUND: Radiofrequency ablation (RFA) is a curative therapy for hepatocellular carcinoma (HCC). In RFA, ultrasonography (US) is most commonly used to guide tumor puncture, while its effects are assessed using dynamic computed tomography or magnetic resonance. The differences in modalities used for RFA and assessment of its effects complicate RFA. We developed a method for assessing the effects of RFA on HCC by combining contrast-enhanced (CE) US and real-time virtual sonography with three-dimensional US data. PATIENTS AND METHODS: Before RFA, we performed a sweep scan of the target HCC nodule and the surrounding hepatic parenchyma to generate three-dimensional US data. After RFA, we synchronized multi-planar reconstruction images derived from stored three-dimensional US data with real-time US images on the same US monitor and performed CEUS and real-time virtual sonography. Using a marking function, we drew a sphere marker along the target HCC nodule contour on pre-treatment US- multi-planar reconstruction images so that the automatically synchronized sphere marker represented the original HCC nodule contour on post-treatment real-time CEUS images. Ablation was considered sufficient when an avascular area with a margin of several millimeters in all directions surrounded the sphere marker on CEUS. RESULTS: This method was feasible and useful for assessing therapeutic effects in 13 consecutive patients with HCC who underwent RFA. In 2 patients who underwent multiple sessions of RFA, HCC-nodule portions requiring additional RFA were easily identified on US images. CONCLUSIONS: This method using advanced US technologies will facilitate assessment of the effects of RFA on HCC.

Influence of HRH2 promoter polymorphism on aberrant DNA methylation of DAPK and CDH1 in the gastric epithelium.

BACKGROUND: Aberrant methylation patterns in CpG island are known to be influential in gene silencing. Histamine plays important physiological roles in the upper gastrointestinal tract and acts via the H2 receptor. We report an investigation into the effect of HRH2 promoter polymorphism (rs2607474 G > A) on the methylation of DAPK and CDH1. METHODS: Non cancerous gastric mucosa samples were obtained from 115 subjects with gastric cancer (GC) and 412 non-cancer subjects (non-GC). Methylation status of genes was determined by MSP. The genotyping of rs2607474 was performed by PCR-SSCP. RESULTS: Methylation of DAPK and CDH1 was observed in 296 and 246 subjects, respectively. The frequency of CDH1 methylation in the subjects with GC was significantly lower in cancer lesion than in non cancerous mucosa, whereas that of DAPK methylation was not different. The allelic distribution of rs2607474 was 401GG, 119GA and 7AA. The GG homozygote was associated with a significantly increased risk for methylation of both DAPK and CDH1 (p < 0.0001 and p = 0.0009, respectively). In the non-GC subjects or more than 60 years of age, GG homozygote was more closely associated with both DAPK and CDH1 methylation. However, this genotype did not show an increased risk for the development of methylation of both genes in patients with GC. In H. pylori negative subjects, GG homozygote showed an increased risk for the methylation of both DAPK and CDH1 (p = 0.0074 and p = 0.0016, respectively), whereas this genotype was associated with an increased risk for the development of DAPK methylation in H. pylori positive subjects (p = 0.0018). In addition, in subjects older than 60 years of age, atrophy and metaplasia scores were significantly higher in the GG homozygote (p = 0.011 and p = 0.039, respectively) and a significant correlation was observed between age and atrophy or metaplasia. CONCLUSIONS: Our results suggest that rs2607474 GG homozygote confers a significantly increased risk for age- and inflammation-related DAPK and CDH1 methylation.

Genetic polymorphisms of SCN10A are associated with functional dyspepsia in Japanese subjects.

BACKGROUND: Visceral sensory impulses are transmitted via C-fibers from the gastrointestinal tract to the central nervous system. The tetrodotoxinresistant (TTX-r) sodium channel, Na(V) 1.8/SNS (sensory-neuron specific), encoded by SCN10A, has been identified on C-fibers. We attempted to clarify the association between functional dyspepsia (FD) and SCN10A non-synonymous polymorphisms (2884 A>G, 3218 C>T and 3275 T>C). METHODS: The study was performed in 642 subjects (345 with no symptoms and 297 with FD). We employed a multiplex polymerase chain reaction single-strand confirmation polymorphism (PCR-SSCP) method to detect the gene polymorphisms. RESULTS: The 3218 CC homozygotes had a reduced risk for the development of FD [odds ratio (OR) 0.589; 95 % confidence interval (CI) 0.402-0.864; p = 0.0067]. In addition, both 2884 A>G and 3275 T>C, which were in linkage disequilibrium, were also associated with the development of FD (p = 0.039 and 0.028, respectively). Each 2884 G carrier, 3218 CC homozygote, and 3275 C carrier had a reduced risk for the development of both epigastric pain syndrome (EPS) and postprandial distress syndrome (PDS). The subjects with the 2884 G allele, 3275 C allele, and no 3218 T allele had a reduced risk for FD (OR 0.618; 95 % CI 0.448-0.853; p = 0.0034). This haplotype was associated with a reduced risk for both EPS and PDS (p = 0.0011 and 0.0056, respectively). In addition, there was a significant association between FD and this haplotype in Helicobacter pylori-negative subjects (OR 0.463; 95 % CI 0279-0.9768; p = 0.0029). CONCLUSION: We conclude that genetic polymorphisms of SCN10A are closely associated with FD (both EPS and PDS), especially in H. pylori-negative subjects, in Japanese.

Association of genetic polymorphisms in IL17A and IL17F with gastro-duodenal diseases.

BACKGROUND & AIM: The important role of IL-17 in inflammatory response to Helicobacter pylori (H. pylori) colonization has been indicated. We investigated the associations between gastro-duodenal diseases and polymorphisms of IL17A (rs2275913 G>A) and IL17F (rs763780 T>C). METHODS: The study was performed in 548 subjects (363 controls and 185 peptic ulcer cases). The multiplex PCR-SSCP method was used to detect gene polymorphisms. RESULTS: Overall, number of rs2275913 A allele was significantly associated with an increased risk for peptic ulcer (OR, 1.50; 95%CI, 1.11-2.01; p=0.0082). The frequency of rs2275913 GA+AA genotype was also significantly higher in ulcer cases than controls (OR, 1.72; 95%CI, 1.09-2.72; p=0.020). The rs2275913 GA+AA genotype conferred an increased risk for the severity of gastric mucosal atrophy in subjects younger than 60 years (OR, 2.83; 95%CI, 1.14-7.04; p=0.025). Both atrophy and metaplasia were increased with age in rs2275913 GA+AA genotype. In NSAIDs/aspirin users, number of rs2275913 A allele was associated with an increased risk for a peptic ulcer (OR, 3.98; 95%CI, 1.48-10.7; p=0.0061). There was no association of rs763780 with the development of peptic ulcer. CONCLUSIONS: Our results provide the evidence that rs2275913 is associated with an increased risk for peptic ulcer and the severity of the gastric mucosal atrophy in comparatively younger subjects. In addition, this allele is also associated with the increased risk for peptic ulcer in NSAIDs/aspirin users.

Association between common genetic variant of HRH2 and gastric cancer risk.

Histamine plays important physiological roles in the upper gastrointestinal tract and acts via the H2 receptor. The -1018 G>A (rs2067474) in an enhancer element of the promoter and non-synonymous rs79385261 (Asn46Thr) were identified in HRH2. We attempted to clarify the associations of these polymorphisms with gastric carcinogenesis. The study was performed in 321 patients with gastric cancer and 599 subjects with no evidence of gastric malignancies on upper gastroduodenal endoscopy. The genotypes were determined using a one-tube multiplex PCR-SSCP method. The degree of gastritis was assessed in 496 subjects and serum pepsinogen (PG) I/II levels were measured in 124 subjects without gastric cancer. The minor allele of Asn46Thr could not be detected. The frequencies of the -1018 A allele in the non-GC and GC groups were 13.5% and 8.26%, respectively (p=0.00077). Overall, -1018 GG homozygotes had an increased risk for developing gastric cancer (OR 1.68; 95% CI 1.17-2.42; p=0.0052), especially intestinal type cancer (OR 1.94; 95% CI 1.23-3.08; p=0.0047). In subjects aged >60 years, the adjusted risk for gastric cancer among individuals who were -1018 GG homozygotes was 1.87 (range 1.19-2.93; p=0.0065) compared with A carriers. In the gastric cancer cases located in the antrum and at comparative advanced stage, -1018 GG homozygosity was a significantly increased risk factor. In subjects >60 years, the metaplasia score was significantly higher in -1018 GG homozygotes than A carriers. Both atrophy and metaplasia scores were significantly increased with age only in -1018 GG homozygotes. The PG I/II ratio was significantly decreased in H. pylori positive GG homozygotes than negative GG homozygotes and positive A carriers. Our results suggest that -1018 GG homozygosity of HRH2 may be associated with the severity of gastric mucosal atrophy. This genotype has an increased risk for the subsequent development of gastric cancer, especially intestinal type, at advanced age.

Genetic polymorphisms of IL17A and pri-microRNA-938, targeting IL17A 3'-UTR, influence susceptibility to gastric cancer.

We report an association between gastric cancer (GC) and polymorphisms in IL17A, rs2275913 (-197 G > A), rs3748067 (*1249 C > T), and pri-miR-938, rs2505901 (T > C). We employed the multiplex PCR-SSCP method to detect gene polymorphisms in 337 GC cases and 587 controls. The minor allele frequency of rs2275913 was significantly higher, and those of rs3748067 and rs2505901 significantly lower, in GC cases than controls. The rs2275913 AA homozygote was associated with an increased risk (OR, 2.38; 95%CI, 1.63-3.46; p < 0.0001) for the development of both intestinal and diffuse types of GC. The rs3748067 T polymorphism was associated with a decreased risk for intestinal GC (OR, 0.511; 95%CI, 0.272-0.962; p = 0.037), whereas rs2505901 C locus carried a decreased risk overall for GC (OR, 0.733; 95%CI, 0.545-0.985; p = 0.039). In addition, rs3748067 T allele was inversely correlated with lymph node metastasis. Our results suggest that polymorphisms in both IL17A and pri-miR-938 contribute to cancer risk susceptibility and therefore can affect the development of gastric cancer.