Liver Tissue Specimens Research Articles

Endoscopic ultrasound-guided liver biopsy using a 20-gauge fine needle biopsy needle with the wet-heparinized suction technique.

The primary aim of this study was to evaluate the histological adequacy of the liver tissue specimens obtained with a 20-gauge fine-needle biopsy needle and the secondary aim was to test the safety endoscopic ultrasound-guided liver biopsy with a 20-gauge fine-needle biopsy needle with the wet-heparinized suction technique. Forty patients who underwent endoscopic ultrasound-guided liver biopsy were included in the study. A 20-gauge fine-needle biopsy needle was used with the wet-heparinized suction technique to make one pass each from the left and the right lobe. Histologic characteristics of the specimens were evaluated, and patients were observed after the procedure in order to intervene in case of an adverse event. The median longest core fragment was 22 mm from the left lobe [first quartile-third quartile 20-25 mm, interquartile range (IQR) 5 mm], and 20 mm (first quartile-third quartile 17-22 mm, IQR 5 mm) from the right lobe. The median cumulative core length per patient was 103 mm (91-108 mm, IQR 17 mm). The median cumulative number of complete portal triads per patient was 69.50 (52.25-82.25, IQR 30). The rate of diagnostic yield was 100%. Post-biopsy self-limiting abdominal pain was reported in two patients (5%). The most common histologic diagnosis was fatty liver disease (25%). Endoscopic ultrasound-guided liver biopsy with the wet-heparinized suction technique using a 20-gauge fine-needle biopsy needle is a safe alternative method in clinical practice.

Gastric banding-associated weight loss diminishes hepatic Tsukushi expression

Obesity has emerged as a substantial global healthcare issue that is frequently associated with insulin resistance and non-alcoholic fatty liver disease (NAFLD). Tsukushi (TSK), a liver-derived molecule, was recently identified as a major driver of NAFLD. Laparoscopic adjustable gastric banding (LAGB) has proven effective in reducing body weight and improving NAFLD. We therefore aimed to investigate the relation between LAGB-induced weight loss and TSK expression. Twenty-six obese patients undergoing LAGB were included in the study and metabolic parameters were assessed before (t0) and six months after LAGB (t6). The expression of TSK in liver and subcutaneous adipose tissue (AT) specimens was determined at both time points. To unravel regulatory mechanisms of TSK expression, human peripheral blood mononuclear cells (PBMCs) were stimulated with pro-inflammatory cytokines and TSK mRNA levels were analyzed by quantitative polymerase chain reaction.LAGB induced pronounced weight loss which was paralleled by amelioration of metabolic disturbances and histologically defined NAFLD. While hepatic TSK expression was markedly decreased after LAGB, adipose tissue TSK expression remained comparable to baseline. The decline in hepatic TSK expression after LAGB positively correlated with weight loss and the reduction in BMI, and negatively correlated with NAFLD activity score (NAS). In human PBMCs, pro-inflammatory cytokines such as IL-1β and TNFα induced the expression of TSK. In conclusion, LAGB-induced weight loss reduces hepatic TSK expression. Inhibiting TSK might represent a promising target for treating NAFLD in the future.

Diagnostic and prognostic roles of circulating miRNA-223-3p in hepatitis B virus-related hepatocellular carcinoma.

Circulating microRNAs (miRNAs) have been shown to dysregulate in many cancer types including hepatocellular carcinoma (HCC). The purpose of this study was to examine the potential diagnostic or prognostic roles of circulating miRNAs in patients with hepatitis B virus (HBV)-related HCC. Paired cancerous and adjacent non-cancerous liver tissue specimens of patients with HBV-related HCC were used as a discovery set for screening 800 miRNAs by a Nanostring quantitative assay. Differentially expressed miRNAs were then examined by SYBR green quantitative RT-PCR in a validation cohort of serum samples obtained from 70 patients with HBV-related HCC, 70 HBV patients without HCC and 50 healthy controls. The discovery set identified miR-223-3p, miR-199a-5p and miR-451a significantly lower expressed in cancerous tissues compared with non-cancerous tissues. In the validated cohort, circulating miR-223-3p levels were significantly lower in the HCC group compared with the other groups. The combined use of serum alpha-fetoprotein and miR-223-3p displayed high sensitivity for detecting early HCC (85%) and intermediate/advanced stage HCC (100%). Additionally, serum miR-223-3p had a negative correlation with tumor size and BCLC stage. On multivariate analysis, serum miR-223-3p was identified as an independent prognostic factor of overall survival in patients with HCC. In contrast, circulating miRNA-199a-5p and miR-451a did not show any clinical benefit for the diagnosis and prognostic prediction of HCC. Our results demonstrated that miR-223-3p was differentially expressed in cancerous compared with paired adjacent non-cancerous tissues. In addition, circulating miRNA-223-3p could represent a novel diagnostic and prognostic marker for patients with HBV-related HCC.

Fine-Needle Aspiration for the Evaluation of Hepatic Pharmacokinetics of Vaniprevir: A Randomized Trial in Patients With Hepatitis C Virus Infection.

Fine-needle aspiration (FNA) for serial hepatic sampling may be an efficient and less invasive alternative to core needle biopsy (CNB), the current standard for liver tissue sampling. In this randomized, open-label trial in 31 participants with hepatitis C virus genotype 1 infection (NCT01678131/Merck protocol PN048), we evaluated the feasibility of using FNA to obtain human liver tissue samples appropriate for measuring hepatic pharmacokinetics (PK), using vaniprevir as a tool compound. The primary end point was successful retrieval of liver tissue specimens with measurable vaniprevir concentrations at two of three specified FNA time points. Twenty-nine patients met the primary end point and, therefore, were included in the PK analyses. Hepatic vaniprevir concentrations obtained with FNA were consistent with known vaniprevir PK properties. The shape of liver FNA and CNB concentration-time profiles were comparable. In conclusion, FNA may be effective for serial tissue sampling to assess hepatic drug exposure in patients with liver disease.

Frequency of somatic mutations in TERT promoter, TP53 and CTNNB1 genes in patients with hepatocellular carcinoma from Southern Italy.

Somatic mutations in the TERT promoter and in the TP53 and CTNNB1 genes are considered drivers for hepatocellular carcinoma (HCC) development. They show variable frequencies in different geographic areas, possibly depending on liver disease etiology and environmental factors. TP53, CTNNB1 and TERT genetic mutations were investigated in tumor and non-tumor liver tissues from 67 patients with HCC and liver tissue specimens from 41 control obese subjects from Southern Italy. Furthermore, TERT expression was assessed by reverse transcription-quantitative PCR. Neither CTNNB1 mutations or TP53 R249S substitution were detected in any case. The TP53 R72P polymorphism was found in 10/67 (14.9%) tumors, but was not found in either non-tumor tissues (P=0.001) or controls (P=0.009). TERT gene promoter mutations were found in 29/67 (43.3%) tumor tissues but were not found in either non-tumor (P<0.0001) or control liver specimens (P<0.0001). The most frequent mutation in the tumors was the known hot spot at −124 bp from the TERT ATG start site (−124G>A, 28 cases, 41.8%; P<0.0001). A new previously never reported TERT promoter mutation (at −297 bp from the ATG, −297C>T) was found in 5/67 (7.5%) tumors, in 0/67 (0%) non-tumor (P<0.0001), and in 0/41 (0%) controls (P=0.07). This mutation creates an AP2 consensus sequence, and was found alone (1 case) or in combination (4 cases) with the −124 bp mutation. The mutation at −124 and −297 bp induced a 33-fold (P<0.0001) and 40-fold increase of TERT expression levels, respectively. When both mutations were present, TERT expression levels were increased >300-fold (P=0.001). A new TERT promoter mutation was identified, which generates a de novo binding motif for AP2 transcription factors, and which significantly increases TERT promoter transcriptional activity.

Safety and efficacy of parenteral glutathione as a promising skin lightening agent: A controlled assessor blinded pharmacohistologic and ultrastructural study in an animal model.

Hyperpigmentation was induced in the skin of experimental animals using UVB at 6 J/cm2 three times a week for three consecutive weeks. Subsequently, glutathione was injected intraperitoneally in the experimental animals at doses of 10, 20, and 40 mg/kg body weight three times a week for three consecutive weeks. At the end of the experiment, blood samples and lung, kidney, liver, and skin tissue specimens were collected from animals for hematological, biochemical, histological, and electron microscopy examination. Glutathione at 40 mg/kg body weight/day reduced skin hyperpigmentation significantly, except at low doses. The skin lightening effect assessed by a chromameter was dose-dependent. There were no statistically significant differences among the mean values of AST, ALT, creatinine, BUN, and CBC counts across the four groups. Lung, kidney, and liver tissue specimens did not show any histological toxic changes. The number of melanin granules was significantly lower in the group treated with the highest dose of glutathione compared to that in the control. Electron microscopy proved that glutathione at 20 and 40 mg/kg body weight/day was able to reduce the number of melanized cells significantly compared to that in the control. Parenteral glutathione was effective as a skin lightening agent and did not provoke any toxic effects in the employed animal model. The limitation of the study was conducted in guinea pigs and was of short-term duration.

Histological grading of hepatic amyloidosis in ducks

Duck farming is very popular in various parts of the world. Even though ducks are highly predisposed to amyloidosis, study on histological grading of amyloidosis and its correlation with gross findings in the visceral organs are scarce. In the current study, we necropsied 112 duck carcasses over a period of one year and the occurrence of hepatic amyloidosis was studied and histological scoring was made. The study revealed amyloidosis in 61.61 per cent carcasses. We analysed the livers of such ducks histologically for the extent and pattern of amyloid deposits. The extent of amyloid deposition was graded as mild, moderate or severe. In severe amyloidosis without cellularity, the liver grossly showed deep yellow discolouration of the parenchyma. In moderate amyloidosis characterised by islands of hepatocytes and erythrocytes, the liver grossly showed pale pink to brownish discolouration. In mild amyloidosis with preserved hepatocellular architecture, the gross discolouration of the liver was minimum. The histological severity of amyloidosis could be directly correlated with the gross features in liver tissue specimens.

Two-Photon Autofluorescence Imaging of Fixed Tissues: Feasibility and Potential Values for Biomedical Applications.

The value of optical redox imaging (ORI) of cells/tissues based on the intrinsic fluorescences of NADH (nicotinamide adenine dinucleotide) and oxidized flavoproteins (containing flavin adenine dinucleotide, i.e., FAD) has been demonstrated for potential biomedical applications including diagnosis, prognosis, and determining treatment response. However, the Chance redox scanner (a 3D cryogenic tissue imager) is limited by spatial resolution (~50μm), and tissue ORI using fluorescence microscopy (single or multi-photon) is limited by the light penetration depth. Furthermore, viable or snap-frozen tissues are usually required. In this project, we aimed to study whether ORI may be achieved for unstained fixed tissue using a state-of-the-art modern Serial Two-Photon (STP) Tomography scanner that can rapidly acquire multi-plane images at micron resolution. Tissue specimens of mouse muscle, liver, and tumor xenografts were harvested and fixed in 4% paraformaldehyde (PFA) for 24h. Tissue blocks were scanned by STP Tomography under room temperature to acquire the autofluorescence signals (NADH channel: excitation 750nm, blue emission filter; FAD channel: excitation 860nm, green emission filter). We observed remarkable signals with significant intra-tissue heterogeneity in images of NADH, FAD and redox ratio (FAD/(NADH+FAD)), which are worthy of further investigation for extracting biological information.

Sample Preparation of Optically Cleared Liver Tissue to Identify Liver Macrophages Using 3D Microscopy.

Histology, or the study of tissue microanatomy, is essential to understanding the in situ function of varying cell types within an organ. How cells are distributed throughout organs provides an indication of how they interact with other cells and structures within the organ microanatomy. The tortuous shape and large size of liver macrophages limits the value of standard tissue thickness of 5-10μm. As a result, imaging of specimens ideally thicker than 100μm is necessary to investigate the liver microanatomy and the how macrophages are distributed within this. Modern methods of microscopy, such as confocal and light sheet microscopy, allow for the analysis of tissue specimens of a thickness well beyond 100μm in the z-dimension. Liver tissue is an opaque tissue, and as a result, different techniques are needed to ameliorate light diffraction within the tissue. These techniques, in conjunction with antibody staining and refractive index matching of the tissue, have allowed researchers to image liver tissue specimens of more than 100μm thickness. Two of these techniques are modified versions of the clearing methods known as clearing-enhanced 3D (Ce3D) and fructose, urea, and glycerol for imaging (FUnGI). Here, we discuss the steps involved in preparing tissue specimens for optically clearing tissue using Ce3D and FUnGI for subsequent analysis of the distribution of macrophages in three dimensions using a confocal microscope.

Increased expression of protease-activated receptors 2 indicates poor prognosis in HBV related hepatocellular carcinoma

ObjectiveTo investigate the potential role of protease-activated receptor 2 (PAR2) in the prognosis of hepatocellular carcinoma (HCC).MethodsA total of 202 HCC patients who underwent liver resections were included. Tissue microarray was established with specimens of both HCC and paired adjacent liver tissues. PAR2 expression was detected by immunohistochemistry (IHC) assays.. A semi-quantification method was used to define the expression level of PAR2. The correlations between PAR2 expression and clinical features of patients with HCC was explored. The association of different PAR2 expressions with both overall survival and disease-free survival was analyzed.ResultsResults showed that the expression of PAR2 in HCC tissues was higher than that in paired para-cancerous liver tissues (4.12 ± 3.55 vs. 2.71 ± 2.56, P < 0.001). Higher expression of PAR2 was associated with poor differentiation (P < 0.001) and advanced tumor-node-metastasis stage (P = 0.015). Kaplan-Meier survival analysis indicated that HCC patients with high PAR2 expression had decreased overall survival (P = 0.033) and disease-free survival (P = 0.043) compared to patients with lower PAR2 expression. Multivariate analysis indicated that PAR2 expression (P = 0.032) was a significant independent prognostic factor for both overall survival and disease-free survival (P = 0.032; P = 0.032, respectively).ConclusionOur data revealed that PAR2 expression was increased in HCC. High PAR2 expression was correlated with both decreased overall survival and disease-free survival in patients with HCC. High PAR2 expression indicated a poor prognosis.

Effect of the elapsed time between sampling and formalin fixation on the N-glycosylation profile of mouse tissue specimens.

Formalin-fixed, paraffin-embedded (FFPE) samples are generally used for histology-study, however, they also possess important molecular diagnostics information. While it has been reported that the N-glycan moieties of glycoproteins is not affected by the FFPE process, no information is available about the effect of the elapsed time between sampling and fixation on the resulting N-glycosylation profile. In this study, lung, brain, heart, spleen, liver, kidney, and intestine mouse tissue specimens were used for N-glycan profiling analysis and the elapsed sampling time effect was investigated with the lung tissue. N-glycan extraction from the tissue samples was performed by glycoprotein retrieval from the FFPE specimens using radioimmunoprecipitation assay (RIPA) buffer followed PNGase F digestion. The released oligosaccharides were fluorophore labeled and analyzed by capillary electrophoresis-laser induced fluorescent detection (CE-LIF). N-glycosylation profiles of freshly collected lung-tissue samples (zero time point), as well as 1 and 2 h after sampling were compared by carbohydrate profiling and exoglycosidase treatment based deep glycomic analysis. It was found that up to two hours of room temperature storage of tissue specimens apparently did not cause changes in the N-glycosylation profiles of complex carbohydrates, but resulted in considerable decrease in the amount of linear glucose oligomers and high mannose type glycans present in the samples.

Zinc nanoparticles coated with Gallic acid potentially ameliorate hepatocellular carcinoma-induced in rats

Materials on the nanoscale are increasingly being targeted to cancer cells with great specificity through both active and passive targeting. In the current study, the potential therapeutic effect of the newly synthesized Zinc nanoparticles coated with Gallic acid (Zn-GANPs) against hepatocellular carcinoma (HCC) - induced in rats were evaluated. Forty rats were divided into four equal groups, group 1: normal control, group 2: received diethylnitrosamine (DEN) (20 mg/Kg b.wt, 5 times per week for 8 weeks), group 3: received Zn-GANPs (20 mg/kg b.wt/day, i.p) from the onset of the 9th week till the end of the experiment (13 weeks), group 4: DEN + Zn-GANPs treated group: Rats were received DEN as group (2) and subsequently treated with Zn-GANPs as group (3) till the end of the experiment (13 weeks).Blood samples and liver tissue specimens were collected for biochemical analysis and histopathological examination. The obtained results revealed that in HCC-induced rats serum aspartate aminotransferase (AST) activity, total bilirubin and creatinine levels were increased, while total protein, albumin concentrations and liver catalase activity were significantly decreased as compared with control normal group. Administration of Zn-GANPs to HCC-induced rats non significantly reduced serum AST activity, total bilirubin and creatinine concentrations. However, serum total protein and albumin levels were non significantly increase and liver catalase activity was markedly increased in Zn-GANPs treated liver cancer group. Ameliorative effect of Zn-GANPs was appreciated with the histopathological alterations observed in DEN groups. These result suggesting the efficacy of Zn-GaNPs administration as an anti-HCC.

Quantitative Expression of RNA from Frozen Organs and Formaldehyde-fixed and Paraffin-embedded Tissues.

Objective Quantitative analysis and comparison of the expression of ribonucleic acid （RNA） from frozen organs and formaldehyde-fixed and paraffin-embedded （FFPE） tissues. Methods Frozen specimens of human brain, myocardium and liver tissues as well as FFPE samples at different postmortem intervals were collected and mass concentration of RNA was extracted and detected. Reverse transcription-quantitative polymerase chain reaction （RT-qPCR） technology was used to analyze the amplification efficiency and relative expression of each RNA marker. Results The mass concentration and integrity of RNA extracted from FFPE samples were relatively low compared with frozen specimens. The amplification efficiency of RNA markers was related with RNA species and the length of amplification products. Among them, glyceraldehyde-3-phosphate dehydrogenase （GAPDH） and β-actin （ACTB） with relatively long amplification products failed to achieve optimal amplification efficiency, whereas 5S ribosomal RNA （5S rRNA） achieved ideal amplification efficiency and showed quite stable expression across various tissues, therefore it was chosen as internal reference marker. The expression quantity of GAPDH and ACTB in frozen specimens with longer postmortem intervals and in FFPE samples with relatively long amplification products was decreased. The expressions of tissue-specific microRNAs （miRNAs）, GAPDH and ACTB with relatively short amplification products had consistency in the same tissues and FFPE samples. Conclusion Through standardizing the RT-qPCR experiment, selecting the appropriate RNA marker and designing primers of appropriate product length, RNA expression levels of FFPE samples can be accurately quantified.

A Novel Automatic Digital Algorithm that Accurately Quantifies Steatosis in NAFLD on Histopathological Whole-Slide Images.

BackgroundAccurate assessment of hepatic steatosis is a key to grade disease severity in non‐alcoholic fatty liver disease (NAFLD).MethodsWe developed a digital automated quantification of steatosis on whole‐slide images (WSIs) of liver tissue and performed a validation study. Hematoxylin–eosin stained liver tissue slides were digitally scanned, and steatotic areas were manually annotated. We identified thresholds for size and roundness parameters by logistic regression to discriminate steatosis from surrounding liver tissue. The resulting algorithm produces a steatosis proportionate area (SPA; ratio of steatotic area to total tissue area described as percentage). The software can be implemented as a Java plug‐in in FIJI, in which digital WSI can be processed automatically using the Pathomation extension.ResultsWe obtained liver tissue specimens from 61 NAFLD patients and 18 controls. The area under the curve of correctly classified steatosis by the algorithm was 0.970 (95% CI 0.968–0.973), P < 0.001. Accuracy of the algorithm was 91.9%, with a classification error of 8.1%. SPA correlated significantly with steatosis grade (Rs = 0.845, CI: 0.749–0.902, P < 0.001) and increased significantly with each individual steatosis grade, except between Grade 2 and 3.ConclusionsWe have developed a novel digital analysis algorithm that accurately quantifies steatosis on WSIs of liver tissue. This algorithm can be incorporated when quantification of steatosis is warranted, such as in clinical trials studying efficacy of new therapeutic interventions in NAFLD. © 2019 The Authors. Cytometry Part B: Clinical Cytometry published by Wiley Periodicals, Inc. on behalf of International Clinical Cytometry Society.

Activation of Hedgehog Signaling in Aggressive Hepatic Hemangioma in Newborns and Infants.

Hepatic hemangiomas (HH) can show an aggressive course with significant complications. Prognostic markers that identify an aggressive course are entirely absent. Since we have showed that Hedgehog signaling is overexpressed in aggressive hemangiomas of the skin. Here, we hypothesize that it is also altered in aggressive HH. Immunohistological staining for GLUT1 and quantitative PCR was performed in seven specimens with aggressive HH. For comparison, we included specimens of kaposiform hemangioendothelioma (KHE), skin hemangioma and normal liver tissue. Overexpression of the Hedgehog signaling components SHH and GLI2 and its target gene FOXA2 in HH were similar to those found in aggressive skin hemangioma and KHE, their expression being significantly higher than in mild skin hemangioma. High expression levels of SHH and FOXA2 positively correlated with HH, but not with normal liver tissue. Hedgehog signaling is up-regulated in aggressive HH. This finding may lead to a biomarker allowing early intervention.

Necrostatin-1 accelerates time to death in a rat model of cecal ligation and puncture and massively increases hepatocyte caspase-3 cleavage.

Necroptosis, a form of regulated necrosis, has been reported to be involved in numerous pathologies, including sepsis. However, a protective effect of the selective inhibitor of necroptosis, necrostatin-1 (Nec-1), against sepsis remains to be confirmed. Animals (rats and mice) were subjected to cecal ligation and puncture (CLP) to mimic clinical sepsis. Nec-1 or its vehicle (control) was administered 20 min before CLP. Survival time was observed up to 72 h after CLP. Specimens of liver tissue and serum were obtained at 6 h, 12 h, and 18 h. Expression of necroptosis-related proteins [receptor-interacting protein kinase (RIP)1, RIP3, and mixed lineage kinase domain-like (MLKL)] was determined by Western blot analysis. The RIP1/RIP3 interaction and the recruitment of MLKL to RIP3 were also analyzed. Liver function, histopathological changes, serum inflammation cytokines, TUNEL staining, and the expression of apoptosis-related protein, including caspase-3, B-cell lymphoma 2 (Bcl-2), and Bcl-2-associated X (Bax), was determined. As expected, Nec-1 administration reduced the expression of necroptosis-related proteins and the RIP1/RIP3 interaction, indicating inhibited necroptosis. Surprisingly, Nec-1 treatment exacerbated the liver injury and shortened survival time of septic rats with increased TUNEL-positive cells, cleaved caspase-3 protein content, and Bax/Bcl-2 ratio. Collectively, these findings show that Nec-1 administration inhibited the hepatocyte necroptosis pathway but accelerated apoptosis via the apoptotic pathway in CLP-induced sepsis rat. NEW & NOTEWORTHY The present study demonstrated that a chemical inhibitor necrostatin-1 (Nec-1) or receptor-interacting protein kinase(RIP1) knock down targeted at necroptosis inhibition accelerated liver injury of following sepsis. For fundamental research, these results warrant further investigation of the potential link between Nec-1 administration and the cellular apoptosis following sepsis induced liver injury. For applied research, these results suggest the potential harmful effect of Nec-1 on future sepsis treatment.

A Potent Antioxidants Property of Moringa Oleifera on Mycotoxin Induced Oxidative Stress in Rats

This work aimed to evaluate the probable protective effect of Moringa Oleifera on mycotoxins induced liver damage and oxidative stress in rats. Thirty male albino rats were classified randomly into three equal groups. Group1 (normal control): Rats fed with ordinary ration. Group2(Mycotoxin): Rats fed with ordinary diet only for first 7 days and from day 8 rats fed mixed ration contain 50 gram of grinded plant (total aflatoxins 4.2 ppb) + 50 gram of grinded fish (total aflatoxins 42.14 ppb) + 50 gram of ordinary diet (free of aflatoxins) for 4 weeks. Group3 (Mycotoxin+ Moringa): Moringa Oleifera (350 mg/kg b.w) was administered orally allover the experimental period (5 weeks). Also, rats were fed with ordinary ration only for first 7 days and from day 8 rats fed mixed ration that mentioned above in group 2. At the end of the experiment liver tissue specimens were isolated and analyzed for the determination of reduced Glutathione (GSH), superoxide dismutase (SOD), catalase (CAT), L-malondialdehyde (L-MDA) and tumor necrosis factor-alpha (TNF-α) gene expression. The obtained results showed a significant up-regulation of TNF-α gene expression level and marked increase in hepatic SOD, CAT activities and L-MDA of mycotoxin rats group. However, liver GSH concentration was markedly decreased as compared to control group. Moringa protected mycotoxins induced liver damage in rats caused a significant improvement of all previous parameters. Conclusively, MoringaOleiferatreatment exerts a protective effect against liver damage and oxidative stress in liver of mycotoxin intoxicated rats through free radical scavenging and anti-inflammatory activities as well as regenerating endogenous antioxidants defense system mechanisms.

Autotaxin and vascular endothelial growth factor receptor-2 and -3 are related to vascular development during the progression of chronic viral hepatitis C.

Vascular endothelial growth factor (VEGF) and autotaxin (ATX) play important roles in embryonic vasculogenesis and cancer progression. This study examines whether these two angiogenic factors cooperate in the mechanism that regulates vascular development during the progression of chronic viral hepatitis C (CVH-C) (Inuyama classification, F1-F4). First, surgical wedge biopsy specimens and needle biopsy specimens were obtained. Immunohistochemical staining for ATX and vascular endothelial growth factor receptor was assessed in serial sections. Immunoelectron microscopy was conducted with a perfusion-fixation method. In normal control liver tissue specimens, ATX was expressed at low levels within the branches of the hepatic artery and hepatic sinusoids. In F1 CVH-C liver tissue specimens, ATX was expressed within the branches of the hepatic artery. Additionally, VEGFR-2 was expressed within the branches of the hepatic artery and capillaries. In F3-F4 CVH-C liver tissue specimens, positive staining for ATX and VEGFR-2 or VEGFR-3 was detected in the branches of the hepatic artery or microlymphatic vessels. ATX-1 reaction products were specifically expressed on the plasma membrane of some microvascular endothelial cells (ECs) in the proliferative capillary artery. VEGFR-2 was expressed on caveolae in ECs and vascular smooth muscle cells. VEGFR-3 immunogold particles were also observed in lymphatic ECs. These results suggest functional interactions among ATX, VEGFR-2, and VEGFR-3 in the modulation of hemovascular and lymphovascular cell activation during vascular development.

Effect of Hepatic Artery Spasm on a Rat Model of Hepatic Ischemia-Reperfusion Injury.

To investigate hemodynamic changes in the hepatic artery after hepatic ischemia-reperfusion injury (IRI) in rats via ultrasound (US) imaging and to discuss the protective effect of phentolamine (PHT) pretreatment on hepatic IRI. Fifty rats were randomly divided into 3 groups: a sham operation group (n = 10), a control ischemia-reperfusion group (n = 20), and a PHT pretreatment group (n = 20). Color Doppler flow imaging and contrast-enhanced US examinations were performed in each group at 30 minutes (n = 10) and 90 minutes (n = 10) after reperfusion. Blood samples were obtained to analyze serum alanine aminotransferase and aspartate aminotransferase levels, and liver tissue specimens were collected for pathologic analysis. Using US, we found that hepatic artery resistance at 30 minutes after reperfusion in the control group was higher than that in the sham group (mean resistive index [RI] ± SD, 0.65 ± 0.09 versus 0.50 ± 0.09; P < .01), which was higher at 30 than 90 minutes (RI, 0.65 ± 0.09 versus 0.50 ± 0.08; P < .01) after reperfusion in the control group. However, the hepatic artery resistance and liver microcirculation in the PHT group were better than those in the control group at 30 minutes after reperfusion (RI, 0.54 ± 0.09 versus 0.65 ± 0.09; P < .05; time to peak, 31.94 ± 2.02 versus 48.34 ± 4.74 seconds; P < .01). Compared to the control group, the aspartate aminotransferase and alanine aminotransferase levels were significantly lower at 30 minutes after reperfusion in the PHT group (P < .05). A pathologic examination revealed a smaller hepatic artery diameter and a depressed vessel wall in the control group. The hepatic artery can undergo a transient spasm during the hepatic IRI process, which can exacerbate liver damage. Phentolamine treatment can alleviate hepatic artery spasms, improve liver perfusion, and reduce liver injury by ameliorating the hepatic microcirculation.

Abstract 3316: Hypermethylation at the SMPD3 promoter is frequently detected in hepatocellular carcinoma tumor tissue and corresponding plasma circulating DNA, and potentially differs by tumor subtype

Abstract Background: Circulating plasma DNAs may exhibit altered methylation reflective of aberrant epigenetics in tumor tissues, which may present a clinical opportunity for non-invasive assessment of tumor molecular phenotype. Hypermethylation at the promoter region of SMPD3 (Sphingomyelin phosphodiesterase 3), a tumor suppressor gene, has been identified in several genome-wide DNA methylation studies involving tissue specimens of hepatocellular carcinoma (HCC) (Shen et al., 2012; Song et al., 2013). The purpose of this study was to measure and compare SMPD3 promoter hypermethylation (specifically at cg10426893) in tumors, adjacent liver tissue, and circulating cell-free DNA (cfDNA) to examine its potential as a biomarker of HCC corresponding with tumor molecular subtype. Methods: Blood and liver tissue specimens were collected following written informed consent from 54 patients undergoing partial liver resection for BCLC stage A HCC. DNA methylation was quantified through pyrosequencing analysis of bisulfite-treated DNAs from corresponding tumor tissue, adjacent liver tissue, plasma, and buffy-coat samples. Results were compared within patients using paired t-test. Transcriptomic molecular sub-classification based on the Hoshida classification system (2009) was performed on a subset of 39 tumors to explore potential associations between SMPD3 promoter hypermethylation and HCC molecular subtype. Results: Levels of SMPD3 promoter methylation were significantly higher in tumor tissue compared to adjacent liver tissue (37.0% vs. 8.8%, p &amp;lt; 0.0001) as well as when compared to cfDNA samples (32.9% vs. 14.3%, p &amp;lt; 0.0001). SMPD3 promoter methylation levels in cfDNA and tumor tissues were also significantly higher than in corresponding buffy-coat samples (p = 0.0146 and p &amp;lt; 0.0001, respectively). Tumors belonging to the S3 molecular sub-class (n = 22) exhibited significantly higher SMPD3 promoter methylation levels than non-S3 tumors (n = 17) (44.1 % vs. 24.1%, p = 0.0166). Conclusions: Significant increases in DNA methylation involving the promoter region of SMPD3 in HCC tumor tissues are reflected in cfDNA, supporting potential clinical liquid biopsy applications. Preliminarily, this epigenetic alteration appears to be associated with the S3 (differentiation) molecular subtype of HCC. Citation Format: Sandi Alexander Kwee, Karolina Peplowska, Maarit Tiirikainen, Min-Ae Song, Linda L. Wong. Hypermethylation at the SMPD3 promoter is frequently detected in hepatocellular carcinoma tumor tissue and corresponding plasma circulating DNA, and potentially differs by tumor subtype [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3316.