Circulating vascular endothelial growth factor and its soluble receptors in patients with liver cirrhosis: possible association with hepatic function impairment

Recent studies provided in vivo evidences of an increased angiogenesis in animal model of portal hypertension and cirrhosis which was linked to increased expression of vascular endothelial growth factor. The aim of study was to evaluate the plasma concentration of VEGF and its receptors in liver cirrhosis and the possible association with the degree of liver insufficiency. Methods. Vascular endothelial growth factor (VEGF) and its soluble receptors: sVEGF-R1, sVEGF-R2 were measured in plasma of 78 patients with liver cirrhosis by ELISA. Results. The significant increase of plasma VEGF and sVEGF-R1 was observed in liver cirrhosis compared to healthy individuals (153.1+/-51.9 vs. 46.8+/-4.1pg/mL, P<0.05; 279.8+/-34.3 vs. 105.1+/-5.9pg/mL, P<0.001, respectively). Plasma VEGF and foremost sVEGF R1 showed significant associations with biochemical indices of liver function. Among clinical parameters, only ascites revealed significant association with plasma VEGR and sVEGF-R1. VEGF and sVEGF-R1 were increased respectively to the degree of liver insufficiency. It was demonstrated through a significant positive correlation with Child-Pugh score and MELD classification. In conclusion, our study suggests that serum VEGF and VEGF-R1 may reflect the hepatic function impairment in liver cirrhosis and seems to be associated with portal hypertension symptoms.     

In situ hybridization of albumin mRNA in normal liver and liver tumors: Identification of hepatocellular origin

In situ hybridization was performed to detect albumin mRNA in normal liver, liver cirrhosis, primary liver tumors and secondary liver neoplasms. In areas of normal liver, and liver cirrhosis, signals for albumin mRNA were present in hepatocytes, whereas no signals were seen in other cells such as endothelial and Küpffer cells, bile duct epithelium and smooth muscle cells. In 53 of 56 hepatocellular carcinomas signals were present in tumor cells but in eight cholangiocarcinomas and 14 metastatic adenocarcinomas from large bowel or pancreas, carcinoma cells were negative for albumin mRNA. In three metastatic tumors (from two neuroendocrine carcinomas and one gastric leiomyosarcoma), tumor cells contained no signals, while the surrounding hepatocytes showed diffuse grains. In 15 of the 84 specimens examined in situ hybridization was applied to routine formalin-fixed and paraffin-embedded blocks and strong signals were obtained for albumin mRNA. We conclude that in situ hybridization of human albumin is a valid tool in the differential diagnosis of hepatocellular carcinoma from cholangiocarcinomas and tumors metastatic to the liver.     

Bone morphogenetic protein receptor 2 in patients with idiopathic portal hypertension

In idiopathic portal hypertension (IPH) typical vascular lesions are present in the branches of the portal vein or in the perisinusoidal area of the liver. Similar histological alterations have been reported in the pulmonary vasculature of patients with idiopathic pulmonary artery hypertension (IPAH). As IPAH is associated with mutations of the bone morphogenetic protein receptor 2 (BMPR2) gene, the aim of this study was to investigate whether this association might also be found in patients with IPH. Twenty-three samples belonging to 21 unrelated caucasian patients with IPH followed in the hepatic haemodynamic laboratory of the Hospital Clinic in Barcelona were included in the study. All patients were studied for the entire open reading frame and splice site of the BMPR2 gene by direct sequencing and multiple ligation probe amplification (MLPA) in order to detect large deletions/duplications. None of the 23 patients had pulmonary artery hypertension. Four patients presented one single nucleotide polymorphism (SNP) in intron 5, four patients had a SNP in exon 12 and a SNP in exon 1 was found in two cases. Two patients had both intron 5 and exon 12 polymorphisms. All SNPs were previously described. Except for these three SNPs, neither mutations nor rearrangements have been identified in the BMPR2 gene in this population. We did not detect mutations or rearrangements in the coding region of the BMPR2 gene in our patients with IPH. These findings suggest that, in contrast to IPAH, mutations in BMPR2 are not involved in the pathogenesis of IPH.     

The Injured Liver Induces Hyperimmunoglobulinemia by Failing to Dispose of Antigens and Endotoxins in the Portal System

Hyperimmunoglobulinemia is frequently observed in patients with chronic liver diseases. However, the exact mechanism underlying the high level of antibody formation is not fully understood. In our study, we provide evidence for the functional role of the liver and the stimulation of plasma cell proliferation in hyperimmunoglobulinemia. We collected sera from patients with chronic liver diseases, and the level of serum immunoglobulins in patients was examined; this was also investigated in animal models of liver cirrhosis and hepatocellular carcinoma. An end-to-side microsurgical portacaval shunt was used to mimic liver dysfunction in rats. We used portal vein serum and inferior vena cava serum to immunize healthy rats and mice in order to confirm the function of the healthy liver in disposing of antigens and endotoxins from the gut. For the analysis of the state of plasma cell activation, plasma cells from mice were stained with PE-conjugated anti-CD138 and FITC-conjugated anti-BrdU for flow cytometry analysis. Hyperimmunoglobulinemia was observed both in patients with chronic liver diseases and in related animal models, and high plasma LPS levels were also observed. There was a significant increase in the activation and proliferation of plasma cell in mice immunized with antigens or LPS-positive serum compared with controls that were immunized with antigens and LPS-negative serum. We confirmed that the healthy liver plays an important role in disposing of antigens and endotoxins derived from the gut. Hyperimmunoglobulinemia in chronic liver diseases mainly arises due to the collateral circulation secondary to portal hypertension, gut antigens and endotoxins that bypass the liver and reach the antibody-producing cells.     

Detection of gastrin and its messenger RNA in Zollinger-Ellison tumors by non-radioactive in situ hybridization and immunocytochemistry.

Gastrin immunocytochemistry and non-radioactive in situ hybridization, using biotinylated oligonucleotide probes, for gastrin mRNA have been used for studying a retrospective material of six gastrin-producing (Zollinger-Ellison) tumors. Hybridization results for gastrin mRNA were positive in all six, while gastrin immunoreactivity could be detected in five tumors. In one of the patients, different areas of the same tumor displayed differences in immunoreactivity to gastrin, but were uniformly hybridization positive. Weak hybridization signals were detected in liver metastases from a necropsy case, while the gastrin immunostaining was more pronounced. The results show that non-radioactive hybridization methods are applicable to routine clinical specimens stored for as long as 16 years and that in situ hybridization may be a useful complement to immunocytochemical diagnosis, particularly in cases where high synthesis and little storage of hormonal products occur.     

Detection of gastrin and its messenger RNA in Zollinger-Ellison tumors by non-radioactive in situ hybridization and immunocytochemistry

Summary Gastrin immunocytochemistry and non-radioactive in situ hybridization, using biotinylated oligonucleotide probes, for gastrin mRNA have been used for studying a retrospective material of six gastrin-producing (Zollinger-Ellison) tumors. Hybridization results for gastrin mRNA were positive in all six, while gastrin immunoreactivity could be detected in five tumors. In one of the patients, different areas of the same tumor displayed differences in immunoreactivity to gastrin, but were uniformly hybridization positive. Weak hybridization signals were detected in liver metastases from a necropsy case, while the gastrin immunostaining was more pronounced. The results show that non-radioactive hybridization methods are applicable to routine clinical specimens stored for as long as 16 years and that in situ hybridization may be a useful complement to immunocytochemical diagnosis, particularly in cases where high synthesis and little storage of hormonal products occur.     

Impaired hepatic removal of interleukin-6 in patients with liver cirrhosis

Systemic concentrations of interleukin-6 (IL-6) are elevated in patients with liver cirrhosis, and impaired hepatic uptake of IL-6 was suggested to contribute to higher levels in these patients. To test this hypothesis IL-6 was measured in portal venous serum (PVS), hepatic venous serum (HVS) and systemic venous serum (SVS) of 41 patients with liver cirrhosis and four patients with normal liver function. IL-6 was higher in PVS than HVS of all blood donors and about 43% of portal vein derived IL-6 was extracted by the healthy liver, and 6.3% by the cirrhotic liver demonstrating markedly impaired removal of IL-6 by the latter. Whereas in patients with CHILD-PUGH stage A IL-6 in HVS was almost 25% lower than in PVS, in patients with CHILD-PUGH stage C IL-6 was similarly abundant in the two blood compartments. Ascites is a common complication in cirrhotic patients and was associated with higher IL-6 levels in all blood compartments without significant differences in hepatic excretion. Hepatic venous pressure gradient did not correlate with the degree of hepatic IL-6 removal excluding hepatic shunting as the principal cause of impaired IL-6 uptake. Furthermore, patients with alcoholic liver cirrhosis had higher IL-6 in all blood compartments than patients with cryptogenic liver cirrhosis. Aetiology of liver cirrhosis did not affect hepatic removal rate indicating higher IL-6 synthesis in patients with alcoholic liver cirrhosis. In summary, the current data provide evidence that impaired hepatic removal of IL-6 is explained by hepatic shunting and liver dysfunction in patients with liver cirrhosis partly explaining higher systemic levels.     

Systemic and hepatic vein galectin-3 are increased in patients with alcoholic liver cirrhosis and negatively correlate with liver function

Recently we demonstrated higher galectin-3 in portal venous serum (PVS) compared to hepatic venous serum (HVS) in a small cohort of patients with normal liver function suggesting hepatic removal of galectin-3. Here, galectin-3 was measured by ELISA in PVS, HVS and systemic venous blood (SVS) of 33 patients with alcoholic liver cirrhosis and a larger cohort of 11 patients with normal liver function. Galectin-3 was cleared by the healthy but not the cirrhotic liver, and subsequently HVS and SVS galectin-3 levels were significantly increased in the patients with liver cirrhosis compared to controls. In healthy liver galectin-3 was produced by cholangiocytes and synthesis by hepatocytes was only observed in cirrhotic liver. Hepatic venous pressure gradient did not correlate with galectin-3 levels excluding hepatic shunting as the principal cause of higher SVS galectin-3. Galectin-3 was elevated in all blood compartments of patients with CHILD-PUGH stage C compared to patients with CHILD-PUGH stage A, and was higher in patients with ascites than patients without this complication. Galectin-3 was negatively associated with antithrombin-3 whose synthesis is reduced with worse liver function. Galectin-3 positively correlated with urea and creatinine, and PVS galectin-3 showed a negative association with creatinine clearance as an accepted measure of kidney function. To summarize in the current study systemic, portal and hepatic levels of galectin-3 were found to be negatively associated with liver function in patients with alcoholic liver cirrhosis and this may in part be related to impaired hepatic removal and/or increased synthesis in cirrhotic liver.     

Use of a transjugular intrahepatic portosystemic shunt combined with autologous bone marrow cell infusion in patients with decompensated liver cirrhosis: an exploratory study

Background aimsCurrently, there is no treatment for decompensated liver cirrhosis except for liver transplantation. The safety and effect on liver function of a transjugular intrahepatic portosystemic shunt (TIPS) with and without autologous bone marrow cell (BMC) infusion in patients with decompensated liver cirrhosis were determined.MethodsTen patients who were diagnosed with decompensated liver cirrhosis during the period from September 2011 to July 2012 were enrolled in this study. The patients underwent TIPS (TIPS group) or combined treatment with TIPS and BMC infusion through the hepatic artery (TIPS+BMC group). All patients were monitored for adverse events, liver function and complications caused by portal hypertension during a period of 52 weeks.ResultsThe number of infused BMCs was 2.65 ± 1.20 ×10⁹. Significant improvements in the serum levels of albumin and total bilirubin and decreased Child-Pugh scores were observed in patients treated with both TIPS and BMCs (P < 0.05), whereas no such changes were observed in the TIPS group. Endoscopic findings showed that varices in the esophagus and the gastric fundus were alleviated after either treatment. All 10 patients showed a complete or partial resolution of ascites at 4 weeks. No major adverse effects were noted during the follow-up period for patients in either group.ConclusionsTIPS combined with BMC infusion is clinically safe; the treatment improved liver function and alleviated complications caused by portal hypertension; therefore, this combination has potential for treatment of patients with decompensated liver cirrhosis.     

Detection of hepatitis C virus RNA in the hearts of patients with hepatogenic cardiomyopathy

We examined the Hepatitis C virus (HCV) genome in the myocardium and liver obtained at autopsy from seven patients with HCV-positive liver cirrhosis and hepatocellular carcinoma (HCC) by in situ hybridization and histopathological studies. The HCV virus genome was detected in the myocardium of one patient as well as in the liver in three out of seven patients. However, Epstein-Barr (EB) virus genome could not be detected in liver or myocardium. In the patient who showed positive reaction to HCV in myocardium, both serum HCV and Hepatitis B virus (HBV) antibodies were positive. It is unknown whether this was related to an immunological abnormality of the host or to an interaction between RNA and DNA viruses. In conclusion, we could identify the HCV genome in the myocardium of a patient with hepatogenic myocardosis.     

Smo 蛋白在肝癌和肝硬化中的表达及临床意义

目的:研究Smoothened(SMO)在肝癌和肝硬化中的表达及临床意义。方法:选取组织标本后,运用石蜡包埋,切片后,HE染色,构建组织芯片,免疫组织化学和原位杂交方法检测Smo蛋白在肝癌和肝硬化中的表达。结果:Smo蛋白在肝癌细胞浆、良性肝肿瘤组织细胞浆、肝硬化组织中强染色,在正常组织中无染色。并且在典型肝硬化中强染色,在中度肝硬化中弱阳性。结论:Smo蛋白表达与肝癌的发生有关,Smo基因的高表达可能激活某种机制而参与诱导肝癌的发生。可能是通过异常激活Sonic hedgchog信号通路,从而诱导肝癌的发生与发展。     

A novel canine model of portal vein stenosis plus thioacetamide administration-induced cirrhotic portal hypertension with hypersplenism

Current large animal models that could closely resemble the typical features of cirrhotic portal hypertension in human have not been well established. Thus, we aimed to develop and describe a reliable and reproducible canine cirrhosis model of portal hypertension. A total of 30 mongrel dogs were randomly divided into four groups: 1 (control; n = 5), 2 (portal vein stenosis [PVS]; n = 5], 3 (thioacetamide [TAA]; n = 5), and 4 (PVS plus TAA; n = 15). After 4-months modeling period, liver and spleen CT perfusion, abdominal CT scans, portal hemodynamics, gastroscopy, hepatic function, blood routine, the bone marrow, liver, and spleen histology were studied. The animals in group 2 (PVS) developed extrahepatic portosystemic collateral circulation, particularly esophageal varices, without hepatic cirrhosis and portal hypertension. Animals from group 3 (TAA) presented mild cirrhosis and portal hypertension without significant symptoms of esophageal varices and hypersplenism. In contrast, animals from group 4 (PVS + TAA) showed well-developed micronodular and macronodular cirrhosis, associated with significant portal hypertension and hypersplenism. The combination of PVS and TAA represents a novel, reliable, and reproducible canine cirrhosis model of portal hypertension, which is associated with the typical characteristics of portal hypertension, including hypersplenism.     

Circulating microRNA-200 Family as Diagnostic Marker in Hepatocellular Carcinoma

Goals

In this clinical study, we aimed to evaluate the role of circulating microRNA-200 family as a non-invasive tool to identify patients with cirrhosis-associated hepatocellular carcinoma (HCC).

Background

Prognosis of HCC remains poor with increasing incidence worldwide, mainly related to liver cirrhosis. So far, no reliable molecular targets exist for early detection of HCC at surgically manageable stages. Recently, we identified members of the microRNA-200 family as potential diagnostic markers of cirrhosis-associated HCC in patient tissue samples. Their value as circulating biomarkers for HCC remained undefined.

Methods

Blood samples and clinicopathological data of consecutive patients with liver diseases were collected prospectively. Expression of the microRNA-200 family was investigated by qRT-PCR in blood serum samples of 22 HCC patients with and without cirrhosis. Serum samples of patients with non-cancerous chronic liver cirrhosis (n = 22) and of healthy volunteers (n = 15) served as controls.

Results

MicroRNA-141 and microRNA-200a were significantly downregulated in blood serum of patients with HCC compared to liver cirrhosis (p<0.007) and healthy controls (p<0.002). MicroRNA-141 and microRNA-200a could well discriminate patients with cirrhosis-associated HCC from healthy volunteers with area under the receiver-operating characteristic curve (AUC) values of 0.85 and 0.82, respectively. Additionally, both microRNAs could differentiate between HCC and non-cancerous liver cirrhosis with a fair accuracy.

Conclusions

Circulating microRNA-200 family members are significantly deregulated in patients with HCC and liver cirrhosis. Further studies are necessary to confirm the diagnostic value of the microRNA-200 family as accurate serum marker for cirrhosis-associated HCC.     

Th17及相关细胞因子与酒精性肝硬化患者肝脏硬度值、肝功能及脂联素水平的相关性分析

摘要 目的：探讨Th17及相关细胞因子与酒精性肝硬化患者肝脏硬度值（LSM）、肝功能及脂联素（APN）水平的相关性分析。方法：以2018年1月~2020年1月本院收治的酒精性肝硬化患者72例作为酒精性肝硬化组,同期在本院进行体检的健康志愿者100例作为健康对照组。对比两组研究对象Th17、白介素-17（IL-17）、LSM、肝功能指标、APN水平的差异,采用Pearson检验评估酒精性肝硬化患者Th17及IL-17与病情相关指标的相关性。结果：酒精性肝硬化组患者的Th17细胞分布比例、IL-17水平、LSM均高于健康对照组（P<0.05）。外周血中谷丙转氨酶（ALT）、谷草转氨酶（AST）、总胆红素（STB）的水平高于健康对照组,白蛋白（ALB）的水平低于健康对照组,血清中APN的水平低于健康对照组（P<0.05）。相关性分析发现,酒精性肝硬化患者Th17细胞分布比例及IL-17水平与LSM、ALT、AST、STB的水平呈正相关,与ALB、APN的水平呈负相关（P<0.05）。结论：酒精性肝硬化患者Th17及其细胞因子IL-17表达水平异常增高,可能在反映病情相关指标异常变化程度方面具有积极作用。     

Detection of Helicobacter in the liver of patients with chronic cholestatic liver diseases.

Helicobacter species were identified in human liver tissues by PCR. Biopsies were obtained from patients with primary sclerosing cholangitis, primary biliary cirrhosis and noncholestatic liver cirrhosis. One set of Helicobacter genus-specific primers and two different primer sets for Helicobacter pylori were used in the PCR-assays. Using Helicobacter genus-specific primers 80% (8/10) of patients with primary sclerosing cholangitis and 90% (9/10) of patients with primary biliary cirrhosis were positive. Seven of these 17 samples were positive using two different primers for H. pylori and Southern blot hybridization. Among the non-cholestatic liver cirrhosis controls, only one sample was positive in the Helicobacter genus-specific PCR-assay. Significantly higher values of alkaline phosphatases and prothrombin complex was found for the patients positive for Helicobacter genus. In conclusion, gene sequences of Helicobacter species and H. pylori were detected in human liver tissue using PCR and DNA hybridization in patients with a cholestatic liver disease, but rarely in noncholestatic liver cirrhosis.     

Wilson's disease: a common liver disorder?

In two sibships 7 of 24 siblings were homozygous for Wilson''s disease. In family A, the largest kindred of this recessively inherited disease thus far reported, the proband presented with chronic active hepatitis, one sibling died of cirrhosis, a second had clinical evidence of chronic liver disease and two others had biochemical and histologic changes in liver biopsy specimens. In family B the proband had cirrhosis and portal hypertension and one sibling had biochemical and histologic evidence of liver disease. All six living patients had low serum concentrations of ceruloplasmin and copper and a high 24-hour urinary excretion of copper, which was greatly increased by administration of D-penicillamine. None showed neurologic abnormalities and only one had Kayser-Fleischer rings (detectable only by slit-lamp examination). Each patient had an erythrocyte sedimentation rate (ESR) of 8 mm/h or less. After 3 and 2 years, respectively, of D-penicillamine therapy the conditions of the two probands had improved. Liver function became normal in three siblings, and no abnormalities developed in the remaining one. Thus, since Wilson''s disease may present with chronic active hepatitis or cirrhosis with a normal ESR and without ocular or neurologic signs, it may be a more common cause of liver disease in young people than has been appreciated.     

Matrix metalloproteinase-9 inhibition or deletion attenuates portal hypertension in rodents

Liver cirrhosis and portal hypertension are accompanied by hyperdynamic circulation, angiogenesis and portosystemic collaterals. Matrix metalloproteinases (MMPs) participate in fibrogenesis and angiogenesis, however, whether they can be targeted in cirrhosis treatment is unclear. Therefore, we performed three series of experiments to investigate this issue. Liver cirrhosis was induced by common bile duct ligation (BDL) in Sprague-Dawley rats. Sham-operated rats served as controls. Rats were randomly allocated to receive vehicle, minocycline (a nonselective MMP inhibitor) or SB-3CT (MMP-2 and −9 inhibitor) for 28 days in the first and second series, respectively. MMP-9 knockout mice were used in the third series. The results showed that minocycline ameliorated portal hypertension, hemodynamic abnormalities, reduced collateral shunting, mesenteric vascular density, plasma VEGF level and alleviated liver fibrosis. SB-3CT attenuated portal hypertension, hemodynamic derangements, reduced shunting, mesenteric vascular density, mesenteric VEGF protein expression, and liver fibrosis. Knockout BDL mice had significantly alleviated portal hypertension, liver fibrosis, liver α-SMA and mesenteric eNOS protein expressions compared to wild-type BDL mice. Liver SMAD2 phosphorylation was down-regulated in all series with MMP inhibition or knock-out. In conclusion, MMP-9 inhibition or deletion ameliorated the severity of cirrhosis, portal hypertension, and associated derangements. MMP-9 may be targeted in the treatment of liver cirrhosis.     

Noninvasive evaluation of portal-systemic shunting by glyceryl trinitrate

Portal-systemic shunting is an important circulatory abnormality in patients with liver cirrhosis. Glyceryl trinitrate (GTN) that is normally subject to first pass elimination, may exhibit higher bioavailability in these patients. This study compares the pharmacodynamic effects of GTN after peroral and sublingual administration for noninvasive assessment of shunting. Six control subjects and 15 patients with cirrhosis were studied after oral and sublingual application of 0.5 mg of GTN. Liver cirrhosis was complicated by portal hypertension in 7 of the patients and 4 patients had surgically implanted portocaval anastomosis. Digital plethysmography, which is highly sensitive and is essentially noninvasive in nature, was used to assess and compare the pharmacodynamic effects of GTN. The following values of the ratio of areas under the pharmacodynamic effects/time curve were obtained: 0.08 +/- 0.06 in healthy subjects, 0.52 +/- 0.21 in patients with uncomplicated cirrhosis, 0.99 +/- 0.34 in patients with portal hypertension and 1.24 +/- 0.43 in patients with portal-systemic shunts. We conclude that increased bioavailability of GTN reflects portal-systemic shunting and might be used providing that the pharmacodynamic data reflect both pharmacokinetic variability and the pharmacokinetic-pharmacodynamic interrelations.