Role of circulating microRNAs in liver diseases

MicroRNAs(miRNAs) are small RNAs regulate gene expression by inhibiting the turnover of their target mRNAs. In the last years, it became apparent that miRNAs are released into the circulation and circulating miRNAs emerged as a new class of biomarkers for various diseases. In this review we summarize available data on the role of circulating miRNAs in the context of acute and chronic liver diseases including hepatocellular and cholangiocellular carcinoma. Data from animal models are compared to human data and current challenges in the field of miRNAs research are discussed.     

Role of neutrophils in acute inflammatory liver injury.

Hepatic infiltration of polymorphonuclear leukocytes (neutrophils) is an early response to tissue injury, cellular stress or systemic inflammation. Neutrophil activation is vital for host-defense and the removal of cell debris but can also cause additional tissue damage or even liver failure. In order to prevent the detrimental effects of neutrophils without compromising host-defense reactions, it is important to understand the mechanisms of neutrophil hepatotoxicity. The first step in the pathophysiology is the priming and recruitment of neutrophils into the liver vasculature by inflammatory mediators, e.g. cytokines, chemokines, or complement factors. Most critical for parenchymal cell damage is the accumulation in sinusoids, which does not depend on cellular adhesion molecules. The next step is the extravasation into the parenchyma. This process requires a chemotactic signal from hepatocytes or already extravasated neutrophils and depends on cellular adhesion molecules on neutrophils (beta(2) or beta(1) integrins) and on endothelial cells (intercellular or vascular cell adhesion molecules). The third step is the direct contact with target cells (hepatocytes), which involves beta(2) integrins and triggers the full activation of the neutrophil with a long-lasting adherence-dependent oxidant stress and degranulation. The oxidants diffuse into hepatocytes and trigger an intracellular oxidant stress, mitochondrial dysfunction and eventually cause oncotic necrotic cell death. Neutrophil-derived proteases facilitate extravasation and are involved in the regulation of inflammatory mediator production. Based on these mechanisms, it appears that strengthening of the intracellular defense mechanisms in hepatocytes may be the most promising therapeutic approach to selectively prevent neutrophil-mediated tissue damage without compromising their host-defense function.     

Role of intrahepatic innervation in regulating the activity of liver cells

Liver innervation comprises sympathetic,parasympathetic and peptidergic nerve fibers,organized as either afferent or efferent nerves with different origins and roles.Their anatomy and physiology have been studied in the past 30 years,with different results published over time.Hepatocytes are the main cell population of the liver,making up almost 80%of the total liver volume.The interaction between hepatocytes and nerve fibers is accomplished through a wealth of neurotransmitters and signaling pathways.In this short review,we have taken the task of condensing the most important data related to how the nervous system interacts with the liver and especially with the hepatocyte population,how it influences their metabolism and functions,and how different receptors and transmitters are involved in this complex process.     

Role of neutrophils in a mouse model of halothane-induced liver injury

Drug-induced liver injury (DILI) is a major safety concern in drug development. Its prediction and prevention have been hindered by limited knowledge of the underlying mechanisms, in part the result of a lack of animal models. We developed a mouse model of halothane-induced liver injury and characterized the mechanisms accounting for tissue damage. Female and male Balb/c, DBA/1, and C57BL/6J mice were injected intraperitoneally with halothane. Serum levels of alanine aminotransferase and histology were evaluated to determine liver injury. Balb/c mice were found to be the most susceptible strain, followed by DBA/1, with no significant hepatotoxicity observed in C57BL/6J mice. Female Balb/c and DBA/1 mice developed more severe liver damage compared with their male counterparts. Bioactivation of halothane occurred similarly in all three strains based on detection of liver proteins adducted by the reactive metabolite. Mechanistic investigations revealed that hepatic message levels of tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta); IL-6, and IL-8 were significantly higher in halothane-treated Balb/c mice compared to DBA/1 and C57BL/6J mice. Moreover, a higher number of neutrophils were recruited into the liver of Balb/c mice upon halothane treatment compared with DBA/1, with no obvious neutrophil infiltration detected in C57BL/6J mice. Neutrophil depletion experiments demonstrated a crucial role for these cells in the development of halothane-induced liver injury. The halothane-initiated hepatotoxicity and innate immune response-mediated escalation of tissue damage are consistent with events that occur in many cases of DILI. In conclusion, our model provides a platform for elucidating strain-based and gender-based susceptibility factors in DILI development.     

循环血microRNA在肝纤维化发生发展中的作用

MiroRNA(miRNA)可通过外泌体等载体分泌,游离于细胞之外,并可在循环血液中保持稳定状态。目前研究表明,循环血中miRNA的变化趋势与疾病的不同病理状态相关,提示循环血中miRNA可作为疾病潜在的诊断和治疗分子靶标。研究表明多种miRNA与肝纤维化的发生发展密切相关,概述了目前关于肝纤维化进程中循环血miRNA表达差异的研究,多种研究提示循环血miRNA可为肝纤维化的无创性早期诊断和治疗提供新的研究方向。     

Asymptomatic bacteriuria in elderly humans is associated with increased levels of circulating TNF receptors and elevated numbers of neutrophils

Low-grade inflammatory activity is strongly associated with age-associated diseases such as atherosclerosis, dementia, type-2 diabetes, sarcopenia, and osteoporosis and predicts mortality risk in elderly populations. The aim of the current study was to investigate if asymptomatic bacteriuria in elderly humans was associated with inflammation. Midstream clean-catch urine culture was collected from consecutive, elderly patients at admission to a department of internal medicine due to functional disability. Forty patients (age 70-91 years) were selected and included in the current study; 20 subjects had positive urine culture and 20 sex- and age-matched subjects had negative urine culture. Inclusion criteria were temperature below 37.8 degrees C, no clinical signs of infection and no current antibiotic treatment. Patients with asymptomatic bacteriuria had significantly increased levels of circulating tumor necrosis factor receptors (sTNFR-I) and a higher number of neutrophils in the blood compared to the group without bacteriuria. Thus, the present study provides some support for the hypothesis that asymptomatic urinary infections are associated with low-grade immune activity in frail, elderly humans.     

Role of neutrophils in innate resistance to Entamoeba histolytica liver infection in mice

In order to define the role of neutrophils in the innate resistance to Entamoeba histolytica liver infection in mice, we examined the pattern of liver lesion induced by direct injection of  E. histolytica trophozoites in normal mice and in neutrophil-depleted mice. A variety of histological lesions were found, the extent of liver damage was considerably higher in the neutrophil-depleted mice. Livers from neutrophil-depleted mice displayed areas of liquefactive (lytic) necrosis containing a large number of amoebae and absence of neutrophils or mononuclear cells. By contrast, in the liver of normal mice, neutrophils were seen associated to E. histolytica at early stages of infection. In both mouse groups, areas of TUNEL-positive dead hepatocytes were observed and a characteristic internucleosomal banding pattern of genomic DNA consistent with apoptosis was detected in DNA harvested from amoebic liver lesions. These data suggest that neutrophils play an important role in the mechanisms of resistance to amoebic liver infection in mice. In addition, our histological analysis suggests that E. histolytica is capable of producing liver damage in the absence of inflammatory cells .     

Role of the liver in clearance and excretion of circulating carcinoembryonic antigen (CEA)

CEA is a glycoprotein with a molecular weight of 200,000 containing 55%–65% carbohydrate. The removal of only two sialic acid residues result in rapid uptake from the circulation by the liver and catabolism in the lysosomes. There is a receptor on the plasma membrane of the hepatocyte (hepatic binding protein) which recognizes galactosyl residues. About 70% of¹²⁵I-labeled intact CEA is cleared by the liver in 1 hr. The exposure of terminal galactose residues by removing sialic acids determines the rate of clearance. CEA is probably initially taken up by Kupffer cells and transferred to hepatocytes. About 10% of CEA added to an isolated perfused liver appears in bile. Biliary duct obstruction and cholestasis may elevate plasma CEA levels due to detergent effects on the liver cell receptors.This investigation was supported by grant CA-04486 from the National Cancer Institute.     

Role of Kupffer cells in arresting circulating tumor cells and controlling metastatic growth in the liver

Metastasis to the liver is a common event in clinical oncology. Blood-borne tumor cells (TCs) arriving to the liver sinusoids run into a special vascular bed. The lining of liver sinusoids is shared by Kupffer cells (KCs) and endothelial cells. KCs, liver-fixed macrophages, are responsible for detection and removal of "non-self" particles. To investigate their role in arresting blood-borne TCs and controlling tumor growth, we injected a syngeneic colon carcinoma cell line into a mesenteric vein of two groups of rats; one group was without Kupffer cells and the other normal controls. We removed the liver of these animals at different time intervals and performed immunohistochemical analysis with monoclonal antibodies (MoAbs) against our tumor cell line, three macrophage subpopulations, natural killer cells, and B and T lymphocytes. Additionally, we showed in vitro spontaneous cytotoxicity of KCs against our tumor cell line. Results suggest that KCs play a relevant role in arresting circulating TCs at the liver sinusoid, although it is limited to a small number of malignant cells. They also seem to play a major role in clearing neoplastic cells from the liver parenchyma, in controlling tumor growth in the very early stages of metastatic development, and in modulating the host immune response to cancer cells. (Hepatology 1996 May;23(5):1224-31)     

Age-dependent removal of circulating glutathione by rat liver: Role of gamma-glutamyl transferase

Total gamma-glutamyl transferase (GGT) activity was found to be 93% greater in the liver of old (60 weeks) rats compared to young (12 weeks) adult animals. In vitro perfused livers of old animals also had shown 92% greater capacity to hydrolyze glutathione (GSH) from the circulation. GSH removal was fully dependent on GGT activity. It increased 50% when glycylglycine was added to the perfusion buffer. There was a positive and highly significant correlation (r=0.984; p<0.005) in the liver of old rats between total GGT activity and the ability of the liver of these animals to remove GSH from the circulation. Results presented here point to a physiological and functional significance to the age-related differences in total liver GGT.     

Role of hepatic gamma-glutamyltransferase in the degradation of circulating glutathione: studies in the intact guinea pig perfused liver

The role of hepatic gamma-glutamyltransferase in the breakdown of circulating glutathione was studied in the perfused guinea pig liver. Hepatic gamma-glutamyltransferase activity in the guinea pig is sevenfold higher than in the rat and is comparable to its activity in man. Guinea pig livers were found to remove, in a single pass, 50% to 90% of glutathione (10 to 50 mumol/L) added to the portal perfusate. Removal of portal glutathione was totally dependent on the activity of gamma-glutamyltransferase and led to the near quantitative appearance of cysteinyl-glycine and cysteine in the caval perfusate. Glutathione removal by the intact liver followed saturation with a Michaelis constant (Km) of 59 mumol/L for glutathione and a maximum velocity of 235 nmol glutathione/min/gm of liver weight. The capacity of the guinea pig liver to remove circulating glutathione was estimated to be sevenfold to 10-fold higher than its net rate of output of glutathione into the circulation. Inhibition of gamma-glutamyltransferase activity in the perfused liver led to threefold to sixfold increases in the hepatic output of glutathione into the circulation, indicating that more than two thirds of glutathione transported extracellularly is broken down. Data obtained demonstrate a major role of hepatic gamma-glutamyltransferase, both in the removal of portally carried glutathione and in the degradation of glutathione molecules released by the liver itself into the sinusoids. These findings suggest the existence of an intraorgan transport of glutathione in the liver, whereby periportal cells could provide glutathione precursors to pericentral cells.     

Expression of adhesion molecules during apoptosis of circulating neutrophils in COPD

STUDY OBJECTIVES: Neutrophil accumulation occurs in the lungs of patients with COPD. This can be due to increased recruitment and/or delayed tissue clearance. Previous studies have described alterations in circulating neutrophils in these patients that can facilitate the former. Dysregulation of neutrophil apoptosis may contribute to the latter. This study investigated the potential abnormalities of the apoptotic process in COPD patients. DESIGN: Prospective study. SETTINGS: Outpatient clinic in a urban, tertiary hospital. PATIENTS: Fourteen stable patients with COPD, 8 smokers with normal lung function, and 8 healthy nonsmoking subjects. MEASUREMENTS AND RESULTS: We cultured circulating neutrophils that had been harvested from the study subjects at 2, 6, and 24 h. Apoptosis was assessed using flow cytometry by annexin binding and CD16 expression. The surface expression of the adhesion molecules Mac-1 (CD11b) and L-selectin (CD62L) also was determined by flow cytometry. The percentage of apoptotic neutrophils increased with time similarly in all groups. However, the surface expression of Mac-1 (CD11b) was higher, and that of L-selectin (CD62L) was lower, during apoptosis in the neutrophils of patients with COPD. CONCLUSIONS: These results show that, quantitatively, in vitro neutrophil apoptosis in COPD patients occurred at a rate similar to that found in healthy individuals and smokers with normal lung function. Qualitatively, however, the increased surface expression of Mac-1 (CD11b) and the decreased surface expression of L-selectin (CD62L) observed in the apoptotic neutrophils of COPD patients indicate increased activation during the apoptotic process. This may be relevant for the pathogenesis of COPD.     

Role of myeloperoxidase in the respiratory burst of human neutrophils

Myeloperoxidase (MPO), a heme enzyme present in the primary granules of polymorphonuclear leukocytes (PMNs), has been demonstrated to participate in the oxygen-dependent microbicidal activity of these cells. Evidence for the importance of MPO in this role comes in part from studies of normal PMNs treated with the heme enzyme inhibitor, sodium azide. MPO has also been suggested to regulate the respiratory activity of PMNs during phagocytosis. The role of MPO in PMN oxygen metabolism was examined by studying parameters of the respiratory burst of PMNs from a number of unrelated MPO-deficient subjects; in addition, the ability of heme enzyme inhibitors to duplicate the MPO-deficient state was studied by treating normal and MPO-deficient cells with these compounds. MPO-deficient PMNs were found to have a time-dependent hypermetabolic response as assessed by measurement of oxygen consumption, superoxide generation, hydrogen peroxide release, and hexose monophosphate shunt activity. Catabolic pathways for hydrogen peroxide were normal, suggesting the increased recovery of oxygen metabolites reflects increased production rather than decreased catabolism of H2O2. These observations support the concept that MPO may play an important role in terminating the respiratory burst of normal PMNs. The three heme enzyme inhibitors studied--sodium azide, potassium cyanide, and 3-aminotriazole--differed greatly in the degree to which they inhibited various enzymatic systems in the PMN. Nonetheless, as a group, they exerted qualitatively similar effects on oxygen metabolism of normal and of MPO-deficient PMNs. This indicates that many of the mechanisms by which heme enzyme inhibitors influence PMN metabolism are independent of the inhibition of MPO. Conclusions from studies using such treatment of PMNs should be interpreted with caution.     

中性粒细胞在慢性阻塞性肺疾病中的作用

慢性阻塞性肺疾病(chronic obstructive pneumonia discase,COPD)是以不完全可逆的气流受限为特征的慢性炎症性疾病.中性粒细胞可能在COPD的发展过程中发挥主要作用.活化的中性粒细胞释放蛋白酶、活性氧和细胞因子,引起肺组织损伤,最终导致气道黏液高分泌,气流受限和肺气肿.     

循环和肿瘤组织内中性粒细胞促肿瘤机制

中性粒细胞是人体内最常见的炎细胞,也是肿瘤微环境中最丰富的细胞种类。正常中性粒细胞来源于骨髓造血干细胞,具有清除病原、组织修复作用;而在肿瘤背景下,中性粒细胞会衍生出骨髓源性抑制细胞(MDSCs)亚群,这是一种未成熟中性粒细胞,具有强烈免疫抑制作用。循环MDSCs通过产生活性氧及精氨酸酶抑制T淋巴细胞抗肿瘤免疫,释放中性粒细胞胞外陷阱促进肿瘤细胞血运转移。肿瘤组织内MDSCs通过表达PD-L1抑制T细胞功能,还可释放血管内皮生长因子促进血管新生,释放金属基质蛋白酶引起上皮-间质转化,加剧肿瘤进展。中性粒细胞在肿瘤发生、发展中起到重要的诱导作用,本文就中性粒细胞的起源、循环和肿瘤组织内中性粒细胞促进肿瘤进展的机制作一综述。     

Role of extracellular matrix in regulating fenestrations of sinusoidal endothelial cells isolated from normal rat liver.

Open fenestrations are a conspicuous feature of sinusoidal endothelial cells and allow free movement of plasma into the space of Disse. In hepatic fibrosis, the number of fenestrations decreases as interstitial collagen increases in the liver, a change that correlates with deposition of extracellular matrix in the space of Disse. In this study, the possibility of a causal relationship between altered fenestral morphology and perisinusoidal matrix has been examined by culturing rat sinusoidal endothelial cells on individual matrix proteins or on a native matrix consisting of human amniotic membrane with interstitial collagen (types I and III) on one side and basement membrane proteins (collagen types IV and V and laminin) on the other. Under culture conditions, individual components of the extracellular matrix failed to maintain fenestrations. A basement-membranelike gel matrix derived from the Engelbreth-Holm-Swarm tumor war similarly ineffective. Fenestral density and porosity (percentage of cell surface occupied by fenestrations) were significantly enhanced, however, when endothelial cells were cultured on the basement-membrane side of human amnion. These data suggest that support of endothelial fenestrations requires a complex matrix. In particular, physiologically derived basement membrane maintains fenestrations, whereas interstitial collagen matrix does not. The loss of fenestrations associated with hepatic fibrosis may be related in part to an accumulation of interstitial collagens in the space of Disse.