CD147 mediates intrahepatic leukocyte aggregation and determines the extent of liver injury.

BACKGROUND: Chronic inflammation is the driver of liver injury and results in progressive fibrosis and eventual cirrhosis with consequences including both liver failure and liver cancer. We have previously described increased expression of the highly multifunctional glycoprotein CD147 in liver injury. This work describes a novel role of CD147 in liver inflammation and the importance of leukocyte aggregates in determining the extent of liver injury. METHODS: Non-diseased, progressive injury, and cirrhotic liver from humans and mice were examined using a mAb targeting CD147. Inflammatory cell subsets were assessed by multiparameter flow cytometry. RESULTS: In liver injury, we observe abundant, intrahepatic leukocyte clusters defined as ≥5 adjacent CD45+ cells which we have termed "leukocyte aggregates". We have shown that these leukocyte aggregates have a significant effect in determining the extent of liver injury. If CD147 is blocked in vivo, these leukocyte aggregates diminish in size and number, together with a marked significant reduction in liver injury including fibrosis. This is accompanied by no change in overall intrahepatic leukocyte numbers. Further, blocking of aggregation formation occurs prior to an appreciable increase in inflammatory markers or fibrosis. Additionally, there were no observed, "off-target" or unpredicted effects in targeting CD147. CONCLUSION: CD147 mediates leukocyte aggregation which is associated with the development of liver injury. This is not a secondary effect, but a cause of injury as aggregate formation proceeds other markers of injury. Leukocyte aggregation has been previously described in inflammation dating back over many decades. Here we demonstrate that leukocyte aggregates determine the extent of liver injury.

Circulating CD147 predicts mortality in advanced hepatocellular carcinoma.

BACKGROUND AND AIM: The glycoprotein CD147 has a role in tumor progression, is readily detectable in the circulation, and is abundantly expressed in hepatocellular carcinoma (HCC). Advanced HCC patients are a heterogeneous group with some individuals having dismal survival. The aim of this study was to examine circulating soluble CD147 levels as a prognostic marker in HCC patients. METHODS: CD147 was measured in 277 patients (110 HCC, 115 chronic liver disease, and 52 non-liver disease). Clinical data included etiology, tumor progression, Barcelona Clinic Liver Cancer (BCLC) stage, and treatment response. Patients with HCC were stratified into two groups based upon the 75th percentile of CD147 levels (24 ng/mL). RESULTS: CD147 in HCC correlated inversely with poor survival (P = 0.031). Increased CD147 predicted poor survival in BCLC stages C and D (P = 0.045), and CD147 levels >24 ng/mL predicted a significantly diminished 90-day and 180-day survival time (hazard ratio [HR] = 6.1; 95% confidence interval [CI]: 2.1-63.2; P = 0.0045 and HR = 2.8; 95% CI: 1.2-12.6; P = 0.028, respectively). In BCLC stage C, CD147 predicted prognosis; levels >24 ng/mL were associated with a median survival of 1.5 months compared with 6.5 months with CD147 levels ≤24 ng/mL (P = 0.03). CD147 also identified patients with a poor prognosis independent from treatment frequency, modality, and tumor size. CONCLUSIONS: Circulating CD147 is an independent marker of survival in advanced HCC. CD147 requires further evaluation as a potential new prognostic measure in HCC to identify patients with advanced disease who have a poor prognosis.

Hepatocytes in liver injury: Victim, bystander, or accomplice in progressive fibrosis?

Chronic liver disease causes significant morbidity and mortality through progressive fibrosis, cirrhosis, and liver cancer. The classical theory of fibrogenesis has hepatic stellate cells (HSCs) as the principal and only significant source of abnormal extracellular matrix (ECM). Further, HSCs have the major role in abnormal ECM turnover. It is the death of hepatocytes, as the initial target of injury, that initiates a sequence of events including the recruitment of inflammatory cells and activation of HSCs. Following this initial response, the ongoing insult to hepatocytes is regarded as perpetuating injury, but otherwise, hepatocytes are regarded as "victims" and "bystanders" in progressive fibrosis. Recent developments, however, challenge this view and suggest the concept of the hepatocyte being an active participant in liver injury. It is clear now that hepatocytes undergo phenotypic changes, adapt to injury, and react to the altered microenvironment. In this review, we describe studies showing that hepatocytes contribute to progressive fibrosis by direct manipulation of the surrounding ECM and through signaling to effector cells, particularly HSCs and intrahepatic immune cells. Together, these findings suggest an active "accomplice" role for the hepatocyte in progressive liver fibrosis and highlight novel pathways that could be targeted for development of future anti-fibrotic therapies.

Hepatocyte produced matrix metalloproteinases are regulated by CD147 in liver fibrogenesis.

BACKGROUND: The classical paradigm of liver injury asserts that hepatic stellate cells (HSC) produce, remodel and turnover the abnormal extracellular matrix (ECM) of fibrosis via matrix metalloproteinases (MMPs). In extrahepatic tissues MMP production is regulated by a number of mechanisms including expression of the glycoprotein CD147. Previously, we have shown that CD147 is expressed on hepatocytes but not within the fibrotic septa in cirrhosis [1]. Therefore, we investigated if hepatocytes produce MMPs, regulated by CD147, which are capable of remodelling fibrotic ECM independent of the HSC. METHODS: Non-diseased, fibrotic and cirrhotic livers were examined for MMP activity and markers of fibrosis in humans and mice. CD147 expression and MMP activity were co-localised by in-situ zymography. The role of CD147 was studied in-vitro with siRNA to CD147 in hepatocytes and in-vivo in mice with CCl4 induced liver injury using ãCD147 antibody intervention. RESULTS: In liver fibrosis in both human and mouse tissue MMP expression and activity (MMP-2, -9, -13 and -14) increased with progressive injury and localised to hepatocytes. Additionally, as expected, MMPs were abundantly expressed by activated HSC. Further, with progressive fibrosis there was expression of CD147, which localised to hepatocytes but not to HSC. Functionally significant in-vitro regulation of hepatocyte MMP production by CD147 was demonstrated using siRNA to CD147 that decreased hepatocyte MMP-2 and -9 expression/activity. Further, in-vivo α-CD147 antibody intervention decreased liver MMP-2, -9, -13, -14, TGF-ß and α-SMA expression in CCl4 treated mice compared to controls. CONCLUSION: We have shown that hepatocytes produce active MMPs and that the glycoprotein CD147 regulates hepatocyte MMP expression. Targeting CD147 regulates hepatocyte MMP production both in-vitro and in-vivo, with the net result being reduced fibrotic matrix turnover in-vivo. Therefore, CD147 regulation of hepatocyte MMP is a novel pathway that could be targeted by future anti-fibrogenic agents.

Novel aspects of the liver microenvironment in hepatocellular carcinoma pathogenesis and development.

Hepatocellular carcinoma (HCC) is a prevalent primary liver cancer that is derived from hepatocytes and is characterised by high mortality rate and poor prognosis. While HCC is driven by cumulative changes in the hepatocyte genome, it is increasingly recognised that the liver microenvironment plays a pivotal role in HCC propensity, progression and treatment response. The microenvironmental stimuli that have been recognised as being involved in HCC pathogenesis are diverse and include intrahepatic cell subpopulations, such as immune and stellate cells, pathogens, such as hepatitis viruses, and non-cellular factors, such as abnormal extracellular matrix (ECM) and tissue hypoxia. Recently, a number of novel environmental influences have been shown to have an equally dramatic, but previously unrecognized, role in HCC progression. Novel aspects, including diet, gastrointestinal tract (GIT) microflora and circulating microvesicles, are now being recognized as increasingly important in HCC pathogenesis. This review will outline aspects of the HCC microenvironment, including the potential role of GIT microflora and microvesicles, in providing new insights into tumourigenesis and identifying potential novel targets in the treatment of HCC.

Generation of hydrogen peroxide-resistant murine neuroblastoma cells: a target discovery platform for novel neuroprotective genes.

Oxidative stress has been suggested to play an important role in the pathogenesis of various neurodegenerative diseases including Alzheimer's disease (AD). Hydrogen peroxide (H2O2), one of the main reactive oxygen species, is converted into the highly toxic ·OH radical in the presence of redox-active transition metals, which then oxidises nucleic acids, lipids and proteins, leading to neurodegeneration and cell death. There is an urgent need to gain more knowledge about relevant therapeutic targets to combat oxidative stress and it neurotoxic effects, and how this knowledge can be utilized to develop novel neuroprotective therapies for AD. One way to identify new mechanisms combating oxidative stress was via the creation of H2O2-resistant cell lines and identification of the mechanisms responsible for their resistance. However, in most cases catalase overexpression or increased glutathione content was identified as the primary mode of H2O2 resistance in these cell lines. In this study, we have generated six different resistant neuronal cell lines or populations (from the same original murine Neuro2a neuroblastoma line) by exposing cells to increasing concentrations of H2O2 and performing continuous selection for survivors over a period of several months, which appear to have acquired H2O2 resistance based on other, novel mechanisms. These six populations showed a significant, but differential resistance against H2O2 when compared with the parental cell line. Using combinations of catalase-, glutathione synthesis- and glutathione peroxidase-inhibitors it was shown that the increased resistance of Neuro2a-HR cells is not solely based on an increased activity of catalase or the glutathione system, suggesting that their resistance might be based on yet unknown, novel defence mechanisms.

Chronic inflammation alters production and release of glutathione and related thiols in human U373 astroglial cells.

Neurons rely on glutathione (GSH) and its degradation product cysteinylglycine released by astrocytes to maintain their antioxidant defences. This is particularly important under conditions of inflammation and oxidative stress, as observed in many neurodegenerative diseases including Alzheimer's disease (AD). The effects of inflammatory activation on intracellular GSH content and the extracellular thiol profile (including cysteinylglycine and homocysteine) of astrocytes were investigated. U373 astroglial cells exposed to IL-1ß and TNF-α for up to 96 h showed a dose-dependent increase in IL-6 release, indicative of increasing pro-inflammatory cellular activation. With increasing concentrations of IL-1ß and TNF-α (0.01-1 ng/ml), an increase in both intracellular and extracellular GSH levels was observed, followed by a return to control levels in response to higher concentrations of IL-1ß and TNF-α. Extracellular levels of cysteinylglycine decreased in response to all concentrations of IL-1ß and TNF-α. In contrast, levels of the neurotoxic thiol homocysteine increased in a dose-dependent manner to IL-1ß and TNF-α-induced activation. Our results suggest that chronically activated astrocytes in the brain might fail to adequately maintain GSH substrate delivery to neurons, thus promoting neuronal vulnerability. They might also explain the elevated levels of homocysteine found in the brains and serum of patients with AD.

Advanced glycation endproducts and their receptor RAGE in Alzheimer's disease.

Alzheimer's disease (AD) is the most common dementing disorder of late life. Although there might be various different triggering events in the early stages of the disease, they seem to converge on a few characteristic final pathways in the late stages, characterized by inflammation and neurodegeneration. In this review, we revisit the hypothesis that advanced glycation endproducts (AGEs) and their receptor RAGE may play an important role in disease pathogenesis. Accumulation of AGEs in cells and tissues is a normal feature of aging, but is accelerated in AD. In AD, AGEs can be detected in pathological deposits such as amyloid plaques and neurofibrillary tangles. AGEs explain many of the neuropathological and biochemical features of AD such as extensive protein crosslinking, glial induction of oxidative stress and neuronal cell death. Oxidative stress and AGEs initiate a positive feedback loop, where normal age-related changes develop into a pathophysiological cascade. RAGE and its decoy receptor soluble RAGE, may contribute to or protect against AD pathogenesis by influencing transport of ß-amyloid into the brain or by manipulating inflammatory mechanisms. Targeted pharmacological interventions using AGE-inhibitors, RAGE-antagonists, RAGE-antibodies, soluble RAGE or RAGE signalling inhibitors such as membrane-permeable antioxidants may be promising therapeutic strategies to slow down the progression of AD.

A versatile high throughput screening system for the simultaneous identification of anti-inflammatory and neuroprotective compounds.

In many chronic neurodegenerative diseases including Frontotemporal Dementia and Alzheimer's disease (AD), microglial activation is suggested to be involved in pathogenesis or disease progression. Activated microglia secrete a variety of cytokines, including interleukin-1beta, interleukin-6, and tumor necrosis factor as well as reactive oxygen and nitrogen species (ROS/RNS). ROS and RNS contribute to alterations in neuronal glucose uptake, inhibition of mitochondrial enzymes, a decrease in mitochondrial membrane potential, impaired axonal transport, and synaptic signaling. In addition, ROS act as signaling molecules in pro-inflammatory redox-active signal transduction pathways. To establish a high throughput screening system for anti-inflammatory and neuroprotective compounds, we have constructed an "Enhanced Green Fluorescent protein" (EGFP) expressing neuronal cell line and set up a murine microglia/neuron co-culture system with these EGFP expressing neuronal cells. We show that microglia activation leads to neuronal cell death, which can be conveniently measured by loss of neuronal EGFP fluorescence. Moreover, we used this system to test selected polyphenolic compounds for their ability to downregulate inflammatory markers and to protect neurons against microglial insult. We suggest that this system might allow accelerated drug discovery for the treatment of inflammation-mediated neurodegenerative diseases.

Cytotoxicity of Maillard reaction products determined with a peptide spot library.

The reaction of reactive carbonyl compounds (RCCs) with lysine and arginine (Maillard reaction) is a common modification of proteins in thermally processed foods. In this study, the toxicity of Maillard reaction products (MRPs) formed from defined amino acids or dipeptides (bound to a cellulose membrane) with ribose, glycerinaldehyde or methylglyoxal was investigated. Murine RAW 264.7 macrophages were cultivated on the cellulose membrane and the effect of MRPs on cell viability was determined. The toxicity of MRPs was dependent on the RCC used and increased in the order of ribose < glycerinaldehyde < methylglyoxal. The dipeptides were more cytotoxic than the amino acids, with Lys-Lys MRPs being the most toxic of all tested MRPs. Cell numbers did not fall below the starting point, indicating that the MRPs rather inhibited proliferation than actually caused cell death. To develop an assay, in which whole membranes with multiple peptide spots could be tested simultaneously, we measured cell numbers on larger cellulose membranes using image analysis of the intracellularly formed formazan crystals. Although this method was technically feasable, it appears that uneven cell attachment on the membrane would require a way to detemine starting cell number by a non-destructive assay to yield more robust data.

Lipoic acid as an anti-inflammatory and neuroprotective treatment for Alzheimer's disease.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder that destroys patient memory and cognition, communication ability with the social environment and the ability to carry out daily activities. Despite extensive research into the pathogenesis of AD, a neuroprotective treatment - particularly for the early stages of disease - remains unavailable for clinical use. In this review, we advance the suggestion that lipoic acid (LA) may fulfil this therapeutic need. A naturally occurring cofactor for the mitochondrial enzymes pyruvate dehydrogenase and alpha-ketoglutarate dehydrogenase, LA has been shown to have a variety of properties which can interfere with the pathogenesis or progression of AD. For example, LA increases acetylcholine (ACh) production by activation of choline acetyltransferase and increases glucose uptake, thus supplying more acetyl-CoA for the production of ACh. LA chelates redox-active transition metals, thus inhibiting the formation of hydroxyl radicals and also scavenges reactive oxygen species (ROS), thereby increasing the levels of reduced glutathione. In addition, LA down-regulates the expression of redox-sensitive pro-inflammatory proteins including TNF and inducible nitric oxide synthase. Furthermore, LA can scavenge lipid peroxidation products such as hydroxynonenal and acrolein. In human plasma, LA exists in an equilibrium of free and plasma protein bound form. Up to 150 muM, it is bound completely, most likely binding to high affinity fatty acid sites on human serum albumin, suggesting that one large dose rather than continuous low doses (as provided by "slow release" LA) will be beneficial for delivery of LA to the brain. Evidence for a clinical benefit for LA in dementia is yet limited. There are only two published studies, in which 600 mg LA was given daily to 43 patients with AD (receiving a standard treatment with choline-esterase inhibitors) in an open-label study over an observation period of up to 48 months. Whereas the improvement in patients with moderate dementia was not significant, the disease progressed extremely slowly (change in ADAScog: 1.2 points=year, MMSE: -0.6 points=year) in patients with mild dementia (ADAScog<15). Data from cell culture and animal models suggest that LA could be combined with nutraceuticals such as curcumin, (-)-epigallocatechin gallate (from green tea) and docosahexaenoic acid (from fish oil) to synergistically decrease oxidative stress, inflammation, Abeta levels and Abeta plaque load and thus provide a combined benefit in the treatment of AD.

Inflammation and the redox-sensitive AGE-RAGE pathway as a therapeutic target in Alzheimer's disease.

Alzheimer's disease (AD) is the most common cause of dementia. Neuritic amyloid plaques and concomitant chronic inflammation are prominent pathological features of AD. beta-amyloid peptide (Abeta), the major component of plaques, and advanced glycation end products (AGEs), post-translational protein modifications, are key activators of plaque-associated inflammation. Abeta, AGEs, S100b, and amphoterin bind to the receptor for AGEs (RAGE), which transmits the signal from RAGE via redox-sensitive pathways to nuclear factor kappa-B (NF-kappaB)-regulated cytokines. RAGE-mediated inflammation caused by glial cells and subsequent changes in neuronal glucose metabolism are likely to be important contributors to neurodegeneration in AD. As long as the neuronal damage is reversible, drugs interfering with the Abeta and AGE-RAGE pathways might be interesting novel therapeutics for the treatment of AD.

Solution structure of the human immunodeficiency virus type 1 p6 protein.

The human immunodeficiency virus type 1 p6 protein represents a docking site for several cellular and viral binding factors and fulfills major roles in the formation of infectious viruses. To date, however, the structure of this 52-amino acid protein, by far the smallest lentiviral protein known, either in its mature form as free p6 or as the C-terminal part of the Pr55 Gag polyprotein has not been unraveled. We have explored the high resolution structure and folding of p6 by CD and NMR spectroscopy. Under membranous solution conditions, p6 can adopt a helix-flexible helix structure; a short helix-1 (amino acids 14-18) is connected to a pronounced helix-2 (amino acids 33-44) by a flexible hinge region. Thus, p6 can be subdivided into two distinct structural and functional domains; helix-2 perfectly defines the region that binds to the virus budding factor AIP-1/ALIX, indicating that this structure is required for interaction with the endosomal sorting complex required for transport. The PTAP motif at the N terminus, comprising the primary late assembly domain, which is crucial for interaction with another cellular budding factor, Tsg101, does not exhibit secondary structure. However, the adjacent helix-1 may play an indirect role in the specific complex formation between p6 and the binding groove in Tsg101. Moreover, binding studies by NMR demonstrate that helix-2, which also comprises the LXXLF motif required for incorporation of the human immunodeficiency virus type 1 accessory protein Vpr into budding virions, specifically interacts with the Vpr binding region, indicating that under the specific solution conditions used for structure analysis, p6 adopted a functional conformation.