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ABSTRACT: The liver plays a vital role in the organism, and thousands of patients suffer and even die from hepatic complications every year. Viral hepatitis is one of the most important causes of liver-related pathological processes. However, sterile liver diseases, such as drug-induced liver injury, cirrhosis and fibrosis, are still a worldwide concern and contribute significantly to liver transplantation statistics. During hepatocyte death, several genuine intracellular contents are released to the interstitium, where they will trigger inflammatory responses that may boost organ injury. Intracellular purines are key molecules to several metabolic pathways and regulate cell bioenergetics. However, seminal studies in early 70s revealed that purines may also participate in cell-to-cell communication, and more recent data have unequivocally demonstrated that the purinergic signalling plays a key role in the recognition of cell functionality by neighbouring cells and also by the immune system. This new body of knowledge has pointed out that several promising therapeutic opportunities may rely on the modulation of purine release and sensing during diseases. Here, we review the most recent data on the physiological roles of purinergic signalling and how its imbalance may contribute to injury progression during sterile liver injury.
Liver international: official journal of the International Association for the Study of the Liver 03/2013; 33(3):353-61. · 3.82 Impact Factor
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Tatyana Y Doktorova,
Reha Yildirimman, Mathieu Vinken,
Mireia Vilardell,
Tamara Vanhaecke,
Hans Gmuender,
Roque Bort,
Gabriella Brolen,
Gustav Holmgren,
Ruoya Li,
Christophe Chesne,
Joost van Delft,
Jos Kleinjans,
Jose Castell,
Petter Bjorquist,
Ralf Herwig,
Vera Rogiers
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ABSTRACT: As the conventional approach to assess the potential of a chemical to cause cancer in humans still includes the 2-year rodent carcinogenicity bioassay, development of alternative methodologies is needed. In the present study, the transcriptomics responses following exposure to genotoxic (GTX) and non-genotoxic (NGTX) hepatocarcinogens and non-carcinogens (NC) in five liver-based in vitro models, namely conventional and epigenetically-stabilized cultures of primary rat hepatocytes, the human hepatoma-derived cell lines HepaRG and HepG2 and human embryonic stem cell-derived hepatocyte-like cells (hES-Heps), are examined. For full characterization of the systems, several bioinformatics approaches are employed including gene-based, ConsensusPathwayDB-based and classification analysis. They provide convincingly similar outcomes, namely that upon exposure to carcinogens, the HepaRG generates a gene classifier(1) able to discriminate the GTX carcinogens from the NGTX carcinogens and NC. The other in vitro models also yield cancer-relevant characteristic gene groups for the GTX exposure, but some genes are also deregulated by the NGTX carcinogens and NC. Irrespective of the tested in vitro model, the most uniformly expressed pathways following GTX exposure are the p53 and those that are subsequently-induced. The NGTX carcinogens triggered no characteristic cancer-relevant gene profiles in all liver-based in vitro systems. In conclusion, liver-based in vitro models coupled with transcriptomics techniques, especially in the case when the HepaRG cell line is used, represent valuable tools for obtaining insight into the mechanism of action and identification of GTX carcinogens.
Carcinogenesis 02/2013; · 5.70 Impact Factor
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ABSTRACT: Abstract Before any drug can be placed on the market, drug efficacy and safety must be ensured through rigorous testing. Animal models are used for this purpose, though currently increasing attention goes to the use of alternative in vitro systems. In particular, liver-based testing platforms that allow the prediction of pharmacokinetic (PK) and pharmacotoxicological properties during the early phase of drug development are of interest. They also enable the screening of potential effects on hepatic drug transporters. The latter are known to affect drug metabolism and disposition, thereby possibly underlying drug-drug interactions, which, in turn, may result in liver toxicity. Clearly, stable in vivo-like functional expression of drug transporters in hepatic in vitro settings is a prerequisite to be applicable in routine PK and pharmacotoxicological testing. In the first part of the article, an updated overview of hepatic drug transporters is provided, followed by a state-of-the-art review of drug-transporter production and activity in primary hepatocyte cultures (PHCs), being the gold-standard in vitro system. Specific focus is hereby put on strategies to maintain long-term functional expression, in casu of drug transporters, in these systems. In the second part, the use of PHCs to assess hepatobiliary transport and transporter-mediated interactions is outlined.
Drug Metabolism Reviews 02/2013; · 6.40 Impact Factor
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Elke Decrock,
Marijke De Bock,
Nan Wang,
Ashish K Gadicherla,
Mélissa Bol,
Tinneke Delvaeye,
Peter Vandenabeele, Mathieu Vinken,
Geert Bultynck,
Dmitri V Krysko,
Luc Leybaert
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ABSTRACT: Research conducted over the past two decades has provided convincing evidence that cell death, and more specifically apoptosis, can exceed single cell boundaries and can be strongly influenced by intercellular communication networks. We recently reported that gap junctions (i.e. channels directly connecting the cytoplasm of neighboring cells) composed of connexin43 or connexin26 provide a direct pathway to promote and expand cell death, and that inositol 1,4,5-trisphosphate (IP(3)) diffusion via these channels is crucial to provoke apoptosis in adjacent healthy cells. However, IP(3) itself is not sufficient to induce cell death and additional factors appear to be necessary to create conditions in which IP(3) will exert proapoptotic effects. Although IP(3)-evoked Ca(2+) signaling is known to be required for normal cell survival, it is also actively involved in apoptosis induction and progression. As such, it is evident that an accurate fine-tuning of this signaling mechanism is crucial for normal cell physiology, while a malfunction can lead to cell death. Here, we review the role of IP(3) as an intracellular and intercellular cell death messenger, focusing on the endoplasmic reticulum-mitochondrial synapse, followed by a discussion of plausible elements that can convert IP(3) from a physiological molecule to a killer substance. Finally, we highlight several pathological conditions in which anomalous intercellular IP(3)/Ca(2+) signaling might play a role.
Biochimica et Biophysica Acta 01/2013; · 4.66 Impact Factor
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Nan Wang,
Elke De Vuyst,
Raf Ponsaerts,
Kerstin Boengler,
Nicolás Palacios-Prado,
Joris Wauman,
Charles P Lai,
Marijke De Bock,
Elke Decrock,
Mélissa Bol, [......],
Vera Rogiers,
Jan Tavernier,
W Howard Evans,
Christian C Naus,
Feliksas F Bukauskas,
Karin R Sipido,
Gerd Heusch,
Rainer Schulz,
Geert Bultynck,
Luc Leybaert
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ABSTRACT: Connexin-43 (Cx43), a predominant cardiac connexin, forms gap junctions (GJs) that facilitate electrical cell-cell coupling and unapposed/nonjunctional hemichannels that provide a pathway for the exchange of ions and metabolites between cytoplasm and extracellular milieu. Uncontrolled opening of hemichannels in the plasma membrane may be deleterious for the myocardium and blocking hemichannels may confer cardioprotection by preventing ionic imbalance, cell swelling and loss of critical metabolites. Currently, all known hemichannel inhibitors also block GJ channels, thereby disturbing electrical cell-cell communication. Here we aimed to characterize a nonapeptide, called Gap19, derived from the cytoplasmic loop (CL) of Cx43 as a hemichannel blocker and examined its effect on hemichannel currents in cardiomyocytes and its influence in cardiac outcome after ischemia/reperfusion. We report that Gap 19 inhibits Cx43 hemichannels without blocking GJ channels or Cx40/pannexin-1 hemichannels. Hemichannel inhibition is due to the binding of Gap19 to the C-terminus (CT) thereby preventing intramolecular CT-CL interactions. The peptide inhibited Cx43 hemichannel unitary currents in both HeLa cells exogenously expressing Cx43 and acutely isolated pig ventricular cardiomyocytes. Treatment with Gap19 prevented metabolic inhibition-enhanced hemichannel openings, protected cardiomyocytes against volume overload and cell death following ischemia/reperfusion in vitro and modestly decreased the infarct size after myocardial ischemia/reperfusion in mice in vivo. We conclude that preventing Cx43 hemichannel opening with Gap19 confers limited protective effects against myocardial ischemia/reperfusion injury.
Archiv für Kreislaufforschung 01/2013; 108(1):309. · 7.35 Impact Factor
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Gustavo André,
Oliveira,
Pedro Elias Marques,
Sylvia Stella Amaral,
Jayane Laís,
Dias Quintão,
Bruno Cogliati,
Maria Lucia,
Zaidan Dagli,
Vera Rogiers,
Tamara Vanhaecke, Mathieu Vinken,
Gustavo Batista Menezes
Liver international: official journal of the International Association for the Study of the Liver 01/2013; · 3.82 Impact Factor
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Mathieu Vinken,
Michaël Maes,
Rachel Cavill,
Dirk Valkenborg,
James K Ellis,
Elke Decrock,
Luc Leybaert,
An Staes,
Kris Gevaert,
André G Oliveira,
Gustavo B Menezes,
Bruno Cogliati,
Maria Lúcia Zaidan Dagli,
Timothy M D Ebbels,
Erwin Witters,
Hector C Keun,
Tamara Vanhaecke,
Vera Rogiers
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ABSTRACT: Freshly established cultures of primary hepatocytes progressively adopt a foetal-like phenotype and display increased production of connexin43. The latter is a multifaceted cellular entity with variable subcellular locations, including the mitochondrial compartment. Cx43 forms hemichannels and gap junctions that are involved in a plethora of physiological and pathological processes, such as apoptosis. The present study was conducted with the goal of shedding more light onto the role of connexin43 in primary hepatocyte cultures. Connexin43 expression was suppressed by means of RNA interference technology, and the overall outcome of this treatment on the hepatocellular proteome and metabolome was investigated using tandem mass tag-based differential protein profiling and (1)H NMR spectroscopy, respectively. Global protein profiling revealed a number of targets of the connexin43 knock-down procedure, including mitochondrial proteins (heat shock protein 60, glucose-regulated protein 75, thiosulphate sulphurtransferase and adenosine triphosphate synthase) and detoxifying enzymes (glutathione S-transferase μ 2 and cytochrome P450 2C70). At the metabolomic level, connexin43 silencing caused no overt changes, though there was some evidence for a subtle increase in intracellular glycine quantities. Collectively, these data could further substantiate the established existence of a mitochondrial connexin pool and could be reconciled with the previously reported involvement of connexin43 signalling in spontaneously occurring apoptosis in primary hepatocyte cultures.
Archive für Toxikologie 12/2012; · 4.67 Impact Factor
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ABSTRACT: The concept of mechanistic toxicogenomics implies that compound-induced changes in gene expression profiles provide valuable information about their mode of action. A growing number of research groups have presented evidence that whole-genome gene expression profiling techniques might be used as tools for in vivo and in vitro generation of gene signatures and elucidation of molecular mechanisms after exposure to toxic compounds. An important issue to be investigated is the in vivo relevance of in vitro-obtained data. In the current study, we compare the gene expression profiles generated in vitro, after exposing conventional and epigenetically stabilized primary rat hepatocytes to well-known genotoxic hepatocarcinogens (aflatoxin B1, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and 2-nitrofluorene) with those derived in vivo after oral exposure of rats to these compounds. Similar statistical tools were applied on both sets of data. The major molecular pathways affected in the in vivo setting were DNA damage, detoxification and cell survival response, as previously described. In the conventional hepatocyte cultures, two of the three genotoxicants showed quite similar responses as in vivo with respect to these pathways. The third compound (2-nitrofluorene) revealed in vitro response which was not observed in vivo. In the epigenetically stabilized hepatocytes, in contrast to what was expected, the responses were less relevant for the in vivo situation. This study highlights the importance of in vitro/in vivo comparison of data that are generated using in vitro models and shows that conventional primary rat hepatocyte cultures represent an appropriate in vitro model to retrieve mechanistic information on the exposure to genotoxicants.
Archive für Toxikologie 09/2012; 86(11):1703-15. · 4.67 Impact Factor
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ABSTRACT: The establishment of an elaborate gap junctional intercellular communication network, especially between hepatocytes, is important for normal liver development. In fact, the production of the gap junction building blocks, the connexins, undergoes several well-defined changes throughout the hepatic differentiation process. This ultimately results in the acquisition of an adult connexin expression pattern which is critical for maintaining the fully differentiated hepatocyte-specific phenotype. Abnormalities of connexin production are observed in a number of pathological conditions, such as during liver cancer. This article provides an overview of these processes with emphasis on the underlying molecular mechanisms.
Cell Communication & Adhesion 09/2012; 19(3-4):55-62. · 1.18 Impact Factor
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ABSTRACT: Plasma membrane hemichannels composed of connexin (Cx) proteins are essential components of gap junction channels but accumulating evidence suggests functions of hemichannels beyond the communication provided by junctional channels. Hemichannels not incorporated into gap junctions, called unapposed hemichannels, can open in response to a variety of signals, electrical and chemical, thereby forming a conduit between the cell's interior and the extracellular milieu. Open hemichannels allow the bidirectional passage of ions and small metabolic or signaling molecules of below 1-2kDa molecular weight. In addition to connexins, hemichannels can also be formed by pannexin (Panx) proteins and current evidence suggests that Cx26, Cx32, Cx36, Cx43 and Panx1, form hemichannels that allow the diffusive release of paracrine messengers. In particular, the case is strong for ATP but substantial evidence is also available for other messengers like glutamate and prostaglandins or metabolic substances like NAD(+) or glutathione. While this field is clearly in expansion, evidence is still lacking at essential points of the paracrine signaling cascade that includes not only messenger release, but also downstream receptor signaling and consequent functional effects. The data available at this moment largely derives from in vitro experiments and still suffers from the difficulty of separating the functions of connexin-based hemichannels from gap junctions and from pannexin hemichannels. However, messengers like ATP or glutamate have universal roles in the body and further defining the contribution of hemichannels as a possible release pathway is expected to open novel avenues for better understanding their contribution to a variety of physiological and pathological processes. This article is part of a Special Issue entitled: The Communicating junctions, roles and dysfunctions (Pt II).
Biochimica et Biophysica Acta 07/2012; · 4.66 Impact Factor
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ABSTRACT: At present, substantial efforts are focused on the development of in vitro assays coupled with "omics" technologies for the identification of carcinogenic substances as an alternative to the classical 2-year rodent carcinogenicity bioassay. A prerequisite for the eventual regulatory acceptance of such assays, however, is the in vivo relevance of the observed in vitro findings. In the current study, hepatocarcinogen-induced gene expression profiles generated after the exposure of conventional cultures of primary rat hepatocytes to three non-genotoxic carcinogens (methapyrilene hydrochloride, piperonyl butoxide, and Wy-14643), three genotoxic carcinogens (aflatoxin B1, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, and 2-nitrofluorene), and two non-carcinogens (nifedipine and clonidine) are compared with previously obtained in vivo data after oral administration for up to 14 days of the same hepatocarcinogens to rats. In addition to the comparison of deregulated genes and functions per compound between in vivo and in vitro models, the major discriminating cellular pathways found in vivo in livers of exposed rats were examined for deregulation in vitro. Further, in vivo-derived gene signatures for the identification of genotoxic versus non-genotoxic carcinogens are used to classify in vitro-tested hepatocarcinogens and non-carcinogens. In the primary hepatocyte cultures, two out of the three tested genotoxic carcinogens mimicked the in vivo-relevant DNA damage response and were correctly assessed. Exposure to the non-genotoxic hepatocarcinogens, however, triggered a relatively weak response in the in vitro system, with no clear similarities to in vivo. This study contributes to the further optimization of toxicogenomics predictive tools when applied in in vitro settings.
Archive für Toxikologie 04/2012; 86(9):1399-411. · 4.67 Impact Factor
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ABSTRACT: Cultures of primary hepatocytes are versatile tools that can serve many in vitro toxicity testing purposes. However, they cope with dedifferentiation, a process that is already initiated during the hepatocyte isolation procedure and that is manifested as the progressive loss of functionality upon subsequent cultivation. A number of strategies to prevent dedifferentiation have been introduced over the last decades, all which aim at re-establishing the in vivo hepatocyte micro-environment in vitro, but that are of merely limited success. Recent mechanistic insight into the mechanisms that underlie hepatocyte dedifferentiation has opened new avenues for the development of novel approaches that target the actual causes of this deteriorative process and thus for the generation of a long-term hepatic in vitro tool. Such experimental system is urgently needed, especially in the light of the stringent European legislative modifications that are currently encountered by the pharmaceutical, chemical and, particularly, the cosmetic industry.
Toxicology in Vitro 04/2012; 26(3):541-4. · 2.78 Impact Factor
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ABSTRACT: Alternative methods, replacing animal testing, are urgently needed in view of the European regulatory changes in the field of cosmetic products and their ingredients. In this context, a joint research initiative called SEURAT was recently raised by the European Commission and COLIPA, representing the European cosmetics industry, with the overall goal of developing an animal-free repeated dose toxicity testing strategy for human safety assessment purposes. Although cosmetic ingredients are usually harmless for the consumer, one of the initial tasks of this research consortium included the identification of organs that could potentially be affected by cosmetic ingredients upon systemic exposure. The strategy that was followed hereof is described in the present paper and relies on the systematic evaluation, by using a self-generated electronic databank, of published reports issued by the scientific committee of DG SANCO responsible for the safety of cosmetic ingredients. By screening of the repeated dose toxicity studies present in these reports, it was found that the liver is potentially the most frequently targeted organ by cosmetic ingredients when orally administered to experimental animals, followed by the kidney and the spleen. Combined listing of altered morphological, histopathological, and biochemical parameters subsequently indicated the possible occurrence of hepatotoxicity, including steatosis and cholestasis, triggered by a limited number of cosmetic compounds. These findings are not only of relevance for the in vitro modeling efforts and choice of compounds to be tested in the SEURAT project cluster, but also demonstrate the importance of using previously generated toxicological data through an electronic databank for addressing specific questions regarding the safety evaluation of cosmetic ingredients.
Archive für Toxikologie 03/2012; 86(3):405-12. · 4.67 Impact Factor
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ABSTRACT: Primary hepatocyte cultures suffer from the progressive occurrence of dedifferentiation followed by spontaneous apoptosis. This is associated with modifications in the expression of connexins (Cxs), which are the building stones of hemichannels that in turn form gap junctions between neighboring cells. Specifically, a shift is observed from the adult hepatocellular Cx32 species toward the fetal Cx43 isoform. The current study was set up to investigate the role of Cx43 in spontaneous apoptosis taking place in primary hepatocyte cultures. For this purpose, freshly isolated adult rat hepatocytes were cultivated in conventional conditions for 4 days with daily monitoring of Cx expression, Cx localization, and gap junction channel and hemichannel functionality. Gap junction activity was low shortly after isolation, whereas the inverse was observed for hemichannel functionality. Both channel types displayed high activity near the end stages of the cultivation period. The Cx32-to-Cx43 switch became progressively manifested at the translational level. At the transcriptional level, a fivefold decrease in Cx32 messenger RNA abundance and a twofold increase in Cx43 expression were noticed within the first 24 h of cultivation. Throughout the cultivation period, Cx32 was mainly located at the plasma membrane surface, whereas Cx43 immunostaining was more diffuse. Application of three Cx43 inhibitors resulted in the downregulation of both hemichannel functionality and gap junction activity. This was paralleled by decreased expression and activity of caspase 3 as well as by reduced expression of Bid. Collectively, these data show that Cx43 signaling actively contributes to the occurrence of spontaneous apoptosis in cultures of primary hepatocytes.
Toxicological Sciences 01/2012; 125(1):175-86. · 4.65 Impact Factor
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ABSTRACT: The 2-year rodent carcinogenicity bioassay evolved more than 40 years ago, and although it is complex, long lasting, expensive, and animal consuming, it is still the only generally accepted test for assessing the carcinogenicity of chemicals. Over time, different alternative approaches have been developed with the final goal to replace the bioassay. Unfortunately, at present, none of these strategies alone provides sufficient assurance of accurate prediction. In this review paper, we discuss the major advantages and pitfalls of the existing alternative methodologies to the carcinogenicity bioassay. Finally, based on the available scientific data in the public domain, we propose what we would like to call a "feasible integrated testing strategy" which incorporates some promising alternatives, providing at the same time information on the mechanism of action and the toxic nature of the compounds tested. It is, however, clear that the adoption of whatever "new" testing scheme should be considered with caution and its effectiveness should be experimentally demonstrated in advance by addressing a reasonable number of chemical carcinogens and non-carcinogens from a variety of structural and functional classes.
Critical Reviews in Toxicology 12/2011; 42(2):91-106. · 5.16 Impact Factor
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Mathieu Vinken
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ABSTRACT: Direct intercellular communication mediated by gap junctions constitutes a major regulatory platform in the control of hepatic homeostasis. Hepatocellular gap junctions are composed of two hemichannels of adjacent cells which are built up by connexin proteins,in casu Cx32. Mathieu Vinken, Pofessor at the Department of Toxicology of the Free University Brussels-Belgium, was one of the first investigators to demonstrate that hepatic connexin expression is controlled by epigenetic mechanisms. In particular, he found that inhibitors of histone deacetylase enzymes enhance Cx32 production and gap junction activity in cultures of primary hepatocytes, a finding that is of importance for liver-based in vitro modelling. Professor Dr. Mathieu Vinken's recent work is focussed on the elucidation of the role of connexin proteins and their channels in the hepatocyte life cycle. Specific attention is paid to apoptosis in this context, whereby it has been found that Cx32 hemichannels control the termination of induced cell death in cultures of primary hepatocytes. Overall, Professor Dr. Mathieu Vinken's research can be considered as an important contribution to the field of hepatic connexin physiology.
World journal of gastrointestinal pathophysiology. 10/2011; 2(5):82-7.
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ABSTRACT: Cellular communication mediated by gap junction channels and hemichannels, both composed of connexin proteins, constitutes two acknowledged regulatory platforms in the accomplishment of tissue homeostasis. In recent years, an abundance of reports has been published indicating functions for connexin proteins in the control of the cellular life cycle that occur independently of their channel activities. This has yet been most exemplified in the context of cell growth and cell death, and is therefore as such addressed in the current paper. Specific attention is hereby paid to the molecular mechanisms that underpin the cellular non-channel roles of connexin proteins, namely the alteration of the expression of tissue homeostasis determinants and the physical interaction with cell growth and cell death regulators. This article is part of a Special Issue entitled: The Communicating junctions, composition, structure and characteristics.
Biochimica et Biophysica Acta 06/2011; 1818(8):2002-8. · 4.66 Impact Factor
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ABSTRACT: Inherent to their pivotal tasks in the maintenance of cellular homeostasis, gap junctions, connexin hemichannels, and pannexin hemichannels are frequently involved in the dysregulation of this critical balance. The present paper specifically focuses on their roles in bacterial infection and disease. In particular, the reported biological outcome of clinically important bacteria including Escherichia coli, Shigella flexneri, Yersinia enterocolitica, Helicobacter pylori, Bordetella pertussis, Aggregatibacter actinomycetemcomitans, Pseudomonas aeruginosa, Citrobacter rodentium, Clostridium species, Streptococcus pneumoniae, and Staphylococcus aureus and their toxic products on connexin- and pannexin-related signaling in host cells is reviewed. Particular attention is paid to the underlying molecular mechanisms of these effects as well as to the actual biological relevance of these findings.
Cellular and Molecular Life Sciences CMLS 06/2011; 68(18):3047-64. · 6.57 Impact Factor
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Marijke De Bock,
Maxime Culot,
Nan Wang,
Mélissa Bol,
Elke Decrock,
Elke De Vuyst,
Anaelle da Costa,
Ine Dauwe, Mathieu Vinken,
Alexander M Simon,
Vera Rogiers,
Gaspard De Ley,
William Howard Evans,
Geert Bultynck,
Geneviève Dupont,
Romeo Cecchelli,
Luc Leybaert
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ABSTRACT: The cytoplasmic Ca(2+) concentration ([Ca(2+)](i)) is an important factor determining the functional state of blood-brain barrier (BBB) endothelial cells but little is known on the effect of dynamic [Ca(2+)](i) changes on BBB function. We applied different agonists that trigger [Ca(2+)](i) oscillations and determined the involvement of connexin channels and subsequent effects on endothelial permeability in immortalized and primary brain endothelial cells. The inflammatory peptide bradykinin (BK) triggered [Ca(2+)](i) oscillations and increased endothelial permeability. The latter was prevented by buffering [Ca(2+)](i) with BAPTA, indicating that [Ca(2+)](i) oscillations are crucial in the permeability changes. Bradykinin-triggered [Ca(2+)](i) oscillations were inhibited by interfering with connexin channels, making use of carbenoxolone, Gap27, a peptide blocker of connexin channels, and Cx37/43 knockdown. Gap27 inhibition of the oscillations was rapid (within minutes) and work with connexin hemichannel-permeable dyes indicated hemichannel opening and purinergic signaling in response to stimulation with BK. Moreover, Gap27 inhibited the BK-triggered endothelial permeability increase in in vitro and in vivo experiments. By contrast, [Ca(2+)](i) oscillations provoked by exposure to adenosine 5' triphosphate (ATP) were not affected by carbenoxolone or Gap27 and ATP did not disturb endothelial permeability. We conclude that interfering with endothelial connexin hemichannels is a novel approach to limiting BBB-permeability alterations.
Journal of cerebral blood flow and metabolism: official journal of the International Society of Cerebral Blood Flow and Metabolism 06/2011; 31(9):1942-57. · 5.46 Impact Factor
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Marijke De Bock,
Maxime Culot,
Nan Wang,
M|[eacute]|lissa Bol,
Elke Decrock,
Elke De Vuyst,
Anaelle da Costa,
Ine Dauwe, Mathieu Vinken,
Alexander M Simon,
Vera Rogiers,
Gaspard De Ley,
William Howard Evans,
Geert Bultynck,
Genevi|[egrave]|ve Dupont,
Romeo Cecchelli,
Luc Leybaert
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ABSTRACT: The cytoplasmic Ca2+ concentration ([Ca2+]i) is an important factor determining the functional state of blood–brain barrier (BBB) endothelial cells but little is known on the effect of dynamic [Ca2+]i changes on BBB function. We applied different agonists that trigger [Ca2+]i oscillations and determined the involvement of connexin channels and subsequent effects on endothelial permeability in immortalized and primary brain endothelial cells. The inflammatory peptide bradykinin (BK) triggered [Ca2+]i oscillations and increased endothelial permeability. The latter was prevented by buffering [Ca2+]i with BAPTA, indicating that [Ca2+]i oscillations are crucial in the permeability changes. Bradykinin-triggered [Ca2+]i oscillations were inhibited by interfering with connexin channels, making use of carbenoxolone, Gap27, a peptide blocker of connexin channels, and Cx37/43 knockdown. Gap27 inhibition of the oscillations was rapid (within minutes) and work with connexin hemichannel-permeable dyes indicated hemichannel opening and purinergic signaling in response to stimulation with BK. Moreover, Gap27 inhibited the BK-triggered endothelial permeability increase in in vitro and in vivo experiments. By contrast, [Ca2+]i oscillations provoked by exposure to adenosine 5′ triphosphate (ATP) were not affected by carbenoxolone or Gap27 and ATP did not disturb endothelial permeability. We conclude that interfering with endothelial connexin hemichannels is a novel approach to limiting BBB-permeability alterations.Keywords: blood–brain barrier; brain edema; brain ischemia; calcium; endothelium
Journal of Cerebral Blood Flow & Metabolism 06/2011; 31(9):1942-1957. · 5.01 Impact Factor
