Thierry Tordjmann

Substitution of Hexon Hypervariable Region 5 of Adenovirus Serotype 5 Abrogates Blood Factor Binding and Limits Gene Transfer to Liver

Liver tropism potentially leading to massive hepatocyte transduction and hepatotoxicity still represents a major drawback to adenovirus (Ad)-based gene therapy. We previously demonstrated that substitution of the hexon hypervariable region 5 (HVR5), the most abundant capsid protein, constituted a valuable platform for efficient Ad retargeting. The use of different mouse strains revealed that HVR5 substitution also led to dramatically less adenovirus liver transduction and associated toxicity, whereas HVR5-modified Ad were still able to transduce different cell lines efficiently, including primary hepatocytes. We showed that HVR5 modification did not significantly change Ad blood clearance or liver uptake at early times. However, we were able to link the lower liver transduction to enhanced HVR5-modified Ad liver clearance and impaired use of blood factors. Most importantly, HVR5-modified vectors continued to transduce tumors in vivo as efficiently as their wild-type counterparts. Taken together, our data provide a rationale for future design of retargeted vectors with a safer profile.

Immediate neuroendocrine signaling after partial hepatectomy through acute portal hyperpressure and cholestasis

BACKGROUND & AIMS Early neuroendocrine pathways contribute to liver regeneration after partial hepatectomy (PH). We investigated one of these pathways involving acute cholestasis, immediate portal hyperpressure, and arginine vasopressin (AVP) secretion. METHODS Surgical procedure (PH, Portal vein stenosis (PVS), bile duct ligation (BDL), spinal cord lesion (SCL)) and treatments (capsaicin, bile acids (BA), oleanolic acid (OA)) were performed on rats and/or wild type or TGR5 (GPBAR1) knock-out mice. In these models, the activation of AVP-secreting supraoptic nuclei (SON) was analyzed, as well as plasma BA, AVP, and portal vein pressure (PVP). Plasma BA, AVP, and PVP were also determined in human living donors for liver transplantation. RESULTS Acute cholestasis (mimicked by BDL or BA injection) as well as portal hyperpressure (mimicked by PVS) independently activated SON and AVP secretion. BA accumulated in the brain after PH or BDL, and TGR5 was expressed in SON. SON activation was mimicked by the TGR5 agonist OA and inhibited in TGR5 KO mice after BDL. An afferent nerve pathway also contributed to post-PH AVP secretion, as capsaicin treatment or SCL resulted in a weaker SON activation after PH. CONCLUSIONS After PH in rodents, acute cholestasis and portal hypertension, via the nervous and endocrine routes, stimulate the secretion of AVP that may protect the liver against shear stress and bile acids overload. Data in living donors suggest that this pathway may also operate in humans.

LIM-Only Protein FHL2 Activates NF-κB Signaling in the Control of Liver Regeneration and Hepatocarcinogenesis

ABSTRACT Four-and-a-half LIM-only protein 2 (FHL2) is an important mediator in many signaling pathways. In this study, we analyzed the functions of FHL2 in nuclear factor κB (NF-κB) signaling in the liver. We show that FHL2 enhanced tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6) activity in transcriptional activation of NF-κB targets by stabilizing the protein. TRAF6 is a binding partner of FHL2 and an important component of the Toll-like receptor–NF-κB pathway. Knockdown of FHL2 in 293-hTLR4/MD2-CD14 cells impaired lipopolysaccharide (LPS)-induced NF-κB activity, which regulates expression of inflammatory cytokines. Indeed, FHL2−/− macrophages showed significantly reduced production of TNF and interleukin 6 (IL-6) following LPS stimulation. TNF and IL-6 are the key cytokines that prime liver regeneration after hepatic injury. Following partial hepatectomy, FHL2−/− mice exhibited diminished induction of TNF and IL-6 and delayed hepatocyte regeneration. In the liver, NF-κB signaling orchestrates inflammatory cross talk between hepatocytes and hepatic immune cells that promote chemical hepatocarcinogenesis. We found that deficiency of FHL2 reduced susceptibility to diethylnitrosamine-induced hepatocarcinogenesis, correlating with the activator function of FHL2 in NF-κB signaling. Our findings demonstrate FHL2 as a positive regulator of NF-κB activity in liver regeneration and carcinogenesis and highlight the importance of FHL2 in both hepatocytes and hepatic immune cells.

Rat hepatocytes express functional P2X receptors

Extracellular ATP regulates many hepatic functions by stimulating purinergic receptors. Only the G protein‐coupled P2Y receptors have been studied in hepatocytes. We investigated the functional expression of P2X receptors, the ATP‐gated channels in rat hepatocytes. P2X4 and P2X7 transcripts and proteins were detected by RT‐PCR and by both Western blotting and immunocytochemistry. High concentrations of ATP, and 2′‐and 3′‐O‐(4‐benzoylbenzoyl)‐ATP the preferring agonist of P2X7, induced membrane blebbing and significant uptake of 4‐[(3‐methyl‐2(3H)‐benzoxazolylidene)methyl]‐1‐[3‐(triethylammonio)propyl]diiodide, both of which were inhibited by oxidised ATP, a blocker of P2X receptors. These results provide evidence that P2X4 and P2X7 receptors are expressed and functional on rat hepatocytes, possibly playing an important role in the purinergic signaling complex in these cells.

The four and a half LIM-only protein 2 regulates liver homeostasis and contributes to carcinogenesis.

BACKGROUND & AIMS The four and a half LIM-only protein 2 (FHL2) is upregulated in diverse pathological conditions. Here, we analyzed the effects of FHL2 overexpression in the liver of FHL2 transgenic mice (Apo-FHL2). METHODS We first examined cell proliferation and apoptosis in Apo-FHL2 livers and performed partial hepatectomy to investigate high FHL2 expression in liver regeneration. Expression of FHL2 was then analyzed by real time PCR in human hepatocellular carcinoma and adjacent non-tumorous livers. Finally, the role of FHL2 in hepatocarcinogenesis was assessed using Apo-FHL2;Apc(lox/lox) mice. RESULTS Six-fold increase in cell proliferation in transgenic livers was associated with concomitant apoptosis, resulting in normal liver mass. In Apo-FHL2 livers, both cyclin D1 and p53 were markedly increased. Evidence supporting a p53-dependent cell death mechanism was provided by the findings that FHL2 bound to and activated the p53 promoter, and that a dominant negative p53 mutant compromised FHL2-induced apoptosis in hepatic cells. Following partial hepatectomy in Apo-FHL2 mice, hepatocytes displayed advanced G1 phase entry and DNA synthesis leading to accelerated liver weight restoration. Interestingly, FHL2 upregulation in human liver specimens showed significant association with increasing inflammation score and cirrhosis. Finally, while Apo-FHL2 mice developed no tumors, the FHL2 transgene enhanced hepatocarcinogenesis induced by liver-specific deletion of the adenomatous polyposis coli gene and aberrant Wnt/β-catenin signaling in Apc(lox/lox) animals. CONCLUSIONS Our results implicate FHL2 in the regulation of signaling pathways that couple proliferation and cell death machineries, and underscore the important role of FHL2 in liver homeostasis and carcinogenesis.

Bile acids and FGF receptors: orchestrators of optimal liver regeneration

Because it fosters our dreams of immortality, regeneration has been for centuries a matter of fascination for countless biologists and clinicians. While in mammals regeneration is obviously less spectacular than in hydra, newt or zebrafish, it allows, in any case, repairing intestine, skin or liver. The latter organ is seen as a paradigmatic regeneration model because, as for an amputated fins zebrafish or newts forelimb, it is indeed possible to follow the complete restoration of a rodent liver mass after a 2/3 partial hepatectomy (PH) within a few days. The caveat is that most of our knowledge about liver regeneration is based on this specific surgical model. The amazing conclusion is that liver regeneration relies mainly on highly differentiated hepatocytes, which, while actively dividing to restore the missing part, maintain their vital functions.1 Hepatocytes are in a quiescent state (G0) in a normal adult liver. Following 2/3 PH, >95% of these parenchymal cells present in the remnant lobes divide in a rather synchronous manner, for one or two rounds of cell division. Other liver cell types, such as macrophages, cholangiocytes or endothelial cells, will divide afterwards. Schematically, one can distinguish two successive main steps allowing proper regeneration. The first phase requires the secretion of cytokines such as tumour necrosis factor-α and interleukin-6 in the very first minutes after PH, which poise hepatocytes to enter G1 phase and to become receptive to growth factors. The second step involves concomitantly metabolic changes, consisting in particular in a transient accumulation of lipid droplets, and in the activation of two growth factor pathways, epidermal growth factor receptor and c-Met. Both pathways then recruit scaffolding proteins and activate multiple intracellular intertwined networks, such as mitogen-activated protein kinases, signal transducer and activator of transcription 3 (STAT3), …

Hippo signalling: Liver size regulation and beyond

After its discovery in Drosophila, the Hippo signalling pathway has been shown to regulate organ size in mammals as well. Based on recent studies, this kinase cascade appears in particular crucial for liver tissue homeostasis, by regulating both cellular proliferation and apoptosis. Thereby, Hippo signalling may appear as a key pathway in liver carcinogenesis.

Innate immunity, purinergic system, and liver regeneration: A trip in complexity†‡

L iver regeneration is a highly integrated process involving a wide array of cellular interactions occurring between the different liver cell types, but also between the liver and the extrahepatic environment. Besides the main growth factors and cytokines acting in a paracrine way, diverse endocrine and neuroendocrine interactions, as well as the recruitment of blood cells, affect liver regeneration. This entire network acts in concert to achieve a fine-tuning of proliferative and hepatoprotective cascades, ultimately leading to liver mass restoration after injury or resection. A study published in this issue of HEPATOLOGY, sheds new light on the complex interactions between immune cells and liver regeneration processes, with the purinergic system as one possible regulator. Among modulators of liver regeneration, the innate immune system constitutes a complex network of interacting cells and cytokines, of which the resulting effect on liver regeneration is not entirely understood. Surprisingly, the complement system as well as cytotoxic cytokines, like tumor necrosis factor alpha, are reported as positive regulators for hepatocyte regeneration. The role of Kupffer cells, the hepatic resident macrophages, is still controversial, because these cells secrete numerous cytokines, stimulating or inhibiting hepatocyte proliferation, after liver injury. As to natural killer (NK) and natural killer T (NKT) cells, the main lymphoid population in human and mouse liver, although the majority of studies reported on their negative effect on liver regeneration, their role still remains controversial. NK cells would inhibit regeneration, in particular through the secretion of interferon-gamma (IFN-c), whereas NKT cells would play a minor role in the normal liver after partial hepatectomy (PH). However, NK and NKT cells may have a positive effect on oval cell-dependent regeneration after acute liver injury when hepatocytes cannot replicate. The study by Graubardt et al. revisited the controversy on NK cells during liver regeneration by introducing a new parameter: the purinergic system, currently recognized as a major component of the inflammatory response after injury. Signaling by extracellular adenosine triphosphate (ATP) acting on plasma membrane purinergic receptors, that is, P2Y1,2,4,6,11-14 (G-protein-coupled receptors) and P2X1-7 (ATP-gated channels with high Ca 2þ permeability), is vital not only in excitable, but also in nonexcitable cells and tissues. Vascular shear stress, organ distension, or cellular injury are known to trigger ATP release from endothelial, epithelial, and other cell types. Once in the extracellular medium, ATP is degraded by the powerful ecto-ATPases expressed on cell surfaces to avoid excessive activation (and desensitization) of purinergic receptors. Adenosine, the breakdown product of ATP, can bind P1 purinergic receptors, with specific downstream biological responses. Thus, in a given tissue, the final purinergic input will be determined by the complex combination of numerous parameters, including mainly mechanisms of ATP release, purinergic receptors expression and distribution, and ecto-ATPases expression. In the liver, each cell type expresses its own repertoire of purinoceptors and ecto-ATPases, and evidence for a crucial effect of purinergic signaling in liver physiology is growing, including modulation of bile secretion and ischemia protection. In vitro studies suggested that extracellular ATP had a positive effect on proliferation of primary rat hepatocytes. Recently, we showed that an extracellular ATP release from the liver occurred immediately after PH, contributing to liver regeneration in the rat. We also observed an immediate ATP release from the liver after PH for living donor transplantation, suggesting that purinergic signaling may be also involved during human liver regeneration. However, the purinoceptors and precise mechanisms involved in ATP-mediated effect on liver regeneration still remain to be defined. The authors of the present study previously reported on a coordinating role of the ectonucleotidase CD39Abbreviations:: ATP, adenosine triphosphate; IFN-c, interferon-gamma; NK, natural killer cells; NKT, natural killer T cells; PH, partial hepatectomy. Address reprint requests to: Thierry Tordjmann, M.D., Ph.D, INSERM U. 757, Universit e Paris Sud, Bât. 443, 91405 Orsay, France. E-mail: thierry.tordjmann@u-psud.fr; fax: (33) 1 69155893. Copyright VC 2013 by the American Association for the Study of Liver Diseases. View this article online at wileyonlinelibrary.com. DOI 10.1002/hep.26312 Potential conflict of interest: Nothing to report.

Bile acids and their receptors during liver regeneration: “Dangerous protectors”

Tissue repair is orchestrated by a finely tuned interplay between processes of regeneration, inflammation and cell protection, allowing organisms to restore their integrity after partial loss of cells or organs. An important, although largely unexplored feature is that after injury and during liver repair, liver functions have to be maintained to fulfill the peripheral demand. This is particularly critical for bile secretion, which has to be finely modulated in order to preserve liver parenchyma from bile-induced injury. However, mechanisms allowing the liver to maintain biliary homeostasis during repair after injury are not completely understood. Besides cytokines and growth factors, bile acids (BA) and their receptors constitute an insufficiently explored signaling network during liver regeneration and repair. BA signal through both nuclear (mainly Farnesoid X Receptor, FXR) and membrane (mainly G Protein-coupled BA Receptor 1, GPBAR-1 or TGR5) receptors which distributions are large in the organism, and which activation elicits a wide array of biological responses. While a number of studies have been dedicated to FXR signaling in liver repair processes, TGR5 remains poorly explored in this context. Because of the massive and potentially harmful BA overload that faces the remnant liver after partial ablation or destruction, both BA-induced adaptive and proliferative responses may stand in a central position to contribute to the regenerative response. Based on the available literature, both BA receptors may act in synergy during the regeneration process, in order to protect the remnant liver and maintain biliary homeostasis, otherwise potentially toxic BA overload would result in parenchymal insult and compromise optimal restoration of a functional liver mass.

Zizyphin modulates calcium signalling in human taste bud cells and fat taste perception in the mouse

Zizyphin, isolated from Zizyphus sps. leaf extracts, has been shown to modulate sugar taste perception, and the palatability of a sweet solution is increased by the addition of fatty acids. We, therefore, studied whether zizyphin also modulates fat taste perception. Zizyphin was purified from edible fruit of Zizyphus lotus L. Zizyphin‐induced increases in [Ca2+]i in human taste bud cells (hTBC). Zizyphin shared the endoplasmic reticulum Ca2+ pool and also recruited, in part, Ca2+ from extracellular environment via the opening of store‐operated Ca2+ channels. Zizyphin exerted additive actions on linoleic acid (LA)‐induced increases in [Ca2+]i in these cells, indicating that zizyphin does not exert its action via fatty acid receptors. However, zizyphin seemed to exert, at least in part, its action via bile acid receptor Takeda‐G‐protein‐receptor‐5 in hTBC. In behavioural tests, mice exhibited preference for both LA and zizyphin. Interestingly, zizyphin increased the preference for a solution containing‐LA. This study is the first evidence of the modulation of fat taste perception by zizyphin at the cellular level in hTBC. Our study might be helpful for considering the synthesis of zizyphin analogues as ‘taste modifiers’ with a potential in the management of obesity and lipid‐mediated disorders.