A novel lipid-based drug carrier targeted to the non-parenchymal cells, including hepatic stellate cells, in the fibrotic livers of bile duct ligated rats

In fibrotic livers, collagen producing hepatic stellate cells (HSC) represent a major target for antifibrotic therapies. We designed liposomes with surface-coupled mannose 6-phosphate (M6P) modified human serum albumin (HSA) to target HSC via the M6P receptor. In this study we determined the pharmacokinetics and target specificity of M6P-HSA-liposomes in a rat model of liver fibrosis. Ten minutes after injection of [³H]-M6P-HSA-liposomes 90% of the dose has cleared the circulation. The blood elimination of these liposomes was counteracted by free M6P-HSA and polyinosinic acid, a competitive inhibitor of scavenger receptors. The M6P-HSA-liposomes accumulated in HSC. However, also Kupffer cells and endothelial cells contributed to the uptake of M6P-HSA-liposomes in the fibrotic livers. Polyinosinic acid inhibited the accumulation of the liposomes in Kupffer cells and liver endothelial cells, but not in HSC. PCR analysis revealed that cultured HSC express scavenger receptors. This was confirmed by Western blotting, although activation of HSC diminishes scavenger receptor protein expression. In conclusion, in a rat model for liver fibrosis M6P-HSA-liposomes can be efficiently targeted to non-parenchymal cells, including HSC. M6P receptors and scavenger receptors are involved in the cellular recognition of these liposomes, allowing multiple pharmacological interference in different pathways involved in the fibrosis.     

Successful targeting to rat hepatic stellate cells using albumin modified with cyclic peptides that recognize the collagen type VI receptor

The key pathogenic event in liver fibrosis is the activation of hepatic stellate cells (HSC). Consequently, new antifibrotic therapies are directed toward an inhibition of HSC activities. The aim of the present study was to develop a drug carrier to HSC, which would allow cell-specific delivery of antifibrotic drugs thus enhancing their effectiveness in vivo. We modified human serum albumin (HSA) with 10 cyclic peptide moieties recognizing collagen type VI receptors (C*GRGDSPC*, in which C* denotes the cyclizing cysteine residues) yielding pCVI-HSA. In vivo experiments showed preferential distribution of pCVI-HSA to both fibrotic and normal rat livers (respectively, 62 +/- 6 and 75 +/- 16% of the dose at 10 min after intravenous injection). Immunohistochemical analysis demonstrated that pCVI-HSA predominantly bound to HSC in fibrotic livers (73 +/- 14%). In contrast, endothelial cells contributed mostly to the total liver accumulation in normal rats. In vitro studies showed that pCVI-HSA specifically bound to rat HSC, in particular to the activated cells, and showed internalization of pCVI-HSA by these cells. In conclusion, pCVI-HSA may be applied as a carrier to deliver antifibrotic agents to HSC, which may strongly enhance the effectiveness and tissue selectivity of these drugs. This approach has the additional benefit that such carriers may block receptors that play a putative role in the pathogenesis of liver fibrosis.     

Uptake of phosphatidylserine-containing liposomes by liver sinusoidal endothelial cells in the serum-free perfused rat liver

We studied the kinetics of hepatic uptake of liposomes during serum-free recirculating perfusion of rat livers. Liposomes consisted of phosphatidylcholine, cholesterol and phosphatidylserine in a 6:4:0 or a 3:4:3 molar ratio and were radiolabelled with [3H]cholesteryl oleyl ether. The negatively charged liposomes were taken up to a 10-fold higher extent than the neutral ones. Hepatic uptake of fluorescently labelled liposomes was examined by fluorescence microscopy. The neutral liposomes displayed a typical Kupffer cell distribution pattern, in addition to weak diffuse staining of the parenchyma, while the negatively charged liposomes showed a characteristic sinusoidal lining pattern, consistent with an endothelial localization. In addition, scattered Kupffer cell staining was distinguished as well as diffuse parenchymal fluorescence. The mainly endothelial localisation of the negatively charged liposomes was confirmed by determining radioactivity in endothelial and Kupffer cells isolated following a 1-h perfusion. Perfusion in the presence of polyinosinic acid, an inhibitor of scavenger receptor activity, reduced the rate of uptake of the negatively charged liposomes twofold, indicating the involvement of this receptor in the elimination mechanism. These results are compatible with earlier in vitro studies on liposome uptake by isolated endothelial cells and Kupffer cells, which showed that in the absence of serum also endothelial cells in situ are able to take up massive amounts of negatively charged liposomes. The present results emphasize that the high in vitro endothelial cell uptake in the absence of serum from earlier observations was not an artifact induced by the cell isolation procedure.     

Uptake of phosphatidylserine-containing liposomes by liver sinusoidal endothelial cells in the serum-free perfused rat liver

We studied the kinetics of hepatic uptake of liposomes during serum-free recirculating perfusion of rat livers. Liposomes consisted of phosphatidylcholine, cholesterol and phosphatidylserine in a 6:4:0 or a 3:4:3 molar ratio and were radiolabelled with [³H]cholesteryl oleyl ether. The negatively charged liposomes were taken up to a 10-fold higher extent than the neutral ones. Hepatic uptake of fluorescently labelled liposomes was examined by fluorescence microscopy. The neutral liposomes displayed a typical Kupffer cell distribution pattern, in addition to weak diffuse staining of the parenchyma, while the negatively charged liposomes showed a characteristic sinusoidal lining pattern, consistent with an endothelial localization. In addition, scattered Kupffer cell staining was distinguished as well as diffuse parenchymal fluorescence. The mainly endothelial localisation of the negatively charged liposomes was confirmed by determining radioactivity in endothelial and Kupffer cells isolated following a 1-h perfusion. Perfusion in the presence of polyinosinic acid, an inhibitor of scavenger receptor activity, reduced the rate of uptake of the negatively charged liposomes twofold, indicating the involvement of this receptor in the elimination mechanism. These results are compatible with earlier in vitro studies on liposome uptake by isolated endothelial cells and Kupffer cells, which showed that in the absence of serum also endothelial cells in situ are able to take up massive amounts of negatively charged liposomes. The present results emphasize that the high in vitro endothelial cell uptake in the absence of serum from earlier observations was not an artifact induced by the cell isolation procedure.     

Uptake of liposomes containing phosphatidylserine by liver cells in vivo and by sinusoidal liver cells in primary culture: in vivo-in vitro differences

The interaction with liver cells of liposomes containing different mol fractions of phosphatidylserine was investigated in vivo and in vitro. Increasing the amount of liposomal phosphatidylserine from 10 to 30 mol% leads to a faster blood disappearance of the liposomes. Within the liver, which is mainly responsible for this elimination, these liposomes are only taken up by the hepatocytes and Kupffer cells. By contrast, sinusoidal endothelial cells, in vitro, do bind and internalize liposomes containing >/=30% phosphatidylserine at least as actively as Kupffer cells. The uptake by endothelial and Kupffer cells is inhibited by poly(inosinic acid) and other anionic macromolecules, suggesting the involvement of scavenger receptors. The lack of liposome uptake by endothelial cells under in vivo conditions can be attributed to plasma effects since addition of various sera caused severe reduction of in vitro uptake of liposomes. In vivo the phosphatidylserine head groups may be masked by plasma proteins adsorbed to the liposomal surface, thus preventing recognition by receptors, which are intrinsically able to recognize phosphatidylserine.     

Addressing liver fibrosis with liposomes targeted to hepatic stellate cells

Liver fibrosis is a chronic disease that results from hepatitis B and C infections, alcohol abuse or metabolic and genetic disorders. Ultimately, progression of fibrosis leads to cirrhosis, a stage of the disease characterized by failure of the normal liver functions. Currently, the treatment of liver fibrosis is mainly based on the removal of the underlying cause of the disease and liver transplantation, which is the only treatment for patients with advanced fibrosis. Hepatic stellate cells (HSC) are considered to be key players in the development of liver fibrosis. Chronically activated HSC produces large amounts of extracellular matrix and enhance fibrosis by secreting a broad spectrum of cytokines that exert pro-fibrotic actions in other cells, and in an autocrine manner perpetuate their own activation. Therefore, therapeutic interventions that inhibit activation of HSC and its pro-fibrotic activities are currently under investigation worldwide. In the present study we applied targeted liposomes as drug carriers to HSC in the fibrotic liver and explored the potential of these liposomes in antifibrotic therapies. Moreover, we investigated effects of bioactive compounds delivered by these liposomes on the progression of liver fibrosis. To our knowledge, this is the first study demonstrating that lipid-based drug carriers can be selectively delivered to HSC in the fibrotic liver. By incorporating the bioactive lipid DLPC, these liposomes can modulate different processes such as inflammation and fibrogenesis in the fibrotic liver. This dual functionality of liposomes as a drug carrier system with intrinsic biological effects may be exploited in new approaches to treat liver fibrosis.     

Formaldehyde treated albumin contains monomeric and polymeric forms that are differently cleared by endothelial and Kupffer cells of the liver: evidence for scavenger receptor heterogeneity.

Formaldehyde treated albumin (F-HSA) was found to consist of a monomeric and a polymeric fraction. Both fractions were primarily endocytosed by rat liver sinusoidal cells. However, immunohistochemical staining of endocytosed material showed that the relative contribution of the endothelial and Kupffer cells in uptake of the monomer and the polymer differed significantly, with the monomer mainly having an endothelial cell- and the polymer predominantly having a Kupffer cell pattern of distribution. To directly confirm these heterogeneous patterns, we injected in vivo the 125I-labeled F-HSA fractions and isolated the endothelial and Kupffer cells by centrifugal elutriation. 73.7% of the monomeric F-HSA was found in endothelial cells and only 14.9% was found in Kupffer cells. In contrast, the polymeric F-HSA (1500 kD) was mainly endocytosed by Kupffer cells (71%), whereas the endothelial cells contributed only for 24% in hepatic uptake. In vivo studies and isolated perfused rat liver experiments showed that endocytosis of both monomer and polymer was inhibited by co-administration of polyinosinic acid, a well known inhibitor for scavenger receptors, indicating that these receptors on endothelial and Kupffer cells are mainly involved in this uptake process.     

Curcumin attenuates angiogenesis in liver fibrosis and inhibits angiogenic properties of hepatic stellate cells

Hepatic fibrosis is concomitant with sinusoidal pathological angiogenesis, which has been highlighted as novel therapeutic targets for the treatment of chronic liver disease. Our prior studies have demonstrated that curcumin has potent antifibrotic activity, but the mechanisms remain to be elucidated. The current work demonstrated that curcumin ameliorated fibrotic injury and sinusoidal angiogenesis in rat liver with fibrosis caused by carbon tetrachloride. Curcumin reduced the expression of a number of angiogenic markers in fibrotic liver. Experiments in vitro showed that the viability and vascularization of rat liver sinusoidal endothelial cells and rat aortic ring angiogenesis were not impaired by curcumin. These results indicated that hepatic stellate cells (HSCs) that are characterized as liver‐specific pericytes could be potential target cells for curcumin. Further investigations showed that curcumin inhibited VEGF expression in HSCs associated with disrupting platelet‐derived growth factor‐β receptor (PDGF‐βR)/ERK and mTOR pathways. HSC motility and vascularization were also suppressed by curcumin associated with blocking PDGF‐βR/focal adhesion kinase/RhoA cascade. Gain‐ or loss‐of‐function analyses revealed that activation of peroxisome proliferator‐activated receptor‐γ (PPAR‐γ) was required for curcumin to inhibit angiogenic properties of HSCs. We concluded that curcumin attenuated sinusoidal angiogenesis in liver fibrosis possibly by targeting HSCs via a PPAR‐γ activation‐dependent mechanism. PPAR‐γ could be a target molecule for reducing pathological angiogenesis during liver fibrosis.     

Addressing Liver Fibrosis with Liposomes Targeted to Hepatic Stellate Cells

Liver fibrosis is a chronic disease that results from hepatitis B and C infections, alcohol abuse or metabolic and genetic disorders. Ultimately, progression of fibrosis leads to cirrhosis, a stage of the disease characterized by failure of the normal liver functions. Currently, the treatment of liver fibrosis is mainly based on the removal of the underlying cause of the disease and liver transplantation, which is the only treatment for patients with advanced fibrosis. Hepatic stellate cells (HSC) are considered to be key players in the development of liver fibrosis. Chronically activated HSC produces large amounts of extracellular matrix and enhance fibrosis by secreting a broad spectrum of cytokines that exert pro-fibrotic actions in other cells, and in an autocrine manner perpetuate their own activation. Therefore, therapeutic interventions that inhibit activation of HSC and its pro-fibrotic activities are currently under investigation worldwide. In the present study we applied targeted liposomes as drug carriers to HSC in the fibrotic liver and explored the potential of these liposomes in antifibrotic therapies. Moreover, we investigated effects of bioactive compounds delivered by these liposomes on the progression of liver fibrosis. To our knowledge, this is the first study demonstrating that lipid-based drug carriers can be selectively delivered to HSC in the fibrotic liver. By incorporating the bioactive lipid DLPC, these liposomes can modulate different processes such as inflammation and fibrogenesis in the fibrotic liver. This dual functionality of liposomes as a drug carrier system with intrinsic biological effects may be exploited in new approaches to treat liver fibrosis.     

In vivo fate of phosphorothioate antisense oligodeoxynucleotides: predominant uptake by scavenger receptors on endothelial liver cells.

Systemically administered phosphorothioate antisense oligodeoxynucleotides can specifically affect the expression of their target genes, which affords an exciting new strategy for therapeutic intervention. Earlier studies point to a major role of the liver in the disposition of these oligonucleotides. The aim of the present study was to identify the cell type(s) responsible for the liver uptake of phosphorothioate oligodeoxynucleotides and to examine the mechanisms involved. In our study we used ISIS-3082, a phosphorothioate antisense oligodeoxynucleotide specific for murine ICAM-1. Intravenously injected [3H]ISIS-3082 (dose: 1 mg/kg) was cleared from the circulation of rats with a half-life of 23.3+/-3.8 min. At 90 min after injection (>90% of [3H]ISIS-3082 cleared), the liver contained the most radioactivity, whereas the second-highest amount was recovered in the kidneys (40.5+/-1.4% and 17.9+/-1.3% of the dose, respectively). Of the remaining tissues, only spleen and bone marrow actively accumulated [3H]ISIS-3082. By injecting different doses of [3H]ISIS-3082, it was found that uptake by liver, spleen, bone marrow, and kidneys is saturable, which points to a receptor-mediated process. Subcellular fractionation of the liver indicates that ISIS-3082 is internalized and delivered to the lysosomes. Liver uptake occurs mainly (for 56.1+/-3.0%) by endothelial cells, whereas parenchymal and Kupffer cells account for 39.6+/-4.5 and 4.3+/-1.7% of the total liver uptake, respectively. Preinjection of polyinosinic acid substantially reduced uptake by liver and bone marrow, whereas polyadenylic acid was ineffective, which indicates that in these tissues scavenger receptors are involved in uptake. Polyadenylic acid, but not polyinosinic acid, reduced uptake by kidneys, which suggests renal uptake by scavenger receptors different from those in the liver. We conclude that scavenger receptors on rat liver endothelial cells play a predominant role in the plasma clearance of ISIS-3082. As scavenger receptors are also expressed on human endothelial liver cells, our findings are probably highly relevant for the therapeutic application of phosphorothioate oligodeoxynucleotides in humans. If the target gene is not localized in endothelial liver cells, the therapeutic effectiveness might be improved by developing delivery strategies that redirect the oligonucleotides to the actual target cells.     

Expression of P2X receptors on immune cells in the rat liver during postnatal development

Single and double-labeling immunofluorescence and RT-PCR expression of P2X receptor proteins and mRNAs were used in a study of the liver of postnatal rats. OX62 and ED1 were used as markers for dendritic and macrophage (Kupffer) cells respectively. The results showed that the P2X₆ receptor subunit was up-regulated by 15-fold on hepatic sinusoid cells during postnatal days P1 to P60. Subpopulations of Kupffer cells co-expressed P2X₄ and P2X₆ receptor subunits and dendritic cells co-expressed P2X₄ and P2X₇ receptor subunits. Lipopolysaccharide (endotoxin) injected into the peritoneal cavity led to increased expression of the P2X₆ receptor on Kupffer cells, suggesting that the P2X₆ receptor subunit may be up-regulated by endotoxin. This study presents the first evidence that P2X receptors are widely distributed in the rat liver immune system and that activation of Kupffer and dendritic cells in the rat liver might be regulated by extracellular ATP.     

Different fate in vivo of oxidatively modified low density lipoprotein and acetylated low density lipoprotein in rats. Recognition by various scavenger receptors on Kupffer and endothelial liver cells

Human low density lipoprotein was oxidized (Ox-LDL) by exposure to 5 microM Cu2+ and its fate in vivo was compared to acetylated low density lipoprotein (Ac-LDL). Ox-LDL, when injected into rats, is rapidly removed from the blood circulation by the liver, similarly as Ac-LDL. A separation of rat liver cells into parenchymal, endothelial, and Kupffer cells at 10 min after injection of Ox-LDL or Ac-LDL indicated that the Kupffer cell uptake of Ox-LDL is 6.8-fold higher than for Ac-LDL, leading to Kupffer cells as the main liver site for Ox-LDL uptake. In vitro studies with isolated liver cells indicated that saturable high affinity sites for Ox-LDL were present on both endothelial and Kupffer cells, whereby the capacity of Kupffer cells to degrade Ox-LDL is 6-fold higher than for endothelial cells. Competition studies showed that unlabeled Ox-LDL competed as efficiently (90%) as unlabeled Ac-LDL with the cell association and degradation of 125I-labeled Ac-LDL by endothelial and Kupffer cells. However, unlabeled Ac-LDL competed only partially (20-30%) with the cell association and degradation of 125I-labeled Ox-LDL by Kupffer cells, while unlabeled Ox-LDL or polyinosinic acid competed for 70-80%. It is concluded that the liver contains, in addition to the scavenger (Ac-LDL) receptor which interacts efficiently with both Ac-LDL and Ox-LDL and which is concentrated on endothelial cells, an additional specific Ox-LDL receptor which is highly concentrated on Kupffer cells. In vivo the specific Ox-LDL recognition site on Kupffer cells will form the major protection system against the occurrence of the atherogenic Ox-LDL particles in the blood.     

Alterations in Fc receptor activity in sinusoidal endothelial cells and Kupffer cells during D-galactosamine (GalN)-induced liver injury in rats. A histological study

Fc receptors in sinusoidal cells and immune complex uptake were studied histologically in D-galactosamine HCl (GalN)-induced liver injury in rats. Kupffer cells and monocytes were distinguished from sinusoidal endothelial cells and from each other by endogenous peroxidase staining. Fc receptors were found along the sinusoidal endothelium throughout the lobules in normal livers. In acute injury caused by 300 or 750 mg/kg of GalN, Fc receptors were preserved within necrotic foci until the foci were infiltrated by inflammatory cells. The endothelial Fc receptor activity altered, as demonstrated by their capacity to bind immune complexes, after GalN injection. The activity decreased from 24 h after injection in the periportal areas in both dose groups, and increased transiently with dose-dependence in the remaining areas. Kupffer cell numbers also showed a transient dose-dependent increase, except in the periphery of lobules where they generally decreased. In chronic injury with 400 mg/kg, Fc receptors were lost and Kupffer cells decreased in the periportal areas. Circulating immune complexes were ingested by Kupffer cells and endothelial cells in normal and injured livers, showing the the same distribution as that of Fc receptors except that the complexes decreased gradually towards the centrilobular zones.     

Effect of human amniotic epithelial cells on pro‐fibrogenic resident hepatic cells in a rat model of liver fibrosis

Myofibroblasts are key fibrogenic cells responsible for excessive extracellular matrix synthesis characterizing the fibrotic lesion. In liver fibrosis, myofibroblasts derive either from activation of hepatic stellate cells (HSC) and portal fibroblasts (PF), or from the activation of fibroblasts that originate from ductular epithelial cells undergoing epithelial–mesenchymal transition. Ductular cells can also indirectly promote myofibroblast generation by activating TGF‐β, the main fibrogenic growth factor, through αvβ6 integrin. In addition, after liver injury, liver sinusoidal cells can lose their ability to maintain HSC quiescence, thus favouring HSC differentiation towards myofibroblasts. The amniotic membrane and epithelial cells (hAEC) derived thereof have been shown to decrease hepatic myofibroblast levels in rodents with liver fibrosis. In this study, in a rat model of liver fibrosis, we investigated the effects of hAEC on resident hepatic cells contributing to myofibroblast generation. Our data show that hAEC reduce myofibroblast numbers with a consequent reduction in fibronectin and collagen deposition. Interestingly, we show that hAEC strongly act on specific myofibroblast precursors. Specifically, hAEC reduce the activation of PF rather than HSC. In addition, hAEC target reactive ductular cells by inhibiting their proliferation and αvβ6 integrin expression, with a consequent decrease in TGF‐β activation. Moreover, hAEC counteract the transition of ductular cells towards fibroblasts, while it does not affect injury‐induced and fibrosis‐promoting sinusoidal alterations. In conclusion, among the emerging therapeutic applications of hAEC in liver diseases, their specific action on PF and ductular cells strongly suggests their application in liver injuries involving the expansion and activation of the portal compartment.     

Antisense strategy against PDGF B-chain proves effective in preventing experimental liver fibrogenesis

Hepatic stellate cells (HSCs) and transdifferentiated myofibroblasts are the principal producers of excessive extracellular matrix in liver fibrosis and cirrhosis. Activation of HSC is regulated by several cytokines and growth factors, including platelet-derived growth factor B-chain (PDGF-B), a potent mitogen for HSC, and overexpressed during hepatic fibrogenesis. Previous studies showed that MAPK and phosphatidylinositol 3' kinase are key signaling pathways involved in PDGF-induced stimulation of HSC. Based on the involvement of PDGF-B in fibrogenesis, reducing ligand stimulation of proliferative cytokine- or growth factor receptors interfering with receptor signaling therefore presents an interesting strategy for hepatic fibrosis prevention or interruption. We therefore generated an adenoviral vector serotype 5 (Ad5) expressing an antisense mRNA of the PDGF B-chain (Ad5-CMV-asPDGF) for application in an experimentally induced liver fibrogenesis model. The transgene clearly showed the ability to down-regulate endogenous PDGF B-chain and PDGFRbeta mRNA in culture-activated HSC and rat livers. The asPDGF mRNA also attenuates experimental liver fibrogenesis indicated by reduced levels of alpha-SMA and collagen type I expression.     

Sinusoidal endothelial COX-1-derived prostanoids modulate the hepatic vascular tone of cirrhotic rat livers

CCl(4) cirrhotic rat liver exhibits a hyperresponse to the alpha(1)-adrenergic agonist methoxamine (Mtx) that is associated with enhanced thromboxane A(2) (TXA(2)) production and is abrogated by indomethacin. To further elucidate the molecular mechanisms involved in the hyperresponse to vasoconstrictors, portal perfusion pressure dose-response curves to Mtx were performed in CCl(4) cirrhotic rats livers after preincubation with vehicle, the cyclooxygenase (COX)-1 selective inhibitor SC-560, and the COX-2 selective inhibitor SC-236. TXA(2) production was determined in samples of the perfusate. COX-1 expression was analyzed and quantified in hepatocytes, Kupffer cells, sinusoidal endothelial cells (SEC), and hepatic stellate cells (HSC) isolated from control and cirrhotic rat livers by double-immunofluorescence staining, with specific markers for each population using flow cytometry or Western blot analysis. COX-1 protein levels were not significantly increased in cirrhotic livers, but COX-2 protein expression was increased. COX-1 inhibition, but not COX-2, significantly attenuated the response to Mtx and prevented the increased production of TXA(2). Cirrhotic livers showed an increased expression of COX-1 in SEC and reduced expression in HSC compared with control livers, whereas COX-1 was similarly distributed in Kupffer cells. Despite abundant hepatic COX-2 expression, the increased response to Mtx of cirrhotic livers is mainly dependent of COX-1. Upregulation of COX-1 in cirrhotic SEC may be responsible for the hyperesponse to Mtx.     

In vitro induction of tumor necrosis factor-α by ochratoxin A (OTA) from rat liver: role of Kupffer cells

Tumor necrosis factor-α (TNF-α) is released from blood-free perfused rat liver by the fungal metabolite ochratoxin A. Here we have identified Kupffer cells as the sole source of OTA-mediated cytokine release. If single cell preparation of Kupffer cells, hepatocytes, or sinusoidal endothelial cells were prepared from rat livers, only Kupffer cells released TNF-α upon incubation with 2.5 μmol/l OTA. OTA failed to induce TNF-α release in the blood-free perfused isolated rat liver when Kupffer cells were blockedin vitro by 15 μmol/l gadolinium chloride. When rats were pretreatedin vivo with the Kupffer cell depleting clodronate liposomes, OTA-mediated TNF-α release was abrogated in the isolated perfused liver model.     

Involvement of the nuclear high mobility group B1 peptides released from injured hepatocytes in murine hepatic fibrogenesis

This study investigated the pro-fibrogenic role of high mobility group box 1 (HMGB1) peptides in liver fibrogenesis. An animal model of carbon tetrachloride (CCl₄)-induced liver fibrosis was used to examine the serum HMGB1 levels and its intrahepatic distribution. The increased serum HMGB1 levels were positively correlated with elevation of transforming growth factor-β1 (TGF-β1) and collagen deposition during fibrogenesis. The cytoplasmic distribution of HMGB1 was noted in the parenchymal hepatocytes of fibrotic livers. In vitro studies confirmed that exposure to hydrogen peroxide and CCl₄ induced an intracellular mobilization and extracellular release of nuclear HMGB1 peptides in clone-9 and primary hepatocytes, respectively. An uptake of exogenous HMGB1 by hepatic stellate cells (HSCs) T6 cells indicated a possible paracrine action of hepatocytes on HSCs. Moreover, HMGB1 dose-dependently stimulated HSC proliferation, up-regulated de novo synthesis of collagen type I and α-smooth muscle actin (α-SMA), and triggered Smad2 phosphorylation and its nuclear translocation through a TGF-β1-independent mechanism. Blockade with neutralizing antibodies and gene silencing demonstrated the involvement of the receptor for advanced glycation end-products (RAGE), but not toll-like receptor 4, in cellular uptake of HMGB1 and the HMGB1-mediated Smad2 and ERK1/2 phosphorylation as well as α-SMA up-regulation in HSC-T6 cells. Furthermore, anti-RAGE treatment significantly ameliorated CCl₄-induced liver fibrosis. In conclusion, the nuclear HMGB1 peptides released from parenchymal hepatocytes during liver injuries may directly activate HSCs through stimulating HSC proliferation and transformation, eventually leading to the fibrotic changes of livers. Blockade of HMGB1/RAGE signaling cascade may constitute a therapeutic strategy for treatment of liver fibrosis.