Hacer Sahin

Antagonism of the chemokine Ccl5 ameliorates experimental liver fibrosis in mice

Activation of hepatic stellate cells in response to chronic inflammation represents a crucial step in the development of liver fibrosis. However, the molecules involved in the interaction between immune cells and stellate cells remain obscure. Herein, we identify the chemokine CCL5 (also known as RANTES), which is induced in murine and human liver after injury, as a central mediator of this interaction. First, we showed in patients with liver fibrosis that CCL5 haplotypes and intrahepatic CCL5 mRNA expression were associated with severe liver fibrosis. Consistent with this, we detected Ccl5 mRNA and CCL5 protein in 2 mouse models of liver fibrosis, induced by either injection of carbon tetrachloride (CCl4) or feeding on a methionine and choline-deficient (MCD) diet. In these models, Ccl5-/- mice exhibited decreased hepatic fibrosis, with reduced stellate cell activation and immune cell infiltration. Transplantation of Ccl5-deficient bone marrow into WT recipients attenuated liver fibrosis, identifying infiltrating hematopoietic cells as the main source of Ccl5. We then showed that treatment with the CCL5 receptor antagonist Met-CCL5 inhibited cultured stellate cell migration, proliferation, and chemokine and collagen secretion. Importantly, in vivo administration of Met-CCL5 greatly ameliorated liver fibrosis in mice and was able to accelerate fibrosis regression. Our results define a successful therapeutic approach to reduce experimental liver fibrosis by antagonizing Ccl5 receptors.

Chemokine Cxcl9 attenuates liver fibrosis‐associated angiogenesis in mice

Recent data suggest that the chemokine receptor CXCR3 is functionally involved in fibroproliferative disorders, including liver fibrosis. Neoangiogenesis is an important pathophysiological feature of liver scarring, but a functional role of angiostatic CXCR3 chemokines in this process is unclear. We therefore investigated neoangiogenesis in carbon tetrachloride (CCl4)‐induced liver fibrosis in Cxcr3−/− and wildtype mice by histological, molecular, and functional imaging methods. Furthermore, we assessed the direct role of vascular endothelial growth factor (VEGF) overexpression on liver angiogenesis and the fibroproliferative response using a Tet‐inducible bitransgenic mouse model. The feasibility of attenuation of angiogenesis and associated liver fibrosis by therapeutic treatment with the angiostatic chemokine Cxcl9 was systematically analyzed in vitro and in vivo. The results demonstrate that fibrosis progression in Cxcr3−/− mice was strongly linked to enhanced neoangiogenesis and VEGF/VEGFR2 expression compared with wildtype littermates. Systemic VEGF overexpression led to a fibrogenic response within the liver and was associated with a significantly increased Cxcl9 expression. In vitro, Cxcl9 displayed strong antiproliferative and antimigratory effects on VEGF‐stimulated endothelial cells and stellate cells by way of reduced VEGFR2 (KDR), phospholipase Cγ (PLCγ), and extracellular signal‐regulated kinase (ERK) phosphorylation, identifying this chemokine as a direct counter‐regulatory molecule of VEGF signaling within the liver. Accordingly, systemic administration of Cxcl9 led to a strong attenuation of neoangiogenesis and experimental liver fibrosis in vivo. Conclusion: The results identify direct angiostatic and antifibrotic effects of the Cxcr3 ligand Cxcl9 in a model of experimental liver fibrosis. The amelioration of liver damage by systemic application of Cxcl9 might offer a novel therapeutic approach for chronic liver diseases associated with increased neoangiogenesis. (HEPATOLOGY 2012)

Functional role of chemokines in liver disease models

Chemokines are a class of small cytokine-like molecules that orchestrate immune cell infiltration into the liver in response to acute and chronic injuries. Apart from their chemotactic effect, however, chemokines seem to mediate many other aspects of liver diseases, including a direct activation of stellate cells, the modulation of hepatocyte proliferation and angiogenesis. The identification of specific biological functions for chemokines in liver diseases has been hampered by the finding that resident and infiltrating cells in the liver are often a source, as well as a target, of chemokines. Furthermore, chemokines might cause differing effects depending on the etiology of liver damage, their local concentrations and their ability to form multimers and heterodimers. Nevertheless, the functions of a number of important chemokines and their associated receptors have been identified in both in vivo and in vitro studies. Indeed, harmful (proinflammatory, profibrogenic) and beneficial (antifibrogenic, antiangiogenic) effects of chemokines have been discovered in experimental liver disease models. In this Review, the current knowledge of chemokines in experimental liver disease models is summarized. Advances that might lead to preclinical applications are discussed, as are the roles of chemokine receptors as promising pharmacologically targetable molecules.

Chemokines in tissue fibrosis

Fibrosis or scarring of diverse organs and tissues is considered as a pathologic consequence of a chronically altered wound healing response which is tightly linked to inflammation and angiogenesis. The recruitment of immune cells, local proliferation of fibroblasts and the consecutive accumulation of extracellular matrix proteins are common pathophysiological hallmarks of tissue fibrosis, irrespective of the organ involved. Chemokines, a family of chemotactic cytokines, appear to be central mediators of the initiation as well as progression of these biological processes. Traditionally chemokines have only been considered to play a critical role in orchestrating the influx of immune cells to sites of tissue injury. However, within the last years, further aspects of chemokine biology including fibroblast activation and angiogenesis have been deciphered in tissue fibrosis of many different organs. Interestingly, certain chemokines appear to mediate common effects in liver, kidney, lung, and skin of various animal models, while others mediate tissue specific effects. These aspects have to be kept in mind when extrapolating data of animal studies to early human trials. Nevertheless, the further understanding of chemokine effects in tissue fibrosis might be an attractive approach for identifying novel therapeutic targets in chronic organ damage associated with high morbidity and mortality. This article is part of a Special Issue entitled: Fibrosis: Translation of basic research to human disease.

Proapoptotic effects of the chemokine, CXCL 10 are mediated by the noncognate receptor TLR4 in hepatocytes

Aberrant expression of the chemokine CXC chemokine ligand (CXCL)10 has been linked to the severity of hepatitis C virus (HCV)‐induced liver injury, but the underlying molecular mechanisms remain unclear. In this study, we describe a yet‐unknown proapoptotic effect of CXCL10 in hepatocytes, which is not mediated through its cognate chemokine receptor, but the lipopolysaccharide receptor Toll‐like receptor 4 (TLR4). To this end, we investigated the link of CXCL10 expression with apoptosis in HCV‐infected patients and in murine liver injury models. Mice were treated with CXCL10 or neutralizing antibody to systematically analyze effects on hepatocellular apoptosis in vivo. Direct proapoptotic functions of CXCL10 on different liver cell types were evaluated in detail in vitro. The results showed that CXCL10 expression was positively correlated with liver cell apoptosis in humans and mice. Neutralization of CXCL10 ameliorated concanavalin A–induced tissue injury in vivo, which was strongly associated with reduced liver cell apoptosis. In vitro, CXCL10 mediated the apoptosis of hepatocytes involving TLR4, but not CXC chemokine receptor 3 signaling. Specifically, CXCL10 induced long‐term protein kinase B and Jun N‐terminal kinase activation, leading to hepatocyte apoptosis by caspase‐8, caspase‐3, and p21‐activated kinase 2 cleavage. Accordingly, systemic application of CXCL10 led to TLR4‐induced liver cell apoptosis in vivo. Conclusion: The results identify CXCL10 and its noncognate receptor, TLR4, as a proapoptotic signaling cascade during liver injury. Antagonism of the CXCL10/TLR4 pathway might be a therapeutic option in liver diseases associated with increased apoptosis. (HEPATOLOGY 2013)

BASIC—a bile acid-sensitive ion channel highly expressed in bile ducts

Brain liver intestine Na+ channel (BLINaC) is an ion channel of the DEG/ENaC gene family of unknown function. BLINaC from rats (rBLINaC) and humans (INaC) is inactive at rest, and its mode of activation has remained unclear. Here, we show that the BLINaC protein localizes to cholangiocytes, epithelial cells that line bile ducts. Moreover, we provide evidence that rBLINaC and INaC are robustly activated by bile acids, in particular chenodeoxycholic acid and hyodeoxycholic acid (EC50=2.1±0.05 mM). Thus, BLINaC appears to be an epithelial cation channel of bile ducts sensitive to physiological concentrations of bile acids. BLINaC is related to acid‐sensing ion channels (ASICs) and to the epithelial Na+ channel (ENaC) and shares ligand activation with ASICs and epithelial localization with ENaC. Therefore, based on the close homology of BLINaC to ASICs and its activation by bile acids, we propose to rename BLINaC bile acid‐sensitive ion channel (BASIC).—Wiemuth, D., Sahin, H., Falkenburger, B. H., Lefèvre, C. M. T., Wasmuth, H. E., Gründer, S. BASIC—a bile acid‐sensitive ion channel highly expressed in bile ducts. FASEB J. 26, 4122–4130 (2012). www.fasebj.org

Impaired biomechanical properties correlate with neoangiogenesis as well as VEGF and MMP-3 expression during rat patellar tendon healing

Recent studies reveal an important role of vascular endothelial growth factor (VEGF)‐induced angiogenesis in degenerative tendon diseases. The way how VEGF influences mechanical properties of the tendons is not well understood yet. We here hypothesized that tendinopathy results in a hypoxia‐mediated stimulation of VEGF and that the mechanical stability of the tendon is impaired in an angiogenic process by VEGF‐induced matrix metalloproteinases (MMPs). A modified in situ freezing model of patellar tendon was used to create a tendinopathy. 0, 7, 14, and 28 days post‐surgical animals were sacrificed and patellar tendons were dissected for biomechanical and immunohistochemical analysis. Native tendons were used as controls. Immunohistochemical staining revealed a peak in HIF‐1α stabilization immediately after surgery. Both VEGF and MMP‐3 were increased 7 days after surgery. Angiogenesis was also abundant 7 days after surgery. In contrast, biomechanical stability of the tendon was decreased 7 days after surgery. The current results reveal a time‐dependent correlation of HIF‐1/VEGF‐induced and MMP‐3‐supported angiogenesis with decreased biomechanical properties during tendon healing. The therapeutical modulation of neoangiogenesis by influencing the level of VEGF and MMP‐3 might be a promising target for new approaches in degenerative tendon diseases.

The Chemokine CCL3 Promotes Experimental Liver Fibrosis in Mice

Liver fibrosis is associated with infiltrating immune cells and activation of hepatic stellate cells. We here aimed to investigate the effects of the CC chemokine CCL3, also known as macrophage inflammatory protein-1α, in two different fibrosis models. To this end, we treated mice either with carbon tetrachloride or with a methionine- and choline-deficient diet to induce fibrosis in CCL3 deficient and wild-type mice. The results show that the protein expression of CCL3 is increased in wild-type mice after chronic liver injury. Deletion of CCL3 exhibited reduced liver fibrosis compared to their wild-type counterparts. We could validate these results by treating the two mouse groups with either carbon tetrachloride or by feeding a methionine- and choline-deficient diet. In these models, lack of CCL3 is functionally associated with reduced stellate cell activation and liver immune cell infiltration. In vitro, we show that CCL3 leads to increased proliferation and migration of hepatic stellate cells. In conclusion, our results define the chemokine CCL3 as a mediator of experimental liver fibrosis. Thus, therapeutic modulation of CCL3 might be a promising target for chronic liver diseases.

Overexpression of Endoglin Modulates TGF-β1-Signalling Pathways in a Novel Immortalized Mouse Hepatic Stellate Cell Line

Hepatic stellate cells (HSCs) play a major role in the pathogenesis of liver fibrosis. Working on primary HSCs requires difficult isolation procedures; therefore we have generated and here characterize a mouse hepatic stellate cell line expressing GFP under control of the collagen 1(I) promoter/enhancer. These cells are responsive to pro-fibrogenic stimuIi, such as PDGF or TGF-β1, and are able to activate intracellular signalling pathways including Smads and MAP kinases. Nevertheless, due to the basal level of activation, TGF-β1 did not significantly induce GFP expression contrasting the TGF-β1 regulated endogenous collagen I expression. We could demonstrate that the accessory TGF-β-receptor endoglin, which is endogenously expressed at very low levels, has a differential effect on signalling of these cells when transiently overexpressed. In the presence of endoglin activation of Smad1/5/8 was drastically enhanced. Moreover, the phosphorylation of ERK1/2 was increased, and the expression of vimentin, α-smooth muscle actin and connective tissue growth factor was upregulated. Endoglin induced a slight increase in expression of the inhibitor of differentiation-2 while the amount of endogenous collagen type I was reduced. Therefore, this profibrogenic cell line with hepatic stellate cell origin is not only a promising novel experimental tool, which can be used in vivo for cell tracing experiments. Furthermore it allows investigating the impact of various regulatory proteins (e.g. endoglin) on profibrogenic signal transduction, differentiation and hepatic stellate cell biology.

Strong activation of bile acid-sensitive ion channel (BASIC) by ursodeoxycholic acid.

Bile acid-sensitive ion channel (BASIC) is a member of the DEG/ENaC gene family of unknown function. Rat BASIC (rBASIC) is inactive at rest. We have recently shown that cholangiocytes, the epithelial cells lining the bile ducts, are the main site of BASIC expression in the liver and identified bile acids, in particular hyo- and chenodeoxycholic acid, as agonists of rBASIC. Moreover, it seems that extracellular divalent cations stabilize the resting state of rBASIC, because removal of extracellular divalent cations opens the channel. In this addendum, we demonstrate that removal of extracellular divalent cations potentiates the activation of rBASIC by bile acids, suggesting an allosteric mechanism. Furthermore, we show that rBASIC is strongly activated by the anticholestatic bile acid ursodeoxycholic acid (UDCA), suggesting that BASIC might mediate part of the therapeutic effects of UDCA.