Role of the stromal-derived factor-1 (SDF-1)-CXCR4 axis in the interaction between hepatic stellate cells and cholangiocarcinoma.

BACKGROUNDS & AIMS: Cholangiocarcinoma (CCA) is highly fatal because of early invasion, widespread metastasis, and lack of an effective therapy. Migration, invasion, and metastasis of CCA cells are modulated by signals received from stromal cells. The SDF-1-CXCR4 axis emerges as a pivotal regulator of migration and survival of different tumor cells. The aim of the present study was to characterize the interaction between CCA cells and human hepatic stellate cells (hHSC) focusing on the role of SDF-1. METHODS: The intrahepatic CCA cell line HuCCT-1 and primary hHSC were used for this study. RNA expression was examined by RTQ-PCR and protein expression by Western blotting. Immunofluorescence microscopy and immunohistochemistry were also employed. Migration of CCA cells was assessed using modified Boyden chambers. RESULTS: CXCR4 was clearly expressed in CCA cells of human CCA liver specimens. SDF-1 and hHSC conditioned medium (CM) promoted HuCCT-1 cell migration, which was abrogated by pre-incubation with AMD3100, a non-peptide antagonist of the CXCR4 receptor. In addition, HuCCT-1 cells silenced for CXCR4 did not migrate in presence of SDF-1. Both P-ERK and p-AKT were implicated in HuCCT-1 migration and showed a biphasic trend under stimulation of SDF-1. Finally, SDF-1 induced apoptotic rescue of HuCCT-1 cells by binding to CXCR4. CONCLUSIONS: Our study demonstrates that CCA cells migration and survival are modulated by the crosstalk between SDF-1, released by hHSC, and HuCCT-1 cells bearing CXCR4.

Lack of CC chemokine ligand 2 differentially affects inflammation and fibrosis according to the genetic background in a murine model of steatohepatitis.

Expression of CCL2 (CC chemokine ligand 2) (or monocyte chemoattractant protein-1) regulates inflammatory cell infiltration in the liver and adipose tissue, favouring steatosis. However, its role in the pathogenesis of steatohepatitis is still uncertain. In the present study, we investigated the development of non-alcoholic steatohepatitis induced by an MCD diet (methionine/choline-deficient diet) in mice lacking the CCL2 gene on two different genetic backgrounds, namely Balb/C and C57/Bl6J. WT (wild-type) and CCL2-KO (knockout) mice were fed on a lipid-enriched MCD diet or a control diet for 8 weeks. In Balb/C mice fed on the MCD diet, a lack of CCL2 was associated with lower ALT (alanine transaminase) levels and reduced infiltration of inflammatory cells, together with a lower generation of oxidative-stress-related products. Sirius Red staining demonstrated pericellular fibrosis in zone 3, and image analysis showed a significantly lower matrix accumulation in CCL2-KO mice. This was associated with reduced hepatic expression of TGF-ß (transforming growth factor-ß), type I procollagen, TIMP-1 (tissue inhibitor of metalloproteinases-1) and α-smooth muscle actin. In contrast, in mice on a C57Bl/6 background, neither ALT levels nor inflammation or fibrosis were significantly different comparing WT and CCL2-KO animals fed on an MCD diet. In agreement, genes related to fibrogenesis were expressed to comparable levels in the two groups of animals. Comparison of the expression of several genes involved in inflammation and repair demonstrated that IL (interleukin)-4 and the M2 marker MGL-1 (macrophage galactose-type C-type lectin 1) were differentially expressed in Balb/C and C57Bl/6 mice. No significant differences in the degree of steatosis were observed in all groups of mice fed on the MCD diet. We conclude that, in experimental murine steatohepatitis, the effects of CCL2 deficiency are markedly dependent on the genetic background.

Mammalian target of rapamycin mediates the angiogenic effects of leptin in human hepatic stellate cells.

Leptin modulates the angiogenic properties of hepatic stellate cells (HSC), but the molecular mechanisms involved are poorly understood. We investigated the pathways regulating hypoxia-inducible factor 1α (HIF-1α) and vascular endothelial growth factor (VEGF) in leptin-stimulated myofibroblastic HSC. Exposure to leptin enhanced the phosphorylation of TSC2 on T1462 residues and of p70 S6 kinase and the translational inhibitor 4E-binding protein-1, indicating the ability of leptin to activate the mammalian target of rapamycin (mTOR) pathway. Similar findings were observed when HSC were exposed to PDGF. Both leptin and PDGF increased the expression of HIF-1α and VEGF in HSC. In the presence of rapamycin, a specific mTOR inhibitor, leptin and PDGF were no longer able to activate mTOR, and expression of VEGF was reduced, whereas HIF-1α abundance was not affected. Moreover, knockdown of Raptor, a component of the mTORC1 complex, reduced the ability of leptin to increase VEGF. mTOR was also necessary for leptin- and PDGF-dependent increase in HSC migration. Leptin increased the generation of reactive oxygen species in HSC, which was reduced by NADP(H) oxidase inhibitors. Both N-acetyl cysteine and diphenylene iodonium, a NADP(H) inhibitor, inhibited the expression of HIF-1α and VEGF stimulated by leptin or PDGF. Finally, conditioned media from HSC treated with leptin or PDGF induced tube formation in cultured human umbilical vein endothelial cells. In conclusion, in HSC exposed to leptin or PDGF, increased expression of VEGF requires both activation of mTOR and generation of reactive oxygen species via NADPH-oxidase. Induction of HIF-1α requires NADP(H) oxidase but not mTOR activation.

Modulation of liver fibrosis by adipokines.

Hepatic fibrosis is an integrated process triggered by chronic liver damage, leading to the accumulation of extracellular matrix. In patients with chronic liver disease, this process is favored by the presence of obesity or overweight, which are also relevant risk factors for the progression of nonalcoholic steatohepatitis. In this paper, we review the available evidence indicating the modulation of the fibrogenic process by adipokines, a group of cytokines secreted primarily by adipose tissue. In particular, we discuss in detail the role of leptin and adiponectin, which favor and limit the fibrogenic process, respectively. The possible involvement of other recently identified adipokines is also briefly outlined.

gp120 modulates the biology of human hepatic stellate cells: a link between HIV infection and liver fibrogenesis.

OBJECTIVE: In patients with hepatitis C virus (HCV)/HIV co-infection, a faster progression of liver fibrosis to cirrhosis has been reported. In this study, an investigation was carried out to determine whether gp120, an HIV envelope protein, modulates the biology of human hepatic stellate cells (HSCs), key cell types in the pathogenesis of fibrosis. METHODS: Myofibroblastic HSCs were isolated from normal human liver tissue. Gene expression was measured by real-time PCR. Cell migration was assessed in Boyden chambers. Intracellular signalling pathways were evaluated using phosphorylation-specific antibodies or by transfection of a reporter plasmid. RESULTS: Transcripts for the chemokine receptors CCR5 and CXCR4, which bind gp120, were detectable in human HSCs. Upon exposure to M-tropic recombinant gp120, which binds CCR5, a significant increase in HSC chemotaxis was observed (1.6+/-0.3-fold, p=0.03). The effects of gp120 were prevented by protein inactivation. gp120 also resulted in a significant increase in secretion (1.5+/-0.3-fold, p=0.03) and gene expression (1.47+/-0.13-fold, p=0.02) of the proinflammatory chemokine monocyte chemoattractant protein-1, and in increased gene expression of tissue inhibitor of metalloprotease-1 and interleukin-6 (2.03+/-0.57-fold, p=0.02). gp120-induced migration required Akt activation. gp120 also induced activation of nuclear factor-kappaB (NF-kappaB) and p38(MAPK). Preincubation of HSCs with TAK779, a CCR5 receptor antagonist, prevented gp120-mediated chemotaxis and monocyte chemoattractant protein-1 secretion. Expression of CCR5 was detectable in areas of inflammation and fibrogenesis in liver biopsies of patients with HCV/HIV co-infection. CONCLUSIONS: This study shows that HIV gp120 modulates different aspects of HSC biology, including directional cell movement and expression of proinflammatory cytokines. These results identify a direct pathway possibly linking HIV infection with liver fibrogenesis via envelope proteins.

Curcumin limits the fibrogenic evolution of experimental steatohepatitis.

Nonalcoholic steatohepatitis is characterized by the association of steatosis with hepatic cell injury, lobular inflammation and fibrosis. Curcumin is known for its antioxidant, anti-inflammatory and antifibrotic properties. The aim of this study was to test whether the administration of curcumin limits fibrogenic evolution in a murine model of nonalcoholic steatohepatitis. Male C57BL/6 mice were divided into four groups and fed a diet deficient in methionine and choline (MCD) or the same diet supplemented with methionine and choline for as long as 10 weeks. Curcumin (25 microg per mouse) or its vehicle (DMSO) was administered intraperitoneally every other day. Fibrosis was assessed by Sirius red staining and histomorphometry. Intrahepatic gene expression was measured by quantitative PCR. Hepatic oxidative stress was evaluated by staining for 8-OH deoxyguanosine. Myofibroblastic hepatic stellate cells (HSCs) were isolated from normal human liver tissue. The increase in serum ALT caused by the MCD diet was significantly reduced by curcumin after 4 weeks. Administration of the MCD diet was associated with histological steatosis and necro-inflammation, and this latter was significantly reduced in mice receiving curcumin. Curcumin also inhibited the generation of hepatic oxidative stress. Fibrosis was evident after 8 or 10 weeks of MCD diet and was also significantly reduced by curcumin. Curcumin decreased the intrahepatic gene expression of monocyte chemoattractant protein-1, CD11b, procollagen type I and tissue inhibitor of metalloprotease (TIMP)-1, together with protein levels of alpha-smooth muscle-actin, a marker of fibrogenic cells. In addition, curcumin reduced the generation of reactive oxygen species in cultured HSCs and inhibited the secretion of TIMP-1 both in basal conditions and after the induction of oxidative stress. In conclusion, curcumin administration effectively limits the development and progression of fibrosis in mice with experimental steatohepatitis, and reduces TIMP-1 secretion and oxidative stress in cultured stellate cells.

Adenosine monophosphate-activated protein kinase modulates the activated phenotype of hepatic stellate cells.

UNLABELLED: Adiponectin limits the development of liver fibrosis and activates adenosine monophosphate-activated protein kinase (AMPK). AMPK is a sensor of the cellular energy status, but its possible modulation of the fibrogenic properties of hepatic stellate cells (HSCs) has not been established. In this study, we investigated the role of AMPK activation in the biology of activated human HSCs. A time-dependent activation of AMPK was observed in response to a number of stimuli, including globular adiponectin, 5-aminoimidazole-4-carboxamide-1-beta-4-ribofuranoside (AICAR), or metformin. All these compounds significantly inhibited platelet-derived growth factor (PDGF)-stimulated proliferation and migration of human HSCs and reduced the secretion of monocyte chemoattractant protein-1. In addition, AICAR limited the secretion of type I procollagen. Knockdown of AMPK by gene silencing increased the mitogenic effects of PDGF, confirming the negative modulation exerted by this pathway on HSCs. AMPK activation did not reduce PDGF-dependent activation of extracellular signal-regulated kinase (ERK) or Akt at early time points, whereas a marked inhibition was observed 24 hours after addition of PDGF, reflecting a block in cell cycle progression. In contrast, AICAR blocked short-term phosphorylation of ribosomal S6 kinase (p70(S6K)) and 4E binding protein-1 (4EBP1), 2 downstream effectors of the mammalian target of rapamycin (mTOR) pathway, by PDGF. The ability of interleukin-a (IL-1) to activate nuclear factor kappa B (NF-kappaB) was also reduced by AICAR. CONCLUSION: Activation of AMPK negatively modulates the activated phenotype of HSCs.

Myostatin regulates the fibrogenic phenotype of hepatic stellate cells via c-jun N-terminal kinase activation.

BACKGROUND & AIMS: Myostatin is mainly expressed in skeletal muscle, where it negatively regulates trophism. This myokine is implicated in the pathophysiology of nonalcoholic steatohepatitis, an emerging cause of liver fibrosis. In this study we explored the effects of myostatin on the biology of hepatic stellate cells. METHODS: The effects of myostatin were assessed both in LX-2 and in human primary stellate cells. Cell migration was determined in Boyden chambers. Activation of intracellular pathways was evaluated by Western blotting. Procollagen type 1 secretion was measured by enzyme immunoassay. The role of c-Jun N-terminal kinase was assessed by pharmacologic and genetic inhibition. RESULTS: Activin receptor-2B was up-regulated in livers of mice with experimental fibrosis, and detectable in human stellate cells. Serum myostatin levels increased in a model of acute liver injury. Myostatin reduced HSC proliferation, induced cell migration, and increased expression of procollagen type1, tissue inhibitor of metalloproteinase-1, and transforming growth factor-ß1. Myostatin activated different signaling pathways, including c-Jun N-terminal kinase and Smad3. Genetic and/or pharmacologic inhibition of c-Jun N-terminal kinase activity significantly reduced cell migration and procollagen secretion in response to myostatin. CONCLUSIONS: Activation of activin receptor-2B by myostatin modulates the fibrogenic phenotype of human stellate cells, indicating that a myokine may be implicated in the pathogenesis of hepatic fibrosis.

Angiotensin II upregulates renin-angiotensin system in human isolated T lymphocytes.

This study was aimed at investigating the effects of Angiotensin (Ang) II stimulation on T lymphocytes mRNA expression of angiotensinogen (AGTN), angiotensin-converting enzyme (ACE) and AT1-receptor (R) and on ACE activity and Ang II content. The effects of Ang II stimulus were studied in lipopolysaccharide (LPS)-stimulated or not stimulated lymphocytes. mRNA expression for interferon-gamma (INF-gamma) was also studied to investigate whether a link between lymphocyte RAS and immunological function might occur. mRNAs for AGTN, ACE and AT1-R were obtained from peripheral blood of 18 healthy subjects and were quantified by real time quantitative transcriptase-polymerase chain reaction (PCR). ACE activity was assayed in cell pellets and supernatants by measuring the hippuric acid formation by high performance liquid chromatography (HPLC) and Ang II cell content was measured by radioimmunoassay (RIA) after HPLC separation. All determination were performed under baseline conditions and after the addition of 10(e- 13) M Ang II to LPS-stimulated or unstimulated lymphocytes. Ang II caused a significant upregulation of T subset lymphocytes gene expression of ACE and AT1-R and of INF gamma, and a marked increase in ACE activity and cell Ang II concentration. AGTN gene was never expressed. All these effects were further enhanced in T lymphocytes presitmulated by LPS and completely inhibited by Irbesartan. Our findings strongly support the evidence of a positive Ang II driven autocrine loop that upregulates cell RAS of isolated lymphocytes and activates the immuno response. The immuno-potentiating effect of Ang II, specifically shown in T subset, can be deleterious when local RAS are disregulated as in cardiovascular atherosclerotic disease.

Myristoylated Alanine-Rich protein Kinase C Substrate (MARCKS) expression modulates the metastatic phenotype in human and murine colon carcinoma in vitro and in vivo.

Several actin-binding proteins have been shown to be altered in metastatic cell lines and tumours and, in particular, Myristoylated Alanine-Rich protein Kinase C substrate (MARCKS) has been implicated in the pathogenesis of various highly metastatic epithelial malignancies. Considering that a large percentage of deaths due to colorectal cancer (CRC) are consequent to hepatic metastasization, aim of this study was to elucidate the involvement and mechanism of MARCKS in CRC by employing in vitro and in vivo approaches. Loss-of and-gain-on function approaches of MARCKS were employed in two human CRC cell lines: Clone A cells expressing MARCKS and LoVo cells known to have a frameshift mutation of MARCKS i.e. typically for MSI-H CRC. The data unveiled that altering MARCKS expression suppresses cell motility and invasion in human colon carcinoma cells when conditioned medium of liver-specific stromal cells (hepatic stellate cells) was used as chemoattractant. Depletion or re-expression of MARCKS inhibited proliferation with a reduction in expression of the mitotic regulator Aurora B kinase (AURKB), whereas AURKB-depletion did not modify MARCKS expression. In murine colon carcinoma CT26 cells, shRNA MARCKS-depletion reduced motility and invasion, and induced an aberrant, prolonged mitotic process. Significantly less metastases were produced in a syngeneic model of colon metastasis by MARCKS-depleted CT26 in comparison to CT26-tumour challenged mice. In conclusion, MARCKS plays an articulated role in the progression of colorectal cancer and might represent a suitable target to interfere and overcome the invasive behaviour of colon carcinoma cells at primary and distant sites.

Ang II Upregulation of the T-lymphocyte renin-angiotensin system is amplified by low-grade inflammation in human hypertension.

BACKGROUND: Low-grade inflammation facilitates the development of essential hypertension and target organ damage (TOD). Recently, human T-lymphocytes were shown to be endowed with a functional active renin-angiotensin system (RAS). We investigated whether in hypertensive patients a selective angiotensin (Ang) II-driven upregulation of T-cell RAS occurs and whether it is differently modulated in presence of low-grade inflammation. METHODS: T-lymphocytes were obtained from 21 hypertensives (I-II World Health Organization class; 16 males, 5 females; 56 ± 11 years). Low-grade inflammation was defined for high sensitive C-reactive protein (hsCRP) > 2 mg/l. Ten healthy subjects formed the age- and sex-matched control group. After T-lymphocytes isolation, mRNAs for angiotensin-converting enzyme (ACE) and angiotensin type 1 receptor (AT1-R) were quantified by reverse-transcriptase PCR with or without 0.1 pmol/l Ang II in addition to T-cells cultures. Cell pellet and supernatant ACE activity and Ang II content were measured. Cardiac and renal TOD-indexes were evaluated. RESULTS: Both in controls and hypertensives, Ang II-stimulation significantly increased ACE and AT1-R mRNA levels (P < 0.05). In patients, the increase was earlier and higher than controls, with the highest values in hypertensives with > 2 mg/l hsCRP. Peak Ang II-induced ACE and AT1-R mRNA levels were positively related to hsCRP, systolic blood pressure and body mass index (BMI) at the univariate analyses. The stepwise regression analyses selected hsCRP (r = 0.47) and left ventricular mass index (LVMI) (r = 0.50) as the variables independently related to peak ace-gene expression, while BMI resulted independently related to peak AT1-R gene expression (P < 0.001). CONCLUSIONS: In hypertension, an Ang II-driven activation of T-cell RAS, further amplified by low-grade inflammation, does occur and is associated to worse TOD. New therapeutic approaches aimed at this specific target might be proposed to control hypertension and hypertensive damage.

Mononuclear cells in liver fibrosis.

Fibrosis is a multicellular wound healing process, where myofibroblasts that express extracellular matrix components extensively cross-talk with other cells resident in the liver or recruited from the bloodstream. Macrophages and infiltrating monocytes participate in the development of fibrosis via several mechanisms, including secretion of cytokines and generation of oxidative stress-related products. However, macrophages are also pivotal in the process of fibrosis resolution, where they contribute to matrix degradation. T lymphocytes modulate the fibrogenic process by direct interaction with myofibroblasts and secreting cytokines. In general, Th2 polarized responses promote fibrosis, while Th1 cytokines may be antifibrogenic. NK cells limit the development of fibrosis and favor its resolution, at least in part via killing of fibrogenic cells. The possible role of NKT cells and B cells is emerging in recent studies. Thus, mononuclear cells represent a critical regulatory system during fibrogenesis and may become an appealing target for therapy.

Prevention of severe toxic liver injury and oxidative stress in MCP-1-deficient mice.

BACKGROUND/AIMS: Administration of carbon tetrachloride determines liver injury, inflammation and oxidative stress, but the molecular mechanisms of damage are only partially understood. In this study, we investigated the development of acute toxic damage in mice lacking monocyte chemoattractant protein-1 (MCP-1), a chemokine which recruits monocytes and activated lymphocytes. METHODS: Mice with targeted deletion of the MCP-1 gene and wild type controls were administered a single intragastric dose of carbon tetrachloride. Serum liver enzymes, histology, expression of different chemokines and cytokines, and intrahepatic levels of oxidative stress-related products were evaluated. RESULTS: Compared to wild type mice, peak aminotransferase levels were significantly lower in MCP-1-deficient animals. This was paralleled by a delayed appearance of necrosis at histology. In addition, MCP-1-deficient mice showed a shift in the pattern of infiltrating inflammatory cells, with a predominance of polymorphonuclear leukocytes. Lack of MCP-1 was also accompanied by reduced intrahepatic expression of cytokines regulating inflammation and tissue repair. The increase in tissue levels of reactive oxygen species and 4-hydroxy-nonenal following administration of the hepatotoxin was also significantly lower in animals lacking MCP-1. CONCLUSIONS: Lack of MCP-1 affords protection from damage and development of oxidative stress in a toxic model of severe acute liver injury.