Role In Fibrogenesis Research Articles

G105 Profibrogenic Chemokines and Viral Evolution Predict Rapid Progression of Hepatitis C to Cirrhosis

The clinical presentation and outcomes of chronic hepatitis C are highly variable, and it is not possible to predict which individuals will rapidly evolve to severe fibrosis and liver failure. Using serial serum samples prospectively collected from 6 patients with transfusion-associated hepatitis C (followed from the day of transfusion for up to 30 years), we identified outcome-specific features that predict long-term disease severity. We found that stable or slowly progressive disease correlated with an earlier ALT peak and antibody seroconversion, transient control of viremia and significant induction of IFN- and MIP-1, all indicative of an effective, albeit insufficient, adaptive immune response. By contrast, rapidly progressive disease was associated with persistent and significant elevations of ALT and the profibrogenic chemokine MCP-1 (CCL-2), a major chemotactic factor for hepatic stellate cells, which play a central role in fibrogenesis. Rapidly progressive disease was also associated with greater viral diversity and divergence, and a higher rate of synonymous substitutions. Strikingly a strong genetic bottleneck occurred in all patients around one year post-infection. This study suggests that the long-term course of chronic hepatitis C is determined early in infection and that disease severity is predicted by the evolutionary dynamics of HCV and the level of MCP-1, a chemokine that appears critical to the induction of progressive fibrogenesis and, ultimately, the ominous complications of cirrhosis. In this proposed model for the pathogenesis of chronic HCV-related liver disease, rapid disease progression may result from an excess of soluble mediators of inflammation and fibrosis rather than from T-cell-mediated hepatotoxicity.

Transient Receptor Potential (TRP) Channels and Cardiac Fibrosis

Cardiac fibrosis is associated with most cardiac diseases. Fibrosis is an accumulation of excessive extracellular matrix proteins (ECM) synthesized by cardiac fibroblasts and myofibroblasts. Fibroblasts are the most prevalent cell type in the heart, comprising 75% of cardiac cells. Myofibroblasts are hardly present in healthy normal heart tissue, but appear abundantly in diseased hearts. Cardiac fibroblasts are activated by a variety of pathological stimuli, such as myocardial injury, oxidative stress, mechanical stretch, and elevated autocrine-paracrine mediators, thereby undergoing proliferation, differentiation to myofibroblasts, and production of various cytokines and ECM proteins. A number of signaling pathways and bioactive molecules are involved and work in concert to activate fibroblasts and myofibroblasts in the fibrogenesis cascade. Fibroblasts and myofibroblasts are not only principal ECM producers, but also play a central role in fibrogenesis and myocardial remodeling in fibrotic heart disease. Thus, understanding the biological processes of cardiac fibroblasts will provide novel insights into the underlying mechanisms of fibrosis and provide potential targets for developing antifibrotic drugs. Recent studies demonstrate that Ca2+ signal is essential for fibroblast proliferation, differentiation, and ECM-protein production. This review focuses on the recent advances in understanding molecular mechanisms of Ca2+ signaling in cardiac fibrogenesis, and potential role of Ca(2+)-permeable channels, in particular, the transient potential (TRP) channels in fibrotic heart disease. TRP channels are highly expressed in cardiac fibroblasts. TRPM7 has been shown to be essential in TGFβ1 mediated fibrogenesis, and TRPC3 has been demonstrated to play an essential role in regulating fibroblast function. Thus, the Ca2+-permeable TRP channels may serve as potential novel targets for developing anti-fibrotic drugs.

Activation of pericytes

Pathological deposition of fibrous matrix in organs is a major problem and contributes to as many as 45% of all natural deaths. Chronic kidney disease affects 8% of the US population, and is characterized by fibrotic processes. It frequently progresses to organ failure and is a major cause of cardiovascular death; yet it lacks therapies. Understanding the pathological mechanisms of fibrosis in the kidney and other organs is central to the development of new therapeutics. Pericytes are mesenchymal cells that partially cover capillary walls. Pericytes play critical roles in micro-vessel formation, maturation and stability. New genetic fate-mapping studies have identified pericytes and the closely related resident fibroblasts as the major progenitors of scar-forming myofibroblasts in multiple organs including the kidney, appearing in response to tissue injury. When pericytes become myofibroblasts they lose pericyte functions. Capillaries become unstable with deleterious consequences for the kidney. The cellular and molecular mechanisms underpinning these processes are starting to unravel, leading to new therapeutics for chronic fibrosing diseases of the kidney and potentially other organs. This review focuses on pericytes in the kidney and other organs, their role in fibrogenesis and their role in regulation of the microvasculature.

Relationship between serum transforming growth factor ß1 and liver collagen content in rats treated with carbon tetrachloride

Transforming Growth Factor ß1 (TGFß1) plays a fundamental role in fibrogenesis, although its importance as a biomarker of liver disease is still matter of debate. Quantify serum TGFß1 and its association to liver collagen content in rats exposed to Carbon Tetrachloride (CCl4). Rats were submitted to a fibrosis model using CCl4 and sacrificed after 6, 10, 12 and 16 weeks of treatment. Serum levels of TGFß1 were measured by ELISA and collagen content was defined by morphometric analysis. Serum levels of TGFß1 increased between 6 and 10 weeks, whereas collagen density increased between 12 and 16 weeks. A negative correlation was observed between liver collagen deposition and serum levels of TGFß1 (r = -0. 48; P<0. 05). Serum levels of TGFß1 were inversely proportional to collagen intensity in cirrhotic livers of rats exposed to CCl4, thus suggesting a limited use as biomarker in advanced liver disease.

Hepatocyte–Stellate Cell Cross-Talk in the Liver Engenders a Permissive Inflammatory Microenvironment That Drives Progression in Hepatocellular Carcinoma

Many solid malignant tumors arise on a background of inflamed and/or fibrotic tissues, features that are found in more than 80% hepatocellular carcinomas (HCC). Activated hepatic stellate cells (HSC) play a critical role in fibrogenesis associated with HCC onset and progression, yet their functional impact on hepatocyte fate remains largely unexplored. Here, we used a coculture model to investigate the cross-talk between hepatocytes (human hepatoma cells) and activated human HSCs. Unsupervised genome-wide expression profiling showed that hepatocyte-HSC cross-talk is bidirectional and results in the deregulation of functionally relevant gene networks. Notably, coculturing increased the expression of proinflammatory cytokines and modified the phenotype of hepatocytes toward motile cells. Hepatocyte-HSC cross-talk also generated a permissive proangiogenic microenvironment, particularly by inducing VEGFA and matrix metalloproteinase (MMP)9 expression in HSCs. An integrative genomic analysis revealed that the expression of genes associated with hepatocyte-HSC cross-talk correlated with HCC progression in mice and was predictive of a poor prognosis and metastasis propensity in human HCCs. Interestingly, the effects of cross-talk on migration and angiogenesis were reversed by the histone deacetylase inhibitor trichostatin A. Our findings, therefore, indicate that the cross-talk between hepatoma cells and activated HSCs is an important feature of HCC progression, which may be targeted by epigenetic modulation.

Increased survivin expression contributes to apoptosis-resistance in IPF fibroblasts

Fibroblasts perform critical functions during the normal host response to tissue injury, but the inappropriate accumulation and persistent activation of these cells results in the development of tissue fibrosis. The mechanisms accounting for the aberrant accumulation of fibroblasts during fibrotic repair are poorly understood, although evidence supports a role for fibroblast resistance to apoptosis as a contributing factor. We have shown that TGF-β1 and endothelin-1 (ET-1), soluble mediators implicated in fibrogenesis, promote fibroblast resistance to apoptosis. Moreover, we recently found that ET-1 induced apoptosis resistance in normal lung fibroblasts through the upregulation of survivin, a member of the Inhibitor of Apoptosis (IAP) protein family. In the current study, we sought to determine the role of survivin in the apoptosis resistance of primary fibroblasts isolated from the lungs of patients with Idiopathic Pulmonary Fibrosis (IPF), a fibrotic lung disease of unclear etiology for which there is no definitive therapy. First, we examined survivin expression in lung tissue from patients with IPF and found that there is robust expression in the fibroblasts residing within fibroblastic foci (the "active" lesions in IPF which correlate with mortality). Next, we show that survivin expression is increased in fibroblasts isolated from IPF lung tissue compared to cells from normal lung tissue. Consistent with a role in fibrogenesis, we demonstrate that TGF-β1 increases survivin expression in normal lung fibroblasts. Finally, we show that inhibition of survivin enhances susceptibility of a subset of IPF fibroblasts to apoptosis. Collectively, these findings suggest that increased survivin expression represents one mechanism contributing an apoptosis-resistant phenotype in IPF fibroblasts.

Conophylline Suppresses Pancreatic Stellate Cells and Improves Islet Fibrosis in Goto-Kakizaki Rats

Activin A is a differentiation factor for β-cells and is effective to promote β-cell neogenesis. Activin A is also an autocrine activator of pancreatic stellate cells, which play a critical role in fibrogenesis of the pancreas. Conophylline (CnP) is a natural compound, which reproduces the effect of activin on β-cell differentiation and promotes β-cell neogenesis when administered in vivo. However, its effect on stellate cells is not known. We therefore investigated the effect of CnP on stellate cells both in vitro and in vivo. Unlike activin A, CnP inhibited activation of cultured stellate cells and reduced the production of collagen. We then analyzed the involvement of stellate cells in islet fibrosis in Goto-Kakizaki (GK) rats, a model of type 2 diabetes mellitus. In pancreatic sections obtained from 6-wk-old GK rats, CD68-positive macrophages and glial fibrillary acidic protein- and α-smooth muscle actin-positive stellate cells infiltrated into islets. Later, the number of macrophages was increased, and the α-smooth muscle actin staining of stellate cells became stronger, indicating the involvement of stellate cells in islet fibrosis in GK rats. When CnP was administered orally for 4 wk, starting from 6 wk of age, invasion of stellate cells and macrophages was markedly reduced and islet fibrosis was significantly improved. The insulin content was twice as high in CnP-treated rats. These results indicate that CnP exerts antifibrotic actions both in vitro and in vivo and improves islet fibrosis in Goto-Kakizaki rats.

Relationship Between Ischemia/Reperfusion Injury and the Stimulus of Fibrogenesis in an Experimental Model: Comparison Among Different Preservation Solutions

Background and Aims Orthotopic liver transplantation (OLT) has been the standard treatment for end-stage acute and chronic liver disease. Ischemia-reperfusion (I/R) injury is one of the major causes of poor graft function early after OLT, and adversely influencing graft and patient survivals. It is unknown whether I/R injury influences liver fibrogenesis. Materials and Methods Livers from 25 adult male Wistar rats were randomly assigned into 5 experimental groups according to the preservation solution: saline solution (SS); University of Wisconsin (UW) solution; Fructose 1, 6-biphosphate (FBP); S-Nitroso-N-Acetylcysteine (SNAC): or UW + SNAC (SNAC+UW). Aspartate aminotransferase (AST), alanine aminotransferase (ALT), and lactic dehydrogenase (LDH) were determined in preservation solution samples at 2, 4, and 6 hours. After 6 hours of cold ischemia, ex situ reperfusion was applied to the liver for 15 minutes. Serum AST, ALT, LDH, and renin levels were determined. Fresh liver slices were processed for histological studies, determination of thiobarbituric acid reactive substances, catalase, and glutathione, and expression of TGF-β1 and angiotensin II AT1 receptor. Results AST was significantly lower during cold storage with UW than with the older media ( P = .001); ALT was lower in the FBP group ( P = .023) and LDH was lower in the FBP and SNAC groups ( P = .007). After reperfusion, serum AST, ALT, LDH, and TBARS showed no significant differences among the groups. Catalase was significantly lower in the SS and FBP groups ( P = .008 and P = .006, respectively). Compared with UW, glutathione concentrations were significantly higher in SS, FBP, and SNAC 200 ( P = .004). Renin levels were significantly lower in the FBP group ( P = .022). No histological signs of preservation injury were observed in the hepatic sample. No expressions were detected of TGF-β1 or AT1 receptor. Conclusion In this experimental model of early reperfusion injury, preservation changes related to higher levels of renin, which suggest its role in fibrogenesis. FBP was associated with lower renin levels than other solutions including UW.

Smad3-Mediated Upregulation of miR-21 Promotes Renal Fibrosis

TGF-β/Smad signaling plays a role in fibrogenesis, but therapies targeting TGF-β are ineffective in treating renal fibrosis. Here, we explored the therapeutic potential of targeting TGF-β-induced microRNA in the progression of renal fibrosis. Microarray analysis and real-time PCR revealed upregulation of miR-21 in tubular epithelial cells (TECs) in response to TGF-β. Lack of Smad3, but not lack of Smad2, prevented cells from upregulating miR-21 in response to TGF-β. In addition, Smad3-deficient mice were protected from upregulation of miR-21 and fibrosis in the unilateral ureteral obstruction model. In contrast, conditional knockout of Smad2 enhanced miR-21 expression and renal fibrosis. Furthermore, ultrasound-microbubble-mediated gene transfer of a miR-21-knockdown plasmid halted the progression of renal fibrosis in established obstructive nephropathy. In conclusion, these data demonstrate that Smad3, but not Smad2, signaling increases expression of miR-21, which promotes renal fibrosis. Inhibition of miR-21 may be a therapeutic approach to suppress renal fibrosis.

Mechanisms of fibrosis: the role of the pericyte

Until recently kidney pericytes were little known. This review will update readers about key new findings concerning pericyte abundance, function in kidney vascular biology and major role in fibrogenesis. Moreover readers will become familiar with the central role of pericyte-endothelial interactions in peritubular capillary health or rarefaction and the pivotal role these may play in renal ischemia. Pericytes are stromal cells that partially cover capillary walls. Pericytes were recently identified as collagen-1α1 producing cells in healthy adult kidney. Kinetic mathematical modeling studies indicated they were the source of scar-forming myofibroblasts. Comprehensive fate mapping of kidney stroma confirmed that pericytes and perivascular fibroblasts, not epithelium, were the major source of myofibroblasts. Blockade of pericyte-endothelial cross-talk in response to renal injury prevents both microvascular rarefaction and interstitial fibrosis. The detachment of pericytes from endothelium under pathological conditions and differentiation into myofibroblasts leads to pericyte deficiency at the microvascular interstitial interface, resulting in unstable microvasculature and thence vessel rarefaction, ultimately leading to nephron ischemia. To develop new therapeutic strategies, a better understanding of not only pericyte detachment and migration from capillaries, but also pericyte-endothelial crosstalk in health and injury is required. This review summarizes the functional role of pericytes during fibrosis, focusing on myofibroblast origins and pericyte-endothelial cross-talk.

LY-6C intermediate and LY-6CLO intrahepatic macrophage subsets orchestrate resolution of hepatic fibrosis following chronic injury

<h3>Introduction</h3> Hepatic fibrosis is potentially reversible. Macrophages are heterogenous and have distinct roles in fibrogenesis and resolution. Studies have identified a pro-inflammatory Ly-6C^hi macrophage subset in mediating fibrogenesis. However, little is known about the identity or phenotype of the restorative hepatic macrophage. Here we identify and characterise the macrophage subset mediating resolution of liver fibrosis following chronic injury. <h3>Methods</h3> Liver fibrosis was induced in adult male mice by twice-weekly CCl₄ injections for 4 weeks, followed by histological assessment or flow cytometry analysis at serial time points following cessation. <h3>Results</h3> Following cessation of CCl₄ distinct phases of injury and recovery could be identified: active inflammation and fibrogenesis (24 h), peak fibrosis (48–72 h), early resolution with dynamic loss of myofibroblasts and remodelling of majority of scar tissue (72–96 h) and late resolution with degradation of remainder of fibrosis (96–256 h). Intrahepatic macrophage subsets demonstrated an increase in Ly-6C^hi cells during inflammation and fibrogenesis. During resolution there is a rapid loss of these Ly-6C^hi cells and emergence of Ly-6C intermediate and Ly-6C^lo macrophage subsets. Using CD11B-DTR transgenic mice we depleted macrophages during early resolution, resulting in a failure to remodel hepatic scar. Critically, depletion was selective for the Ly-6C^int and Ly-6C^lo liver macrophage subsets, and the degree of depletion of these cells correlated significantly with the amount of persistent fibrosis, thus confirming their role in scar resolution. Adoptive transfer and tracking experiments demonstrated that during early resolution the Ly-6C^int cells derive from Ly-6C^hi circulating monocytes, indicating a phenotypic switch from pro-inflammatory to early pro-resolution macrophages. During late resolution Ly-6C^lo monocytes are recruited to replenish resident ‘Kupffer’ cells. Gene expression analysis on FACS sorted pro-resolution Ly-6C^int macrophages compared with pro-fibrotic Ly-6C^hi macrophages demonstrated reduced expression of pro-inflammatory mediators such as IL-1α, IL-1β, IL-6, CXCL2 and MCP-1 and an increase in expression of matrix degrading enzymes such as MMP-12 and MMP-9. <h3>Conclusion</h3> We have identified novel Ly-6C^int and Ly-6C^lo intrahepatic macrophage subsets as central orchestrators in the resolution of liver fibrosis. Critically, during early resolution the Ly-6C^int cells derive from a phenotypic switch in pro-fibrotic Ly-6C^hi cells, resulting in a change in macrophage gene expression from one promoting fibrogenesis to one favouring fibrosis resolution.

A Novel Small Compound That Promotes Nuclear Translocation of YB-1 Ameliorates Experimental Hepatic Fibrosis in Mice

Transforming growth factor-β (TGF-β) is considered to be a major factor contributing to liver fibrosis. We have previously shown that nuclear translocation of YB-1 antagonizes the TGF-β/Smad3 signaling in regulating collagen gene expression. More recently, we have demonstrated that the novel small compound HSc025 promotes nuclear translocation of YB-1, resulting in the improvement of skin and pulmonary fibrosis. Here, we presented evidence as to the mechanism by which HSc025 stimulates nuclear translocation of YB-1 and the pharmacological effects of HSc025 on a murine model of hepatic fibrosis. A proteomics approach and binding assays using HSc025-immobilized resin showed that HSc025 binds to the amino acid sequence within the C-tail region of YB-1. In addition, immunoprecipitation experiments and glutathione S-transferase pulldown assays identified poly(A)-binding protein (PABP) as one of the cytoplasmic anchor proteins of YB-1. HSc025 directly binds to YB-1 and interrupts its interaction with PABP, resulting in accelerated nuclear translocation of YB-1. Transfection of cells with PABP siRNA promoted nuclear translocation of YB-1 and subsequently inhibited basal and TGF-β-stimulated collagen gene expression. Moreover, HSc025 significantly suppressed collagen gene expression in cultured activated hepatic stellate cells. Oral administration of HSc025 to mice with carbon tetrachloride-induced hepatic fibrosis improved liver injury as well as the degree of hepatic fibrosis. Altogether, the results provide a novel insight into therapy for organ fibrosis using YB-1 modulators.

Intraperitoneal administration of chitosan/DsiRNA nanoparticles targeting TNFα prevents radiation-induced fibrosis

Background and purpose One of the most common and dose-limiting long-term adverse effects of radiation therapy is radiation-induced fibrosis (RIF), which is characterized by restricted tissue flexibility, reduced compliance or strictures, pain and in severe cases, ulceration and necrosis. Several strategies have been proposed to ameliorate RIF but presently no effective one is available. Recent studies have reported that tumor necrosis factor-α (TNFα) plays a role in fibrogenesis. Material and methods Male CDF1 mice were radiated with a single dose of 45 Gy. Chitosan/DsiRNA nanoparticles targeting TNFα were intraperitoneal injected and late radiation-induced fibrosis (RIF) was assessed using a modification of the leg contracture model. Additionally, the effect of these nanoparticles on tumor growth and tumor control probability in the absence of radiation was examined in a C3H mammary carcinoma model. Results We show in this work, that targeting TNFα in macrophages by intraperitoneal administration of chitosan/DsiRNA nanoparticles completely prevented radiation-induced fibrosis in CDF1 mice without revealing any cytotoxic side-effects after a long-term administration. Furthermore, such TNFα targeting was selective without any significant influence on tumor growth or irradiation-related tumor control probability. Conclusion This nanoparticle-based RNAi approach represents a novel approach to prevent RIF with potential application to improve clinical radiation therapeutic strategies.

PSP/reg inhibits cultured pancreatic stellate cell and regulates MMP/ TIMP ratio

Pancreatic stellate cells (PSC) play a central role in fibrogenesis associated with acute and chronic pancreatitis. Pancreatic stone protein/regenerating protein (PSP/reg) belongs to a family of secretory stress proteins (SSP) that are constitutively synthesized by pancreatic acinar cells and upregulated dramatically during acute and chronic pancreatitis. Assuming a protective role of this stress protein, we investigated its effects on human PSC. Pancreatic stellate cells were obtained by outgrowth from fibrotic human pancreas tissue. PSP/reg was expressed in the yeast Pichia pastoris and purified from medium supernatants. PSP/reg was added at concentrations of 100 ng/mL to cultured PSC. Cell proliferation was determined by bromodeoxyuridine incorporation. PSC migration was assessed by a wound healing assay. Extracellular matrix (collagen type I and fibronectin), matrix metalloproteinases (MMPs) and tissue inhibitors of matrix metalloproteinases (TIMPs) were demonstrated on protein level. Pancreatic stone protein/regenerating protein inhibited PSC proliferation and migration. Soluble collagen I and fibronectin were reduced after the addition of PSP/reg. PSP/reg slightly decreased the synthesis of MMP-1 and MMP-2 and strongly decreased TIMP-1 and TIMP-2 concentrations in PSC supernatants. Our work describes a novel aspect that in vitro PSP/reg reduces PSC activity (proliferation and migration) and stimulates fibrolysis by increasing MMP/TIMP ratio. The findings suggest that PSP/reg might have a protective function in the repair phase of acute and chronic pancreatitis by promoting resolution of fibrosis. We highlight PSP/reg as an antifibrogenic protein in pancreatic injury.

Deficiency of complement factor c5 prevents the progression of biliary fibrosis in bile duct-ligated mice

Aims: Fibrogenesis represents the universal response of the liver to chronic liver injury. Growing evidence indicates disease specific mechanisms of fibrosis initiation. Recently, C5 has been linked to fibrogenesis in a rodent model as well as in human chronic hepatitis C. C5 may also play a central role in fibrogenesis of chronic cholestatic disorders, since FXR has been characterized as an activator of the C3 gene. Increased levels of complement activation products have been detected in human cholestatic disorders. Aim of this study is to further elucidate complement activation in the pathogenesis of biliary fibrosis in the BDL model.

Resolved

Epithelial-mesenchymal transition (EMT) is a mechanism for generating primitive mesenchymal cells during gastrulation or mobile tumor cells during cancer metastasis. For 15 years, EMT has also been viewed as a principal source of fibroblasts in tissue fibrosis. Because several recent studies question its role in fibrogenesis, it seems like a good time for debate.

Yin and Yang Part Deux: CCN5 inhibits the pro‐fibrotic effects of CCN2

There is no treatment for fibrotic disease is a significant cause of mortality. CCN2 Members of the CCN family of matricellular proteins have a characteristic four domain structure. CCN2 (connective tissue growth factor) is believed to play an essential role in fibrogenesis. In a recent paper, data are provided that CCN5 (wisp2), which lacks the carboxy-terminal heparin-binding domain shared by the other CCN proteins, may act as a dominant-negative protein to suppress CCN2-mediated fibrogenesis. These data are consistent with the notion that different CCN proteins may enhance or suppress each other's action and also suggest that CCN5, may be used as a novel anti-fibrotic therapy.

Organ-, inflammation- and cancer specific transcriptional fingerprints of pancreatic and hepatic stellate cells

BackgroundTissue fibrosis is an integral component of chronic inflammatory (liver and pancreas) diseases and pancreatic cancer. Activated pancreatic- (PSC) and hepatic- (HSC) stellate cells play a key role in fibrogenesis. To identify organ- and disease-specific stellate cell transcriptional fingerprints, we employed genome-wide transcriptional analysis of primary human PSC and HSC isolated from patients with chronic inflammation or cancer.MethodsStellate cells were isolated from patients with pancreatic ductal adenocarcinoma (n = 5), chronic pancreatitis (n = 6), liver cirrhosis (n = 5) and liver metastasis of pancreatic ductal adenocarcinoma (n = 6). Genome-wide transcriptional profiles of stellate cells were generated using our 51K human cDNA microarray platform. The identified organ- and disease specific genes were validated by quantitative RT-PCR, immunoblot, ELISA, immunocytochemistry and immunohistochemistry.ResultsExpression profiling identified 160 organ- and 89 disease- specific stellate cell transcripts. Collagen type 11a1 (COL11A1) was discovered as a novel PSC specific marker with up to 65-fold higher expression levels in PSC compared to HSC (p < 0.0001). Likewise, the expression of the cytokine CCL2 and the cell adhesion molecule VCAM1 were confined to HSC. PBX1 expression levels tend to be increased in inflammatory- vs. tumor- stellate cells. Intriguingly, tyrosine kinase JAK2 and a member of cell contact-mediated communication CELSR3 were found to be selectively up-regulated in tumor stellate cells.ConclusionsWe identified and validated HSC and PSC specific markers. Moreover, novel target genes of tumor- and inflammation associated stellate cells were discovered. Our data may be instrumental in developing new tailored organ- or disease-specific targeted therapies and stellate cell biomarkers.

Abstract 1013: Hepatic stellate cells promote progression of cholangiocarcinoma in vitro and in vivo

Abstract Background: Although tumor-stroma interaction has been discussed, the role of hepatic stellate (HS) cells against cancer, especially cholangiocarcinoma, has not been clarified. Although hepatic stellate cells have been known to have a pivotal role in fibrogenesis in liver, the influence of them on liver cancer has been poorly investigated. We previously reported hepatic stellate cells may exist in cancer stroma of intrahepatic cholangiocarcinoma and interact with cancer cells in vitro. The aim of this study was to investigate the effect of HS cells on cholangiocarcinoma cells progression in vitro and in vivo and to identify key proteins which regulate their interaction. Methods: The effects of cholangiocarcinoma cells’ conditioned medium (CC-CM) on activation and proliferation of HS cells (LI90 and LX-2), the influences of HS cells conditioned medium (HS-CM) on in vitro proliferation and invasion of cholangiocarcinoma cells (HuCCT-1 and RBE), and the effects of their interaction on human umbilical venous endothelial cells (HUVECs) tube formation were assessed using each of conditioned medium. The effect of HS cells on tumor growth was examined in vivo by subcutaneous co-injection of them. To clarify the factors inducing their interaction, cytokine array was performed to assess the secreted proteins induced by co-culture of cholangiocarcinoma and HS cells. Results: CC-CM activated HS cells and increased their proliferation. HS-CM dose-dependently increased cholangiocarcinoma cell proliferation and invasion. Chemotherapy to cholangiocarcinoma cells was less effective treated with HS-CM. HS-CM activated the mitogen-activated protein kinase (MAPK) and Akt pathways in tumor cells. The indirect interaction of cholangiocarcinoma and HS cells promote the tube formation of HUVECs. Subcutaneous co-injection of tumor cells with HS cells in nude mouse resulted in increased tumor size and weight. There found to be several proteins including interleukin-1β (IL1β) that remarkably increased by co-culture of them in culture medium and they were thought to be key proteins which regulated tumor-stromal interaction. Conclusions: This study indicated that HS cells have an important role in accelerating cholangiocarcinoma progression. Several proteins including IL1β induced by co-culture model may be therapeutic targets which focus tumor-stromal interaction in cholangiocarcinoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1013.

Histochemical and Immunohistochemical Staining for Markers of Fibrosis in Embalmed Cadavers

Liver fibrosis is a significant liver disease, but its incidence in the elderly is not known. Embalmed cadavers used for teaching human anatomy provide liver tissue for analysis of fibrosis in this population. Livers from 32 cadavers (age 64–100 yrs) with diverse causes of death were embedded in paraffin, sectioned and stained for markers of fibrosis: Sirius red stain for collagen, resorcin fuchsin and van Gieson stains for elastin and collagen, and á‐smooth muscle actin (á‐SMA) antibody, followed by Sirius red, for á‐SMA expression by hepatic stellate cells (HSCs) and myofibroblasts. As determined by collagen staining and morphometry, central vein fibrosis was seen in 21 livers, perisinusoidal fibrosis in 25, portal tract fibrosis in 16, short and long fibrous septa in 12, bridging septa (portal‐portal, portal‐central, central‐central) in 10 and cirrhosis in 1. Only 3 livers showed minimal change. Although absent in the space of Disse, elastic fibers co‐distributed with collagen in the portal tracts, septa, central veins and walls of blood vessels. á‐SMA‐stained HSCs localized to collagen fibers in the space of Disse, while myofibroblasts were found with collagen and elastin in portal tracts, septa and central veins, consistent with their role in fibrogenesis. Conclusions: Liver fibrosis, from early to late stages, is prevalent in older adults, and this pathology may have exacerbated the health of this population.