Grant A. Ramm

Desmoplastic reaction in pancreatic cancer: role of pancreatic stellate cells.

Objectives: Pancreatic cancer has a very poor prognosis, largely due to its propensity for early local and distant spread. Histopathologically, most pancreatic cancers are characterized by a prominent stromal/fibrous reaction in and around tumor tissue. The aims of this study were to determine whether (1) the cells responsible for the formation of the stromal reaction in human pancreatic cancers are activated pancreatic stellate cells (PSCs) and (2) an interaction exists between pancreatic cancer cells and PSCs that may facilitate local and distant invasion of tumor. Methods: Serial sections of human pancreatic cancer tissue were stained for desmin and glial fibrillary acidic protein (stellate cell selective markers) and &agr;-smooth muscle actin (&agr;SMA), a marker of activated PSC activation, by immunohistochemistry, and for collagen using Sirius Red. Correlation between the extent of positive staining for collagen and &agr;SMA was assessed by morphometry. The cellular source of collagen in stromal areas was identified using dual staining methodology, ie, immunostaining for &agr;SMA and in situ hybridization for procollagen &agr;1I mRNA. The possible interaction between pancreatic cancer cells and PSCs was assessed in vitro by exposing cultured rat PSCs to control medium or conditioned medium from 2 pancreatic cancer cell lines (PANC-1 and MiaPaCa-2) for 24 hours. PSC activation was assessed by cell proliferation and &agr;SMA expression. Results: Stromal areas of human pancreatic cancer stained strongly positive for the stellate cell selective markers desmin and GFAP (indicating the presence of PSCs), for &agr;SMA (suggesting that the PSCs were in their activated state) and for collagen. Morphometric analysis demonstrated a close correlation (r = 0.77; P < 0.04; 8 paired sections) between the extent of PSC activation and collagen deposition. Procollagen mRNA expression was localized to &agr;SMA-positive cells in stromal areas indicating that activated PSCs were the predominant source of collagen in stromal areas. Exposure of PSCs to pancreatic cancer cell secretions in vitro resulted in PSC activation as indicated by significantly increased cell proliferation and &agr;SMA expression. Conclusions: Activated PSCs are present in the stromal reaction in pancreatic cancers and are responsible for the production of stromal collagen. PSC function is influenced by pancreatic cancer cells. Interactions between tumor cells and stromal cells (PSCs) may play an important role in the pathobiology of pancreatic cancer.

Activation of pancreatic stellate cells in human and experimental pancreatic fibrosis.

The mechanisms of pancreatic fibrosis are poorly understood. In the liver, stellate cells play an important role in fibrogenesis. Similar cells have recently been isolated from the pancreas and are termed pancreatic stellate cells. The aim of this study was to determine whether pancreatic stellate cell activation occurs during experimental and human pancreatic fibrosis. Pancreatic fibrosis was induced in rats (n = 24) by infusion of trinitrobenzene sulfonic acid (TNBS) into the pancreatic duct. Surgical specimens were obtained from patients with chronic pancreatitis (n = 6). Pancreatic fibrosis was assessed using the Sirius Red stain and immunohistochemistry for collagen type I. Pancreatic stellate cell activation was assessed by staining for alpha-smooth muscle actin (alphaSMA), desmin, and platelet-derived growth factor receptor type beta (PDGFRbeta). The relationship of fibrosis to stellate cell activation was studied by staining of serial sections for alphaSMA, desmin, PDGFRbeta, and collagen, and by dual-staining for alphaSMA plus either Sirius Red or in situ hybridization for procollagen alpha(1) (I) mRNA. The cellular source of TGFbeta was examined by immunohistochemistry. The histological appearances in the TNBS model resembled those found in human chronic pancreatitis. Areas of pancreatic fibrosis stained positively for Sirius Red and collagen type I. Sirius Red staining was associated with alphaSMA-positive cells. alphaSMA staining colocalized with procollagen alpha(1) (I) mRNA expression. In the rat model, desmin staining was associated with PDGFRbeta in areas of fibrosis. TGFbeta was maximal in acinar cells adjacent to areas of fibrosis and spindle cells within fibrotic bands. Pancreatic stellate cell activation is associated with fibrosis in both human pancreas and in an animal model. These cells appear to play an important role in pancreatic fibrogenesis.

TWEAK and LTβ signaling during chronic liver disease

Chronic liver diseases (CLD) such as hepatitis B and C virus infection, alcoholic liver disease, and non-alcoholic steatohepatitis are associated with hepatocellular necrosis, continual inflammation, and hepatic fibrosis. The induced microenvironment triggers the activation of liver-resident progenitor cells (LPCs) while hepatocyte replication is inhibited. In the early injury stages, LPCs regenerate the liver by proliferation, migration to sites of injury, and differentiation into functional biliary epithelial cells or hepatocytes. However, when this process becomes dysregulated, wound healing can progress to pathological fibrosis, cirrhosis, and eventually hepatocellular carcinoma. The other key mediators in the pathogenesis of progressive CLD are fibrosis-driving, activated hepatic stellate cells (HSCs) that usually proliferate in very close spatial association with LPCs. Recent studies from our group and others have suggested the potential for cytokine and chemokine cross-talk between LPCs and HSCs, which is mainly driven by the tumor necrosis factor (TNF) family members, TNF-like weak inducer of apoptosis (TWEAK) and lymphotoxin-β, potentially dictating the pathological outcomes of chronic liver injury.

Lipid peroxidation in hepatic steatosis in humans is associated with hepatic fibrosis and occurs predominately in acinar zone 3.

Hepatic steatosis has been shown to be associated with lipid peroxidation and hepatic fibrosis in a variety of liver diseases including non‐alcoholic fatty liver disease. However, the lobular distribution of lipid peroxidation associated with hepatic steatosis, and the influence of hepatic iron stores on this are unknown. The aim of this study was to assess the distribution of lipid peroxidation in association with these factors, and the relationship of this to the fibrogenic cascade.

Ferritin functions as a proinflammatory cytokine via iron-independent protein kinase C zeta/nuclear factor kappaB-regulated signaling in rat hepatic stellate cells.

Circulating ferritin levels reflect body iron stores and are elevated with inflammation in chronic liver injury. H‐ferritin exhibits a number of extrahepatic immunomodulatory properties, although its role in hepatic inflammation and fibrogenesis is unknown. Hepatic stellate cells respond to liver injury through production of proinflammatory mediators that drive fibrogenesis. A specific receptor for ferritin has been demonstrated on activated hepatic stellate cells, although its identity and its role in stellate cell activation is unclear. We propose that ferritin acts as a cytokine regulating proinflammatory function via nuclear factor kappaB (NF‐κB)–regulated signaling in hepatic stellate cell biology. Hepatic stellate cells were treated with tissue ferritin and iron‐free apoferritin, recombinant H‐ferritins and L‐ferritins, to assess the role of ferritin versus ferritin‐bound iron in the production of proinflammatory mediators of fibrogenesis, and to determine whether signaling pathways act via a proposed H‐ferritin endocytosis receptor, T cell immunoglobulin‐domain and mucin‐domain 2 (Tim‐2). This study demonstrated that ferritin activates an iron‐independent signaling cascade, involving Tim‐2 independent phosphoinositide 3 (PI3)‐kinase phosphorylation, protein kinase C zeta (PKCζ) and p44/p42‐mitogen‐activated protein kinase, resulting in p50/p65‐NF‐κB activation and markedly enhanced expression of hepatic proinflammatory mediators interleukin‐1β (IL‐1β), inducible nitric oxide synthase (iNOS), regulated on activation normal T cell expressed and secreted (RANTES), inhibitor of kappa Bα (IκBα), and intercellular adhesion molecule 1 (ICAM1). Conclusions:This study has defined the role of ferritin as a proinflammatory mediator of hepatic stellate cell biology acting through the NF‐κB signaling pathway, and suggests a potential role in the inflammatory processes associated with hepatic fibrogenesis. (HEPATOLOGY 2009;49:887–900.)

The Role of Hepatic Stellate Cells and Transforming Growth Factor-β1 in Cystic Fibrosis Liver Disease

Liver disease causes significant morbidity and mortality from multilobular cirrhosis in patients with cystic fibrosis. Abnormal bile transport and biliary fibrosis implicate abnormal biliary physiology in the pathogenesis of cystic fibrosis-associated liver disease (CFLD), yet the mediators linking biliary events to fibrosis remain unknown. Activated hepatic stellate cells (HSCs) are the pre-eminent mediators of fibrosis in a range of hepatic disorders. The dominant stimulus for matrix production by HSCs is the cytokine transforming growth factor (TGF)-beta(1). In CFLD, the role of HSCs and the source of TGF-beta(1) have not been evaluated. Liver biopsy tissue obtained from 38 children with CFLD was analyzed. Activated HSCs, identified by co-localization of procollagen alpha(1)(I) mRNA and alpha-smooth muscle actin, were demonstrated as the cellular source of excess collagen production in the fibrosis surrounding the bile ducts and the advancing edge of scar tissue. TGF-beta protein and TGF-beta(1) mRNA expression were shown to be predominantly expressed by bile duct epithelial cells. TGF-beta(1) expression was significantly correlated with both hepatic fibrosis and the percentage of portal tracts showing histological abnormalities associated with CFLD. This study demonstrates a definitive role for HSCs in fibrogenesis associated with CFLD and establishes a potential mechanism for the induction of HSC collagen gene expression through the production of TGF-beta(1) by bile duct epithelial cells.

PATHOPHYSIOLOGY OF IRON TOXICITY

There are several inherited and acquired disorders that can result in chronic iron overload in humans, and the major clinical consequences are hepatic fibrosis, cirrhosis, hepatocellular cancer, cardiac disease, and diabetes. It is clear that lipid peroxidation occurs in experimental iron overload if sufficiently high levels of iron within hepatocytes are achieved. Lipid peroxidation is associated with hepatic mitochondrial and microsomal dysfunction in experimental iron overload, and lipid peroxidation may underlie the increased lysosomal fragility that has been detected in liver samples from both iron-loaded human subjects and experimental animals. Reduced cellular ATP levels, impaired cellular calcium homeostasis, and damage to DNA may all contribute to hepatocellular injury in iron overload. Long-term dietary iron overload in rats can lead to increased collagen gene expression and hepatic fibrosis, perhaps due to activation of hepatic lipocytes. The mechanisms whereby lipocytes are activated in iron overload remain to be elucidated; possible mediators include aldehydic products of iron-induced lipid peroxidation produced in hepatocytes, tissue ferritin, and/or cytokines released by activated Kupffer cells.

Fibrogenesis in pediatric cholestatic liver disease: Role of taurocholate and hepatocyte‐derived monocyte chemotaxis protein‐1 in hepatic stellate cell recruitment

Cholestatic liver diseases, such as cystic fibrosis (CF) liver disease and biliary atresia, predominate as causes of childhood cirrhosis. Despite diverse etiologies, the stereotypic final pathway involves fibrogenesis where hepatic stellate cells (HSCs) are recruited, producing excess collagen which initiates biliary fibrosis. A possible molecular determinant of this recruitment, monocyte chemotaxis protein‐1 (MCP‐1), an HSC‐responsive chemokine, was investigated in CF liver disease and biliary atresia. The bile‐duct‐ligated rat and in vitro coculture models of cholestatic liver injury were used to further explore the role of MCP‐1 in HSC recruitment and proposed mechanism of induction via bile acids. In both CF liver disease and biliary atresia, elevated hepatic MCP‐1 expression predominated in scar margin hepatocytes, closely associated with activated HSCs, and was also expressed in cholangiocytes. Serum MCP‐1 was elevated during early fibrogenesis. Similar observations were made in bile‐duct‐ligated rat liver and serum. Hepatocytes isolated from cholestatic rats secreted increased MCP‐1 which avidly recruited HSCs in coculture. This HSC chemotaxis was markedly inhibited in interventional studies using anti‐MCP‐1 neutralizing antibody. In CF liver disease, biliary MCP‐1 was increased, positively correlating with levels of the hydrophobic bile acid, taurocholate. In cholestatic rats, increased MCP‐1 positively correlated with taurocholate in serum and liver, and negatively correlated in bile. In normal human and rat hepatocytes, taurocholate induced MCP‐1 expression. Conclusion: These observations support the hypothesis that up‐regulation of hepatocyte‐derived MCP‐1, induced by bile acids, results in HSC recruitment in diverse causes of cholestatic liver injury, and is a key early event in liver fibrogenesis in these conditions. Therapies aimed at neutralizing MCP‐1 or bile acids may help reduce fibro‐obliterative liver injury in childhood cholestatic diseases. (HEPATOLOGY 2008.)

A novel mutation in ferroportin1 is associated with haemochromatosis in a Solomon Islands patient

Background: A severe form of iron overload with the clinicopathological features of haemochromatosis inherited in an autosomal dominant manner has been described in the Solomon Islands. The genetic basis of the disorder has not been identified. The disorder has similarities to type 4 haemochromatosis, which is caused by mutations in ferroportin1. Aims: The aims of this study were to identify the genetic basis of iron overload in a patient from the Solomon Islands. Patient and methods: Genomic DNA was isolated from peripheral blood leucocytes of a Solomon Islands man with severe iron overload. The entire coding region and splice sites of the ferroportin1 gene was sequenced. Results and conclusions: A novel missense mutation (431A>C; N144T) was identified in exon 5 of the ferroportin1 gene. A novel restriction endonuclease based assay which identifies both the N144T and N144H mutations was developed which will simplify the diagnosis and screening of patients for iron overload in the Solomon Islands and other populations. This is the first identified mutation associated with haemochromatosis in the Solomon Islands population.

Evidence that myofibroblast-like cells are the cellular source of capsular collagen in hepatocellular carcinoma

BACKGROUND/AIMS The prognosis for patients with hepatocellular carcinoma is poor although tumour encapsulation has been associated with improved survival and disease-free rates. While the source of the tumour capsule is unclear, the major role that activated hepatic stellate cells play in the deposition of liver matrix in normal and diseased states suggests the possible involvement of these cells in tumour encapsulation. METHODS Twenty-four liver tumours (seven encapsulated HCC, seven non-encapsulated HCC, 10 colorectal metastases) were studied. Activated hepatic stellate cells were identified by immunohistochemistry for alpha-smooth muscle actin (alpha-SMA) and in situ hybridization for pro-collagen alpha1 (I) mRNA. Collagen deposition was localized using Massons trichrome stain. RESULTS Pro-collagen alpha1 (I) mRNA co-localized to alpha-SMA positive hepatic stellate cells within the region of increased collagen deposition in (i) the tumour capsule of encapsulated HCC, and (ii) the tumour junction of non-encapsulated HCC and colorectal metastasis. In addition, there was marked peritumour expression of alpha-SMA and procollagen alpha1 (I) mRNA, which diminished with distance away from the tumour in all tumour groups. The degree of expression was greatest with encapsulated HCC, less with non-encapsulated HCC and least with colorectal metastasis. This contrasted with the absence of alpha-SMA expression in normal liver from the same patients. Within the tumours, colorectal metastases differed from HCC by demonstrating marked alpha-SMA expression and collagen deposition in the septa. CONCLUSIONS Our findings demonstrate that activated hepatic stellate cells (i) are responsible for increased peritumour collagen production in non-encapsulated HCC and colorectal metastasis, and (ii) may be implicated in tumour capsule formation in HCC and metastasis stroma development. Thus, stellate cells may influence the local hepatic invasion by these tumours.