Timo Rath

Activation of hepatic stellate cells is associated with cytokine expression in thioacetamide-induced hepatic fibrosis in mice

The pathophysiological mechanisms of thioacetamide (TAA)-induced hepatic fibrogenesis are not yet fully understood. In particular, the role of hepatic stellate cells (HSCs) remains unclear. We therefore examined proliferation and transdifferentiation of HSC as well as the underlying molecular mechanisms in TAA-induced fibrosis. Hepatic fibrogenesis was induced in mice by addition of TAA to drinking water. Liver damage was determined by assessment of alanine aminotransferase and aspartate aminotransferase levels, and measurement of collagen deposition. Additionally, expression patterns of α-smooth muscle actin, glial fibrillary acidic protein (GFAP, specific hepatic biomarker for HSC), cysteine- and glycine-rich protein 2 (CRP2, specific marker of HSC transdifferentiation), tissue inhibitor of metalloproteinases-1, matrix metalloproteinase-9 (MMP-9), interleukins (IL-1β, IL-6), platelet-derived growth factors (PDGF-B, PDGF-D) , tumor necrosis factor (TNF)-α, and (transforming growth factor (TGF)-β1 were assessed by real-time PCR. Transcription of GFAP and CRP2 were transiently upregulated during TAA-induced fibrogenesis (punctum maxima (p.m.) week 10 for GFAP and week 14 for CRP2). Similar transient expression patterns were demonstrated for IL-1β, IL-6, TGF-β1, and PDGF-B (p.m. week 12) whereas TNF-α and PDGF-D continuously increased with ongoing liver injury. In particular, not only neutrophil granulocytes, but also macrophages and leukocytes served as a major source for MMP-9 expression. GFAP and CRP2 expression patterns demonstrated transiently increased HSC-activation during TAA-induced hepatic fibrogenesis. The rate of increase of transcription of GFAP correlated best with PDGF-B, whereas CRP2 levels correlated with PDGF-B, PDGF-D, and IL-1β expression. This study demonstrates for the first time that transiently increased activation patterns of HSC are observed in toxically induced hepatic fibrosis. Thus, TAA in drinking water is an effective and elegant model to induce reproducible states of liver fibrosis without parenchymal damage in mice.

Bone marrow transplantation demonstrates medullar origin of CD34+ fibrocytes and ameliorates hepatic fibrosis in Abcb4−/− mice

Bone marrow (BM)‐derived stem cells and CD34+ fibrocytes are associated with fibrogenesis in several organs. In an Abcb4−/− mouse model for sclerosing cholangitis alpha‐smooth muscle actin‐positive (α‐SMA+) myofibroblasts are thought to play a pivotal role in hepatic fibrogenesis. The aim of this study was 2‐fold: (1) to demonstrate that the origin of an important fibrogenetic cell population is the BM; and (2) to investigate whether transplantation of BM (BM‐Tx) affects liver function, staging, and grading. Surrogate markers for fibrogenesis and regulation of hepatic stellate cells (HSC) as well as progenitor‐cell‐derived fibrocytes in liver tissue were analyzed by quantitative real‐time polymerase chain reaction (PCR) and immunohistology. After lethal irradiation of recipient mice, BM‐Tx was carried out by way of tail vein injection of BM cells from marker protein donors (green fluorescent protein, GFP+) or Abcb4−/− mice as control (syngeneic Tx). Parameters of liver function were assessed serologically and histologically. Activated HSC of α‐SMA+/CRP2+ phenotype were expressed in ≈50% of proliferating bile ducts, whereas fibrotic liver parenchyma showed no expression thereof. Epithelial mesenchymal transfer (EMT) was visualized in the areas of proliferating bile ducts. The hematopoietic origin of CD34+ fibrocytes was demonstrated immunohistologically in livers of BM chimeric mice. These CD34+ cells infiltrated hepatic lobules from portal fields and developed a desmin+ phenotype expressing collagen type I in fibrotic parenchyma as well as in vitro after isolation by magnetic cell separation. Transplantation of GFP+/Abcb4+ BM improved liver function and staging compared with sham transplantation, but no significant differences were noticed among allogeneic and syngeneic Tx. Conclusion: The present study is the first to identify that both BM‐derived fibrocytes and HSC are involved in biliary fibrogenesis in Abcb4−/− mice. Our data suggest that changes in immunity subsequent to BM‐Tx may alter hepatic fibrosis. (HEPATOLOGY 2009.)

Enhanced expression of MMP-7 and MMP-13 in inflammatory bowel disease: a precancerous potential?

&NA; Matrix metalloproteinases (MMPs) are responsible for the turnover and degradation of extracellular matrix. They play a crucial role in the growth and migration of colorectal carcinoma cells. Colorectal carcinomas are characterized by enhanced expression of MMP‐2, MMP‐9, MMP‐7, and MMP‐13. The aim of this study was to determine the expression levels of MMP‐2, MMP‐9, MMP‐7, MMP‐13, and MMP‐14 and their specific inhibitor TIMP‐1 in inflammatory bowel diseases and precancerous lesions of the colon, i.e., Crohns disease and ulcerative colitis, and in adenomatous polyps (APs) for comparison. Biopsy samples of pathological and healthy tissue were obtained from 40 patients with inflammatory bowel disease (ulcerative colitis, n = 17; Crohns disease, n = 23) and from 19 patients with APs. mRNA was measured by quantitative real‐time polymerase chain reaction to study MMP and TIMP‐1 gene expression in both pathological and normal mucosal specimens. For MMP‐2, MMP‐9, and TIMP‐1, protein expression also was quantified with sandwich enzyme‐linked immunosorbent assay. In biopsy specimens of Crohns disease and ulcerative colitis, significantly increased levels of MMP‐2, MMP‐7, and MMP‐13 mRNA were found. MMP‐2 and MMP‐9 showed enhanced secretion on the protein level. AP revealed an increased transcription of MMP‐7 and MMP‐13 genes. MMP‐14 mRNA was decreased in APs. MMPs, especially MMP‐7 and MMP‐13, which are expressed primarily on the tumor cell surface, are elevated in inflammatory bowel disease, which may have more chance to evolve into malignancy than normal tissue. In APs, increased expression of MMP‐7 and MMP‐13 may serve as an early indicator for colorectal carcinogenesis.

Serum matrix metalloproteinases in adult CF patients: Relation to pulmonary exacerbation

BACKGROUND Matrix metalloproteinases (MMPs) virtually degrade all components of the extracellular matrix and are mainly expressed in tissues following inflammation or remodeling. Increased expression of certain MMPs in sputum and bronchoalveolar lavage of patients with cystic fibrosis (CF) is related to impaired lung function. We investigated whether a panel of serum MMPs is associated with both, impairment of pulmonary function and occurrence of pulmonary exacerbations (PEx) in adult patients with CF. METHODS Serum concentrations of MMP-1, MMP-2, MMP-3, MMP-7, MMP-8, MMP-9, MMP-12, MMP-13, and TIMP-1 were determined by ELISA in 54 adult CF patients. PEx was defined based on a score established by Rosenfeld in 2001. MMP-1, MMP-8, MMP-9 and TIMP-1 were assessed in 7 CF patients with PEx before and after systemic antibiotic therapy. RESULTS Of the 54 CF patients, PEx was diagnosed in 16 different CF patients. Compared to healthy controls, MMP-1, MMP-8, and MMP-9, serum levels were elevated in CF patients and correlated with PEx. MMP-8 expression was associated with impaired lung function. For MMP-2, we observed a decreased expression and an association of MMP-2 decline with PEx. Antibiotic treatment of CF patients with PEx led to a decrease of MMP-1, MMP-8 and active MMP-9 protein concentration. CONCLUSIONS Increased serum expression of certain MMPs is associated with occurrence of PEx and impaired lung function in CF. Hence, these MMPs might serve as surrogate markers for PEx.

Cellular sources of MMP-7, MMP-13 and MMP-28 in ulcerative colitis.

Abstract Objective. Matrix metalloproteinases (MMPs) are considered the predominant proteases in the pathogenesis of mucosal ulcerations associated with inflammatory bowel disease (IBD). Whether the malignancy associated MMP-7 and MMP-13 or the recently cloned MMP-28 convey a certain meaning for intestinal homeostasis and pathogenesis of IBD is currently unknown. We therefore set off to analyze regulation patterns and cellular origins of these MMPs in mucosal tissues of patients with ulcerative colitis (UC). Material and methods. Biopsy samples of affected and healthy tissues were obtained from 35 Norwegian patients with UC. RNA was quantified by quantitative real-time polymerase chain reaction to study MMP gene expression in both pathological and healthy mucosal specimens. Cellular origins were determined by immunohistology using surrogate markers for inflammation, neovascularization, and epithelial structures. Protein expression of MMP-7 and MMP-13 was quantified using enzyme-linked immunosorbent assay. Results. MMP-7 and MMP-13 gene expression was significantly increased in UC affected colonic mucosa whereas MMP-28 showed a decreased expression in inflamed mucosa. Endothelial cells and infiltrating leukocytes were identified as the major cellular sources of MMP-7 and MMP-13 in UC. Enterocytes represented the major cellular source of MMP-28 in healthy and inflamed mucosa. Conclusions. MMP-7 and MMP-13 expression in inflammatory and endothelial cells indicate a role of these MMPs for both colitis associated neoangiogenesis and inflammatory changes. Decreased MMP-28 expression in UC is most likely the result of colitis associated epithelial destruction and loss of cryptal architecture.

Innovative immunohistochemistry identifies MMP-9 expressing macrophages at the invasive front of murine HCC

AIM To investigate the proteolytic contribution of tumor-associated macrophages (TAM) in tumor invasion, we analyzed whether TAM at the invasive front of small HCC in Abcb4(-/-)-mice show an enhanced expression of MMP-9. METHODS Liver cryosections of the hepatocellular carcinoma (HCC) invasive front from 12 mo old Abcb4(-/-)-mice were stained for collagen type I and MMP-9 using Alexa488 and Alexa568 labeled secondary antibodies. Afterwards, the Alexa568 dye was bleached and the macrophage marker F4/80 was visualized using Alexa568 labeled secondary antibodies. Finally, photographs of the invasive tumor front were digitally overlaid and analyzed. RESULTS After complete bleaching of the primary dye, specific fluorescence staining of a third antigen, here F4/80, was successfully performed on the same histological section. With this method, we were able to identify conglomerates of matrix metalloproteinase (MMP-9) expressing macrophages within the tumor capsule of HCC. CONCLUSION MMP-9 expressing macrophages are involved in matrix remodelling at the invasive tumor front of HCC. The described staining protocol provides a simple yet powerful extension of conventional immuno-histochemistry, facilitating visualization of at least three different antigens plus nuclei in one single histological section.

Serum Proteome Profiling Identifies Novel and Powerful Markers of Cystic Fibrosis Liver Disease

Background and Aims Cystic Fibrosis associated liver disease (CFLD) develops in approximately 30% of CF patients. However, routine sensitive diagnostic tools for CFLD are lacking. Within this study, we aimed to identify new experimental biomarkers for the detection of CFLD. Methods 45 CF patients were included in the study and received transient elastography. Differential regulation of 220 different serum proteins was assessed in a subgroup of patients with and without CFLD. Most interesting candidate proteins were further quantified and validated by ELISA in the whole patient cohort. To assess a potential relation of biomarker expression to the degree of hepatic fibrosis, serum biomarkers were further determined in 18 HCV patients where liver histology was available. Results 43 serum proteins differed at least 2-fold in patients with CFLD compared to those without liver disease as identified in proteome profiling. In ELISA quantifications, TIMP-4 and Endoglin were significantly up-regulated in patients with CFLD as diagnosed by clinical guidelines or increased liver stiffness. Pentraxin-3 was significantly decreased in patients with CFLD. Serum TIMP-4 and Endoglin showed highest values in HCV patients with liver cirrhosis compared to those with fibrosis but without cirrhosis. At a cut-off value of 6.3 kPa, transient elastography compassed a very high diagnostic accuracy and specificity for the detection of CFLD. Among the biomarkers, TIMP-4 and Endoglin exhibited a high diagnostic accuracy for CFLD. Diagnostic sensitivities and negative predictive values were increased when elastography and TIMP-4 and Endoglin were combined for the detection of CFLD. Conclusions Serum TIMP-4 and Endoglin are increased in CFLD and their expression correlates with hepatic staging. Determination of TIMP-4 and Endoglin together with transient elastography can increase the sensitivity for the non-invasive diagnosis of CFLD.

YKL-40 and transient elastography, a powerful team to assess hepatic fibrosis

Abstract Objective. Transient elastography (TE) is a non-invasive and accurate method for the diagnosis of severe hepatic fibrosis and cirrhosis (F = 3 and F = 4). However, the assessment of significant fibrosis (F = 2) by TE is impaired due to a high variation in the diagnostic accuracy. Within this study, we aim to compare the diagnostic value of TE and experimental biomarkers of liver fibrosis. Material and methods. A total of 55 patients with chronic liver disease of different etiologies were included in the study. Among them, patients with HCV infection represented the largest cohort (n = 25). Liver fibrosis was evaluated according to the Desmet/Scheuer score. All patients received TE. Serum concentrations of YKL-40, hyaluronic acid (HA), Laminin, C-terminal procollagen I peptide, MMP-9, TIMP-1, TIMP-2 and MMP-9/TIMP-1 complex were determined by ELISA. Results. In the total patient population, areas under the receiver operator characteristic curve (AUROC) for TE were 0.798 (F ≥ 2), 0.880 (F ≥ 3) and 1 (F = 4). Among the serum markers, highest diagnostic accuracies were calculated for YKL-40 for F ≥ 2 (0.792) and F ≥ 3 (0.914) and for YKL-40 and HA for F = 4 (both 0.936). In the subgroup of HCV patients, the following AUROCs for TE were calculated: 0.802 (F ≥ 2), 0.798 (F ≥ 3) and 0.998 (F = 4). YKL-40 exhibited the highest diagnostic accuracy of all biomarkers in the HCV population (0.880, 0.854 and 0.986, respectively). Conclusions. YKL-40 is a powerful fibrosis marker with high diagnostic accuracy, in particular in HCV-associated liver disease. Its determination may confirm and improve the diagnostic accuracy of TE especially in early stages of liver fibrosis.

Presence of intestinal Mycobacterium avium subspecies paratuberculosis(MAP) DNA is not associated with altered MMP expression in ulcerative colitis

BackgroundMycobacterium avium subspecies paratuberculosis (MAP) is suspected to be a causative agent in human Crohns disease (CD). Recent evidence suggests that pathogenic mycobacteria and MAP can induce the expression of Matrix Metalloproteinases (MMP), which are the main proteases in the pathogenesis of mucosal ulcerations in inflammatory bowel disease (IBD). Within this study we assessed the prevalence of intestinal MAP specific DNA in patients with Crohns disease, ulcerative colitis (UC), and healthy controls. We further analysed regulation patterns of MMPs in mucosal tissues of UC patients with and without intestinal MAP DNA detection.MethodsColonic biopsy samples were obtained from 63 Norwegian and German IBD patients and 21 healthy controls. RNA was quantified by quantitative real-time polymerase chain reaction (PCR) to study MMP gene expression in both pathological and healthy mucosal specimens. The presence of MAP DNA in colonic mucosa was examined using MAP specific PCR.ResultsMAP DNA was detected in 20% of UC patients and 33% of healthy controls but only in 7% of patients with CD. UC patients treated with corticosteroids exhibited a significantly increased frequency of intestinal MAP DNA compared to those not receiving corticosteroids. Expression of MMP-1, -2, -7, -9, -13, -19, -28 and TNF-α did not differ between UC patients with presence of intestinal MAP DNA compared to those without. MMP-2, MMP-9 and MMP-13 were significantly decreased in UC patients receiving corticosteroids.ConclusionsThe presence of intestinal MAP specific DNA is not associated with altered MMP expression in UC in vivo. Corticosteroids are associated with increased detection of intestinal MAP DNA and decreased expression of certain MMPs. Frequent detection of MAP DNA in healthy controls might be attributable to the wide environmental distribution of MAP and its presence in the food-chain.

Downregulation of Breast Cancer Resistance Protein in colon adenomas reduces cellular xenobiotic resistance and leads to accumulation of a food-derived carcinogen

Several molecular changes in colorectal adenomas provide the basis of the adenoma–carcinoma sequence. We investigated the expression of xenobiotic ATP‐binding cassette (ABC) transporters in humans and in ApcMin mice and conducted functional studies estimating the importance of the expression changes. Twenty‐nine adenomas from 21 patients and eight adenomas from four ApcMin mice were analyzed using Western blotting and quantitative Real‐time polymerase chain reaction (RT‐PCR). Adjacent healthy tissue served as control for each polyp. Breast cancer resistance protein (BCRP) was significantly downregulated in human colorectal adenomas (to 28 ± 35% of adjacent healthy tissue). This was in line with data from ApcMin mice adenomas, where downregulation was significant as well (to 58 ± 34%). In parallel, quantitative RT‐PCR showed BCRP mRNA downregulation in human adenomas (to 17 ± 31%). Basal multidrug resistance‐associated protein 2 expression was low and did not change in adenomas; multidrug resistance transporter 1 expression also did not differ between adenomas and healthy tissue. In a functional study, ApcMin mice received radioactively labelled 2‐amino‐1‐methyl‐6‐phenylimidazo[4,5‐β] pyridine (PhIP), a food colon carcinogen and substrate of BCRP, by oral gavage with analysis of PhIP accumulation and DNA adduct formation 48 hr later. In this setting, we could demonstrate a higher carcinogen concentration in adenomas of ApcMin mice (181 ± 113% of normal tissue) including immunohistochemical detection of PhIP–DNA adducts. We conclude that significant transcriptional downregulation of BCRP/Bcrp leads to higher carcinogen concentrations in colorectal adenomas of mice and men. This might promote the adenoma–carcinoma sequence by higher genotoxic effects. The results indicate a possible role of transporter deficiencies in susceptibility for colon carcinoma.