Aims: CYR61 (CCN1), CTGF (CCN2) and NOV (CCN3) belong to the CCN protein family. They share a modular structure with a secretary signal peptide, an insulin-like growth factor binding protein-like domain, a von Willebrand factor type C and a Thrombospondin type-1 repeat module and a cysteine knot containing module. These cysteine-rich proteins are secreted and play an essential role in fibrogenesis [1]. Recently it was demonstrated that NOV and CTGF appear to act in a Yin/Yang manner to regulate extracellular matrix (ECM) metabolism [2]. Here we aimed to analyse the synergy of CTGF and TGF-β in the regulation of ECM synthesis in the hepatic stellate cell line GRX and cell proliferation in EA·hy 926 endothelial cells.

BACKGROUND/AIMS: The activation of hepatic stellate cells (HSC) and their transdifferentiation into myofibroblasts (MFB) is the key step for development of liver fibrosis. Over the past several years, significant progress has been made in the understanding of the critical pathways involved incells undergoing activation. Cellular activation in the course of transdifferentiation involves, among other biochemical modifications, functionally relevant changes in the control of gene expression. These include the up-regulation of transcription factors, different extracellular matrix proteins, cell adhesion molecules, smooth muscle specific genes, and proteins involved in matrix remodelling, or cytoskeletal organization. The corresponding regulatory elements of these genes have afforded us the opportunity to express transgenes with antifibrotic potential in a cell type- and/or transdifferentiation-dependent manner. METHODS: In the present study, we have tested several promoters for their ability to mediate cell-specific expression, including those for CSRP2, SM22alpha, and TIMP-1 (CSRP2, gene encoding the LIM domain protein CRP2; SM22alpha, smooth muscle-specific gene encoding a 22-kDa protein; TIMP-1, gene encoding the tissue inhibitor of metalloproteinases-1), which in liver are specifically expressed in HSC or become strongly activated during the acute remodelling into MFB. We constructed adenoviral reporter vectors in which relevant portions of the promoters were fused to the green fluorescent protein. RESULTS AND CONCLUSION: Our experiments demonstrate that each of these promoters is sufficient to achieve strong or partially selective expression in vitro but none is able to direct a specific or inducible expression of transgenes in HSC/MFB in vivo.

At the cellular level, the activation and transdifferentiation of quiescent hepatic stellate cells (HSC) into myofibroblasts is the key process involved in hepatic fibrogenesis that is associated with an increased and altered deposition of extracellular matrix components in the liver. The temporal sequence of molecular events associated with stellate cell activation turned out to be appropriately mimicked when HSC isolated from normal livers are cultured on uncoated plastic surface. Therefore, cultured primary cells isolated from rodents and human beings are common in vitro models in investigations addressing these issues of hepatic stellate biology and function. However, the limited supply, cost-effective isolation procedure and the ever growing need have resulted in efforts to establish immortalized stellate cell lines having the advantage of virtually unlimited access. They allow rapid screening for disease-associated factors and restrict the necessary number of animal experiments. From the first description of an immortal HSC line in 1986, a huge number of studies were conducted with these established cell lines. However, differences in morphology, growth characteristics and anomalies of chromosome number and structure make the applications of these models questionable. Here, we summarize the history and cellular characteristics of respective cell lines and discuss the differences of continuous HSC lines and their primary counterparts.

Fewer than 1% of live births are complicated by a life-threatening form of preeclampsia known as HELLP (hemolysis, elevated liver enzymes, low platelets) syndrome. About 15% of women with eclampsia or preeclampsia exhibit all aspects of HELLP syndrome, signifying an elevated risk of perinatal morbidity and mortality for both the fetus and mother. Evidence is increasing that hypertensive complications of pregnancy are associated with inherited or acquired thrombophilias. Using a case-control design, the investigators retrospectively estimated the prevalence of three inherited thrombophilic mutations (factor V Leiden, the prothrombin 20210G>A mutation, the methylenetetrahydrofolate reductase [MTHFR] 677C>T polymorphism) in 71 Caucasian mother-child pairs with HELLP syndrome and 79 control pairs with uncomplicated pregnancies. The mutations were genotyped using LightCycler technology. Maternal heterozygosity for factor V Leiden was significantly more prevalent in the HELLP group than in the control group. The odds ratio was 4.45, with a 95% confidence interval of 1.31–15.31. In contrast, no significant association with HELLP syndrome was observed for either the maternal prothrombin mutation or the MTHER polymorphism. The fetal genotype was not associated with HELLP syndrome for any of the three mutations. No differences were found when analyzing gene-gene interactions or genotype-phenotype correlation with regard to clinical parameters or perinatal outcomes. The clinical variables examined included gestational age at delivery, birth weight, systolic and diastolic blood pressure, the platelet count, liver enzyme levels, and hemolysis. These results affirm that women who are heterozygous for factor V Leiden, one of the most common inherited thrombophilias, are at increased risk of developing HELLP syndrome. Gene-gene interactions appear not to have major influence on the role of thrombophilic mutations in the causality of HELLP syndrome.

Hepatic stellate cells (HSC) are prominent cellular effectors of liver fibrosis, which become activated during liver injury and transdifferentiate from quiescent, fat storing cells into a proliferative myofibroblastic cell type (MFB). This process is accompanied by modulations of gene expression. A common feature of activated HSC is upregulation of genes encoding known markers of smooth muscle cells (SMC), e.g. α-smooth muscle actin (α-SMA). In SMC, these genes are regulated by the ubiquitous transcription factor serum response factor (SRF) in cooperation with specific co-factors, especially myocardin. As SRF becomes strongly expressed in activated HSC, we looked for its transcriptional activity at different time points of differentiation and factors involved in this process.

Background/Aims: Liver fibrogenesis is the consequence of a dysbalance of synthesis and degradation of extracellular matrix components. During hepatic fibrogenesis, hepatic stellate cells (HSC) are the major producer of these constituents. Therefore, HSC are in the focus of antifibrotic therapies. A previous report from us have demonstrated that the thymidine kinase/Ganciclovir system directed under transcriptional control of the tissue inhibitor of metalloproteinases–1 (TIMP–1) promoter is suitable to induce apoptosis in culture-activated HSC [1]. We now extended our studies and tested if a selective induction of HSC apoptosis with this system is achievable in vivo.

Die in der Leber produzierte Plasma-Serinprotease FSAP spielt eine Schlüsselrolle bei gefäßverengenden Erkrankungen1. FSAP spaltet und inaktiviert PDGF-BB, wodurch die Proliferation und Migration von Vascular Smooth Muscle Cells und somit die Neointima-Bildung in den erkrankten Gefäßen verringert wird. Während der hepatischen Fibrogenese wird die Transdifferenzierung Hepatischer Sternzellen (HSCs) zu einem pro-fibrogenen Myofibroblasten-ähnlichen Phenotyp durch PDGF-BB stimuliert2. In der vorliegenden Studie sollte der Einfluss von FSAP auf die PDGF-stimulierte p42/p44 MAPK Aktivierung in primären HSC untersucht werden. Weiterhin wurde die Wirkung von FSAP auf die PDGF-BB stimulierte Migration und Proliferation von HSC analysiert.

Transdifferentiation of hepatic stellate cells (HSC) into myofibroblast-like cells is a key event in liver fibrogenesis accompanied by differential gene regulation. Antifibrotic strategies focus on manipulation of this relevant cell type, e.g. induction of HSC apoptosis, but to date a specific and easy-to-handle targeting of activated HSC is not reported. As demonstrated by Lemken et al. [1] a targeting of reporter genes to liver using enhancer elements of hepatocyte specific genes fused to a minimal promoter was successful. Therefore, we proofed if short promoter fragments with essential cis-acting regulatory elements from genes upregulated during transdifferentiation are able to direct HSC specific reporter gene expression. Four different DNA-fragments, each ˜120 bp in length, were amplified from the promoters of rat α-smooth muscle actin and SM22α genes by PCR (‘modulesrsquor;) and cloned into the pGL3 promoter vector. Transcriptional efficiency and specificity of 20 different module combinations were determined by luciferase expression levels after transfection into CFSC and HepG2 cells and selected combinations were further verified by transfection into primary rat HSC and hepatocytes. We could select 3 constructs that were able to direct high reporter gene activity in CFSC as well as in HSC. Two of these constructs contain modules of the SM22α gene promoter in different combinations with a TCE element or SP1 binding site, respectively. The most promising module combination (SP1+TCE) mediated a 10-fold higher luciferase expression in CFSC compared to HepG2, while the other combinations revealed an expression level that was high in both cell lines. Principally, we could demonstrate that combination of small promoter modules with known regulatory elements from genes upregulated during transdifferentiation enables the targeting of HSC. This allows the easy-to-handle expression of diverse transgenes in contrast to a recent publication [2] showing that a targeting of HSC was only successfully by delivery of 2 different adenoviral constructs. We think that our approach implicate the potential to enhance expression efficiency and specificity by easy recombination with additional promoter modules together with an exchange of the used SV40 minimal promoter with a more HSC related proximal gene promoter, e.g. TIMP–1.