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The induction of experimental obstructive cholestasis is a reliable model for cholestatic liver diseases in rodents. Bile duct ligation (BDL) in mice provokes typical time-dependent morphological and structural changes in the liver, ranging from liver cell injury and elevated serum enzyme levels after several days, to a severe inflammatory response in the liver after 5–7 days, up to an advanced hepatic fibrosis as soon as three to four weeks after surgical ligation of the common biliary duct. Upon BDL induction, hepatic stellate cells become activated and transdifferentiate into myofibroblasts that produce extracellular matrix proteins such as collagen. In principle, the periportal fibrosis induced by BDL in rat livers is reversible. After the relief of a biliary obstruction, the liver has the capacity to revert to a nearly normal histological architecture and a fully normal biochemical function. When BDL surgery is performed by an experienced scientist, this model has very high reproducibility among all fibrotic models. All these factors corroborate the outstanding value of this model for basic and translational research in biomedicine and hepatology. Nevertheless, this model can result in significant variations when surgery is carried out by untrained personnel or when unconscious modifications are implemented that affect the quality of the intervention. A detailed protocol is provided here for the provision of reliable and reproducible BDL in mice.

Hepatic stellate cells (HSCs) are found in the perisinusoidal space of the liver (i.e., the space of Dissé). They represent 5-8% of the total number of liver cells. In normal liver, these cells have a quiescent phenotype and are characterized by numerous fat vacuoles that store vitamin A in a form of retinyl ester. In injured liver, these cells transdifferentiate into a myofibroblast phenotype, become highly proliferative and are responsible for excess collagen synthesis and deposition during fibrosis. Due to their exceptional pathophysiological relevance, several isolation and purification protocols of primary HSCs have been established that provide the basis for studying HSC biology in vitro. We here describe a method for high-purity isolation of HSCs from mice. This protocol includes the enzymatic digestion of the liver tissue by pronase and collagenase, cellular enrichment by centrifugation of the crude cell suspension through a Nycodenz density gradient, and a final (optional) flow cytometric enrichment that allows generating ultrapure HSC fractions.

Hepatic stellate cells (HSCs) are found in the perisinusoidal space of the liver (i.e., the space of Disse). They represent 5-8% of the total number of liver cells. In normal liver, these cells have a quiescent phenotype and are characterized by numerous fat vacuoles that store vitamin A in a form of retinyl ester. In injured liver, these cells transdifferentiate into a myofibroblast phenotype, become highly proliferative and are responsible for excess collagen synthesis and deposition during fibrosis. Due to their exceptional pathophysiological relevance, several isolation and purification protocols of primary HSCs have been established that provide the basis for studying HSC biology in vitro. We here describe a method for high-purity isolation of HSCs from mice. This protocol includes the enzymatic digestion of the liver tissue by pronase and collagenase, cellular enrichment by centrifugation of the crude cell suspension through a Nycodenz density gradient, and a final (optional) flow cytometric enrichment that allows generating ultrapure HSC fractions.

The Factor VII-activating protease (FSAP) is a plasma serine protease that acts as an activator of factor VII, in- dependently of tissue factor, promoting the coagulation cascade and it activates pro-urokinase in the fibrinolytic pathway. Two single nucleotide polymorphisms (SNPs) in the coding region of the FSAP gene that lead to amino acid substitutions within the serine protease domain are presently discussed to be involved in the formation of atherosclerosis leading to ca- rotid stenosis, cardiovascular diseases, and thromboembolic disorders. The G534E polymorphism, also known as Marburg I, is a guanine to adenine substitution that is found in about 5% of the population and impairs the in vitro capacity to acti- vate pro-urokinase while the biological effect of the second variant (E393Q), also known as the Marburg II, has not yet been identified. Based on the properties to impair the pro-urokinase activity and their association with the incidence and progression of carotid stenosis it is conceivable that genotyping of this allelic variants is potentially interesting for routine genotyping of risk patients. We here describe the development of a novel LightCycler-based methodology allowing simul- taneous genotyping of both allelic variants. The different genotypes could be identified easily and clearly by the genera- tion of characteristic fluorescence melting peaks. The outlined methodology will be helpful for routine genotyping of these FSAP variants.

Hepatic injury and regeneration of the liver are associated with activation of hepatic stellate cells (HSC). Fibroblast growth factors (FGFs) and their receptors are important regulators of repair in various tissues. HSC express FGFR3IIIc as well as FGFGR4 and different spliced FGFR1IIIc and FGFR2IIIc isoforms which differ in the presence or absence of the acid box and of the first Ig-like domain. Expression of FGF9, known to be capable to activate the HSC FGFR2/3-isoforms, was increased in HSC in liver slice cultures after exposition to carbon tetrachloride, as an acute liver injury model. FGF9 significantly stimulated 3-H thymidine incorporation of hepatocytes, but failed to induce DNA synthesis in HSC despite the fact that FGF9 induced a sustained activation of extracellular signal-related kinases (ERK) 1/2. FGF9 induced an increased phosphorylation of Tyr436 of the fibroblast growth factor receptor substrate (FRS) 2, while phosphorylation of Tyr196 which is required for efficient Grb2 recruitment remained unchanged. Our findings suggest that HSC FGF9 provide a paracrine mitogenic signal to hepatocytes during acute liver injury, while the autocrine FGF9 signaling appears to be not sufficient to induce cell proliferation.

In most vertebrates, the liver produces bile that is necessary to emulsify absorbed fats and enable the digestion of lipids in the small intestine as well as to excrete bilirubin and other metabolic products. In the liver, the experimental obstruction of the extrahepatic biliary system initiates a complex cascade of pathological events that leads to cholestasis and inflammation resulting in a strong fibrotic reaction originating from the periportal fields. Therefore, surgical ligation of the common bile duct has become the most commonly used model to induce obstructive cholestatic injury in rodents and to study the molecular and cellular events that underlie these pathophysiological mechanisms induced by inappropriate bile flow. In recent years, different surgical techniques have been described that either allow reconnection or reanastomosis after bile duct ligation (BDL), e.g., partial BDL, or other microsurgical methods for specific research questions. However, the most frequently used model is the complete obstruction of the common bile duct that induces a strong fibrotic response after 21 to 28 days. The mortality rate can be high due to infectious complications or technical inaccuracies. Here we provide a detailed surgical procedure for the BDL model in mice that induce a highly reproducible fibrotic response in accordance to the 3R rule for animal welfare postulated by Russel and Burch in 1959.

Analytical cellular pathology 2015, 417023 (2015). doi:10.1155/2015/417023, Published by PubMed Central, Bethesda, Md.

Endothelial cells line the blood vessel and precursor endothelial cells appear to have a pivotal effect on the organ formation of the heart, the embryonic development of the kidney, and the liver. Several growth factors including the fibroblast growth factors (FGF) seem to be involved in these processes. Ligands such as basic FGF produced and secreted by endothelial cells may also coordinate cellular migration, differentiation, and proliferation under pathological conditions including wound healing, tumorgenesis, and fibrogenesis in the adult. Recently we demonstrated the expression of two secreted FGFs, FGF16, and FGF18, in HUVEC and in rat aortic tissue. In the present report, we confirmed by RT-PCR analysis that FGF18 is wildly expressed in the cardiovascular tissue, while FGF16 showed a more restricted expression pattern. HUVEC clearly demonstrated chemotaxis towards FGF16 and FGF18. Both FGFs also enhanced cell migration in response to mechanical damage. However, recombinant FGF16 and FGF18 failed to induce endothelial cell proliferation or sprouting in a three-dimensional in vitro angiogenesis assay. Fgf18 expression was earlier reported in the liver, and we detected FGF18 expression in liver vascular and liver sinusoidal endothelial cells (LSECs), but not in hepatic parenchymal cells. Recombinant FGF18 stimulated DNA synthesis in primary hepatocytes, suggesting, that endothelial FGF18 might have a paracrine function in promoting growth of the parenchymal tissue. Interestingly, FGF2, which is mitogenic on endothelial cells and hepatocytes stimulates a sustained MAPK activation in both cell types, while FGF18 causes a short transient activation of the MAPK pathway in endothelial cells but a sustained activation in hepatocytes. Therefore, the difference in the time course of MAPK activation by the different FGFs appears to be the cause for the different cellular responses.

There is a high demand for the isolation of primary endothelial cells for biomaterial endotheliazation studies, tissue engineering, and artificial organ development. Further, biomarkers for monitoring the response of endothelial cells in biomaterials science are required. We systematically compared two strategies for isolating liver sinusoidal endothelial cells (LSEC) from mouse liver. We demonstrate that fluorescence-activated cell sorting results in a considerably higher purity (~ 97%) compared to magnetic-assisted cell sorting (~ 80%), but is associated with a lower yield and recovery rate. Cell repellent polyethylene glycol (PEG) substrates affected the morphology of primary LSEC in culture and significantly downregulated the intracellular adhesion molecule (ICAM) and upregulated the vascular cell adhesion molecule (VCAM). This molecular response could partially be reverted by further modification with arginylglycylaspartic acid (RGD). Thus, usage of PEGylated materials may reduce, while applying RGD may support endotheliazation of materials, and we could relate LSEC attachment to their expression of ICAM and VCAM mRNA, suggesting their usage as biomarkers for endothelialization.

Latent transforming growth factor beta binding protein (LTBP), a high-molecular-weight glycoprotein of the large latent TGF-beta complex is suggested to serve as an anchor for latent TGF-beta in the extracellular matrix and as a component of microfibrillar structures. Proteolytic cleavage of LTBP is supposed to be a prerequisite for the release and generation of bioactive (mature) TGF-beta. We investigated the expression of LTBP isoforms in normal and fibrotic rat liver and in cultured rat hepatic stellate cells (HSC) transdifferentiating to myofibroblasts (MFB). We further determined their interaction with the matrix and some of their basic functions. Immunostainings of normal and fibrotic livers demonstrate intense signals for LTBP-1 and -2, preferably in parenchymal, but also nonparenchymal, cells and in fibrotic extracellular matrix. However, in situ hybridization points to a restriction of transcripts to nonparenchymal cells from fibrotic livers, whereas hepatocytes were always devoid of LTBP transcripts. The findings were confirmed by real-time quantitative reverse-transcription polymerase chain reaction (RT-PCR), which showed isoform-specific increases of LTBP transcripts in cultured stellate cells transdifferentiating to MFB and by Northern blot analyses showing the absence of LTBP-1 mRNA in freshly isolated hepatocytes. Using a cell enzyme-linked immunosorbent assay (ELISA), a differential increase of partly deoxycholate (DOC)-resistant, matrix-bound LTBP-1 and -2 was measured in cultured stellate cells. Treatment with plasmin generated soluble LTBP-1 and bioactive TGF-beta, which was able to induce Smad7 expression in an autocrine fashion. Our data propose (transdifferentiating) stellate cells, respectively MFB, as the major source of LTBP in normal and fibrotic liver, which here probably fulfills structural and TGF-beta-regulating functions as suggested for nonhepatic tissues.

Two point mutations in the hemochromatosis gene (HFE) are considered responsible for the development of hereditary hemochromatosis (HH), an autosomal recessive iron overload disease. One of these mutations produces a cysteine-to-tyrosine amino acid substitution at position 282 of the HFE protein (Cys282Tyr), caused by a G-to-A transition at nucleotide position 845. A homozygous Cys282Tyr mutation is present in 80–100% of hemochromatosis patients (1)(2). This mutation prevents interaction of HFE protein with β2-microglobulin, leading to an inability to bind to the transferrin receptor, a process that is necessary for iron resorption (3)(4). A second mutation, which changes histidine at position 63 to aspartic acid (His63Asp), caused by a C187G transversion does not affect this binding but possibly blocks the interaction with other ligands (3)(5). In contrast to the Cys282Tyr mutation, the role of the His63Asp mutation in the pathogenesis of the disease is uncertain. A heterozygous His63Asp mutation is often observed in Cys282Tyr heterozygous patients. This genotype combination is called compound heterozygosity and is observed in 4–5% of HH cases. Therefore, it must be considered, like the homozygous Cys282Tyr mutation, as a genetic disposition for HH. However, it seems to play a subordinate role and seems to be important only in combination with the Cys282Tyr mutation (6). Approximately 3% of the population is homozygous for the His63Asp mutation, and thus is not considered in the risk group for HH (6)(7). In rare HH cases, none of these known HFE mutations are present. Other mutations, possibly not associated with HFE , might contribute to the development of hemochromatosis (2). There are many current methods available for genotyping the two known hemochromatosis-causing mutations, including oligonucleotide ligation assay (8), single-strand conformation polymorphism (9), allele-specific PCR (10)(11), and PCR-restriction fragment length …

PLoS ONE 11(6), e0158104 (2016). doi:10.1371/journal.pone.0158104, Published by PLoS, Lawrence, Kan.

Cysteine-rich FGF receptor (CFR) was originally identified as a FGF2 receptor and found to be identical to Golgi complex-localized glycoprotein-1 (GLG1), also known as MG-160, and to a murine E-selectin ligand-1 (ESL-1). Although CFR is a 150-kDa integral membrane glycoprotein that is primarily located in the cis-medial Golgi complex, a substantial proportion of CFR is secreted but the underlying mechanism is unknown. CFR contains several possible furin-like proprotein convertase (PC) and matrix metalloproteinase cleavage sites. Cells expressing CFR were treated with the furin protease inhibitor decanoyl-Arg-Val-Lys-Arg-chloromethylketone (decCMK) or the MMP-inhibitor GM6001. In the presence of furin-like PC inhibitor, secretion of CFR was almost completely inhibited. Secretion was not affected by the GM6001 inhibitor. The secreted forms were further characterized by creating different mutant CFR proteins with N-terminal and C-terminal tags. Immunoblot analysis and immunofluorescence indicated, that successive endoproteolytical processing of CFR which takes place in the Golgi complex is essential for secretion. Secreted CFR bound to heparan sulphate proteoglycan (HSPG) could trap FGFs and thereby directly competing with tyrosine kinase receptors for FGF binding.

Leukocytes and tumor cells use E-selectin binding ligands to attach to activated endothelial cells expressing E-selectin during inflammation or metastasis. The cysteine-rich fibroblast growth factor receptor (CFR) represents the main E-selectin ligand (ESL-1) on granulocytes and its expression is exclusively modified by alpha(1,3)-fucosyltransferases IV or VII (FucT4 and FucT7). Hepatic stellate cells (HSC) are pericytes of liver sinusoidal endothelial cells. The activation of HSC and transdifferentiation into a myofibroblastic phenotype is involved in the repair of liver tissue injury, liver regeneration and angiogenesis of liver metastases. In the present study, we demonstrated that HSC expressed CFR together with FucT7 and exhibited a functional E-selectin binding activity on their cell surface. Since HSC appear to be oxygen-sensing cells, the expression of E-selectin binding activity was analyzed in HSC under a hypoxic atmosphere. While the expression of the glycoprotein CFR was unaffected by hypoxia, the cell-associated E-selectin binding activity decreased. However, under the same conditions, mRNA expression of the modifying enzyme FucT7 increased. The loss of E-selectin binding activity, therefore, appears to be neither the result of a reduced expression of the modifying transferase nor the expression of the backbone glycoprotein. After the transient transfection of HSC with CFR cDNA, the E-selectin binding activity (ESL-1) was efficiently released into the supernatant. Therefore, we hypothesize that under hypoxia, ESL-1 is shed from activated HSC. Our findings provide a novel perspective on the function of HSC in liver metastasis and inflammatory liver diseases.

Genetic risk factors play an important role for the progression of liver fibrosis in chronic hepatitis C virus (HCV) infection, but functional data on specific alleles and their related proteins are limited. Platelet-derived growth factor BB (PDGF-BB) is one of the strongest mitogens for hepatic stellate cells and is considered as a critical soluble mediator of liver fibrosis in vitro and in vivo. The biological activity of PDGF-BB is dependent on its degradation by the factor VII–activating protease (FSAP). Here, we demonstrate that a coding polymorphism (G534E) in the gene for FSAP is significantly associated with severe HCV-induced liver fibrosis (odds ratio, 2.59; P = 0.017), which is independent of age, gender, and presence of diabetes in multivariate analysis. These genetic findings were replicated in a cohort of patients with liver transplantation due to HCV-induced cirrhosis (OR, 2.56; P = 0.011). Functional dissection of the association demonstrates that the single amino acid change encoded by G534E in the FSAP protein does not influence PDGFβ receptor or α-smooth muscle actin expression but completely abrogates FSAP-mediated inhibition of PDGF-BB–induced proliferation of primary stellate cells in vitro. Conclusion: The G534E variant of FSAP is a risk locus for HCV-induced liver fibrosis and cirrhosis by determining PDGF-BB–mediated hepatic stellate cell proliferation through a single amino acid substitution in FSAP. FSAP G534E might be useful for risk stratification in patients with HCV infection. (HEPATOLOGY 2009.)

Background Adult-type hypolactasia is a genetically determined inability to digest lactose after weaning. Two single-nucleotide polymorphisms ( C-13910T , G-22018A ) located upstream of the lactase gene ( LCT ) within the gene MCM6 are associated with the lactase persistence/non-persistence trait in patients of European descent. Therefore, the genotyping of these SNPs has been established as a diagnostic tool for adult-type hypolactasia. We have recently shown that several novel allelic variants located in close proximity to the C-13910T SNP interfere with the diagnostic accuracy of real-time PCR-based genotyping methods. Methods We describe here the validation of a comprehensive reverse-hybridization teststrip-based assay for the detection of common and novel LCT SNPs ( C-13907G , C-13910T , T-13913C , G-13914A , T-13915G , and G-22018A ). This assay is based on multiplex DNA amplification and ready-to-use membrane teststrips containing variant-specific oligonucleotide probes immobilized as an array of parallel lines. Results We evaluated the novel reverse-hybridization StripAssay on 125 DNA samples in comparison to LightCycler analysis and sequencing. The outcome of StripAssay genotyping was found to be completely concordant with that obtained by sequencing. Conclusions The StripAssay represents an accurate and robust screening tool to identify multiple LCT/MCM6 variants in a rapid manner. It overcomes diagnostic pitfalls that were reported and allows the simultaneous genotyping of closely spaced LCT variant sites in a single-step diagnostic approach .

Hepatic stellate cells (HSCs) are pericytes of liver sinusoidal endothelial cells (LSECs) and activation of HSC into a myofibroblast-like phenotype (called transdifferentiation) is involved in several hepatic disease processes including neovascularization during liver metastasis, chronic and acute liver injury. While early smooth muscle cell (SMC) differentiation markers including SM alpha-actin and SM22alpha are expressed in a variety of non-SMC, expression of late-stage markers is far more restricted. Here, we found that in addition to early SMC markers, activated rat HSC express a large panel of characteristic late vascular SMC markers including SM myosin heavy chain, h1-calponin and h-caldesmon. Furthermore, myocardin, which is present exclusively in SMCs and cardiomyocytes and controls the transcription of a subset of early and late SMC markers, is highly expressed in activated HSC. We further studied activated HSC in a functional three-dimensional spheroidal co-culture system together with endothelial cells (EC). Co-culture spheroids of EC and SMC differentiate spontaneously and organize into a core of SMC and a surface layer of EC representing an inside-outside model of the physiological assembly of blood vessels. Replacing SMC by in vitro activated HSC resulted in a similar organized spheroid with differentiated, von-Willebrand factor producing, surface lining quiescent human umbilical vein endothelial cell and a core of HSC. In an in vitro angiogenesis assay, activated HSC induced quiescence in vascular EC-the hallmark of vascular SMC function. Co-spheroids of LSEC and activated HSC formed capillary-like sprouts in gel angiogenesis assays expressing the vascular EC marker VE-cadherin. Our findings indicate that activated HSC are capable to adapt a functional SMC phenotype and to induce formation of tubular sprouts by LSEC and vascular endothelial cells. Since tumors and tumor metastasis induce HSC activation, HSC may take part in tumor-induced neoangiogenesis by adapting SMC-like functions.

There is growing evidence that hypertensive pregnancy complications and other adverse pregnancy outcomes are associated with the presence of inherited or acquired thrombophilias. As hemolysis, elevated liver enzymes, low platelets (HELLP) syndrome is one of the most severe forms of pre-eclampsia we aimed to assess the prevalence of the factor V Leiden, the prothrombin 20210GA mutation and the methylenetetrahydrofolate reductase (MTHFR) 677CT polymorphism in women with HELLP syndrome and in their fetuses from the same index pregnancy.The study was performed retrospectively in a case-control design.Seventy-one mother-child pairs with HELLP syndrome and 79 control mother-child pairs with uncomplicated pregnancies were included in the study.Genotyping of the three thrombophilic mutations was performed using the LightCycler technology. The chi-squared test was used for statistical analysis. Main outcome measures were maternal and fetal genotypes and their correlation with clinical parameters.Maternal heterozygosity for factor V Leiden was significantly more prevalent in the HELLP group than in controls (OR 4.45, 95% CI 1.31-15.31). No significant association was observed for maternal prothrombin mutation or MTHFR polymorphism (p=0.894, p=0.189, respectively). The fetal genotype was not associated with HELLP syndrome for any of the three mutations investigated. Analysis of gene-gene interactions and genotype-phenotype correlation with respect to clinical parameters and perinatal outcome revealed no further differences.Our study confirms that women heterozygous for factor V Leiden have an increased risk of developing HELLP syndrome, while the most frequent mutations of the prothrombin and MTHFR gene do not play a major role in the pathogenesis of HELLP syndrome.

Background: Patients presenting with symptoms of lactose intolerance are in some centres routinely tested for a single-nucleotide polymorphism C-13910T, which is located upstream of the lactase gene (LCT) and is tightly associated with genetically determined lactase persistence/non-persistence. Typing of this polymorphism enables differential diagnosis for genetic versus secondary causes of lactose intolerance. Several PCR-based methods have been established as tests for this SNP. In particular, automated genotyping assays conducted on LightCycler platforms provide a rapid, labour-saving means for routine high-throughput analysis of this variant. Recently, several novel allelic variants have been identified in non-European populations. Three of these variants occur in close proximity to C-13910T, but their effect on the genetic test is unknown.Methods: Here we analyse whether the occurrence of C-139076, T-13913C, and T-13915G, affect the diagnostic accuracy of C-13910T typings obtained using the LightCycler MutaREAL lactase real-time PCR kit.Results: Genotyping of DNA samples harbouring respective variants or combinations thereof significantly influenced the LightCycler analysis. Some allelic combinations generated melting profiles that prevented the correct assignment of C-13910T.Conclusions: We conclude that genotyping of the C-13910T variant with the MutaREAL lactase real-time PCR kit in non-Europeans is prone to error and should be omitted. (c) 2007 Elsevier B.V. All rights reserved.

The common −13910T/C exchange located 13910-bp upstream of the lactase gene ( LCT ) is associated with the persistence/nonpersistence trait of adult-type hypolactasia, which leads to symptoms of lactose malabsorption, including bloating, flatulence, diarrhea, and abdominal pain. The typing of this variant is used as a genetic test for lactase (non)persistence. We used the MutaReal Lactase real-time PCR test (Immundiagnostik) with the Roche LightCycler capillary system to analyze genomic DNA and observed in 1 sample an aberrant melting curve profile attributable to a novel base replacement, −13914G>A, in the vicinity of the common −13910T/C lactase gene variation. Lactose intolerance results primarily from the physiological decline in activity of the LCT gene-encoded enzyme lactase-phorozin hydrolase, which breaks down milk sugar. Use of linkage disequilibrium and haplotype analysis of affected Finnish families revealed a defined DNA transition (−13910T/C) proximal to the LCT gene in intron 13 of the adjacent minichromosome maintenance type 6 gene ( MCM6 ), which is associated with verified lactase nonpersistence and reduced bone mineral density predisposing for bone fractures (1)(2). Persons homozygous …

Pleiotrophin (PTN) is a secretory heparin binding protein with various biological activities including mitogenesis, angiogenesis, and tissue repair after injury. Recent studies have shown that PTN is a strong mitogen of hepatocytes and involved in liver regeneration. In adult liver cells Ptn gene is mainly expressed by quiescent hepatic stellate cells (HSCs). Although we have been able to demonstrate mRNA and protein expression of the anaplastic lymphoma kinase-the receptor tyrosine kinase for PTN-on HSCs, PTN did not act as a mitogen of HSCs in contrast to hepatocytes. PTN immunoreactivity was markedly increased in experimental fibrogenesis by common bile duct ligation and observed in sinusoidal HSCs. In primary HSC cultures, Ptn transcription was significantly increased by PDGF-BB, and under hypoxic atmosphere. Mechanistically, hypoxia and PDGF mediated induction of PTN expression in sinusoidal HSCs may provide a strong mitogenic signal for hepatocytes to limit the damage to the parenchymal cells in biliary-type liver fibrogenesis.

Fibroblast growth factors (FGFs) are important angiogenic growth factors. While basic FGF (FGF2) is well established as a potent inducer of angiogenesis much less is known about other FGFs possibly expressed by EC. We investigated the expression of all known FGFs, their main tyrosine kinase receptors and antagonists by RT-PCR analysis in human umbilical vascular endothelial cells (HUVECs) to obtain a complete expression profile of this important growth factor system in model endothelial cells (EC). In addition to FGFR1IIIc, which is considered as the major FGF receptor in EC, HUVECs express similar levels of FGFR3IIIc, detectable amounts of FGFR2IIIc and a new FGF receptor without an intracellular kinase domain (FGFR5). HUVECs express several secreted FGFs, including FGF5, 7, 8, 16 and 18 and two members of the fibroblast growth factor homologous factors (FHFs), not yet reported to be expressed in EC. The expression panel was compared with that obtained from human vascular smooth muscle cells (VSMCs) and human aortic tissue. Human umbilical artery smooth muscle cells (HUASMCs) and HUVECs express the identical FGF receptor and ligand panel implicating that both cell types act, according the FGF signals more as an entity than as individual cell types. Expression of Fgf1, 2, 7, 16 and 18 and the antagonists Sprouty 2,3 and 4 was demonstrated for all analysed cDNAs. The IIIc isoforms of FGFR1 and 2 and the novel FGFR5 were expressed in the aorta, but expression of the FGF receptor 3 was not detected in cDNAs derived from aortic tissue. In the VSMC of rat aortic tissue and in HUASM cultured cells we could demonstrate FGF18 immunoreactivity in the nucleus of the cells. The expression of several secreted FGFs by EC may focus the view more on their paracrine effects on neighbouring cells during tissue regeneration or tumor formation.

AIM: Considerable attention is focused on polymorphisms in the gene encoding transforming growth factor-β1 (TGF-β1), a multifunctional cytokine that is in turn a potent growth inhibitor involved in wound healing and differentiation. In humans, it promotes the pathogenesis of organ fibrosis, atherosclerosis, cancer, autoimmune and inflammatory diseases, keloid disease, and hypertrophic scarring. For this reason, much emphasis has been placed on studies elucidating the impact of TGF-β1 and its gene variations for the susceptibility and pathogenesis of these diseases. Unfortunately, some studies have serious limitations. METHODS: We have recently described a high-throughput method for investigation the Arg25Pro polymorphism of human TGF-β1 gene and showed that the frequency of the Pro25 allele is significantly associated with hepatic fibrogenesis. In this report, we describe two novel LightCycler (LC) techniques that facilitate the examination of the two other known alterations in the coding region of TGF-β1. We investigated whether these polymorphisms contribute to hepatitis-induced progression of fibrogenesis in Chinese and Caucasians. RESULTS: In the Chinese ancestry, the gene polymorphisms at codons 25 and 263 were not found and the genetic variant at codon 10 is unlikely to confer susceptibility to hepatic fibrosis. Contrarily, in Caucasians TGF-β1 allelic variations are more frequent and the presence of prolines either in codon 25 or 10 is associated with the interindividual variability in developing more severe fibrosis during chronic hepatitis C infection. CONCLUSION: In summary, these results confirm the hypothesis that TGF-β1 polymorphisms are associated with fibrosis progression in Caucasians chronically infected with hepatitis C.

The polymorphism at position 25 of the gene encoding transforming growth factor-beta1 (TGF-beta1), which changes the amino acid sequence of the signal peptide sequence (arginine to proline), is causing a variation in TGF-beta1 production. The homozygous genotype (Arg25Arg) is associated with higher TGF-beta1 production than the heterozygous (Arg25Pro) genotype. Therefore, the possible involvement of this genetic variation in the TGF-beta1 gene for induction and progression of various diseases is under close investigation. At present, several labor-intensive established assays ranging from amplification refractory mutation system (ARMS)-PCR methodologies, sequence specific oligonucleotide probing (SSOP), restriction fragment length polymorphism (RFLP) analysis, 5' nuclease assays, and specialized fingerprinting protocols are applied to analyze the polymorphism in question. We developed a novel approach for analyzing this polymorphism in a LightCycler system and determined the allele frequency distributions between patients with different degrees of hepatic fibrosis induced by chronic hepatitis C virus infection. In patients with severe hepatic fibrosis (METAVIR-score 3-4), the Pro25 allele was twice as frequent compared to patients with mild fibrosis (METAVIR-score 0-2). However, we found no association of necroinflammatory activity and genotype distribution. This suggests that the stage of hepatic fibrosis, rather than the grade (inflammation), is influenced by the presence of proline at codon 25 in patients with chronic hepatitis C.

Transdifferentiation of hepatic stellate cells (HSC) to collagen producing myofibroblasts (MFB) is a principal event in liver fibrogenesis. In our studies we investigated if tissue transglutaminase (tTG) from these cell types may play a role in liver fibrosis. Separation of cytosol and membrane components showed membrane associated tTG and during transdifferentiation an upregulation of total tTG on mRNA and protein level was found, but no modulation during stimulation with TGF-β1. In HSC and fully differentiated MFB a significant amount of the total tTG synthesised during transdifferentiation is found to be membrane-associated whereas the remaining portion is cytosol-associated and only very little is found within the extracellular matrix (ECM). The data implicate that tTG in this cell type seems to play an important role in liver fibrogenesis.

Objectives: Real time polymerase chain reaction followed by melting curve analysis using hybridization probes has become an important tool in routine diagnosis of the HFE mutations, which are associated with hereditary hemochromatosis. Design and methods: We used the LightCycler technology for simultaneous detection of the H63D and C282Y mutations of the HFE gene in patients with a higher prevalence for hemochromatosis. Results: In our cohort we identified two siblings with a variant pattern of the HFE -LightCycler melting profiles preventing allelic discrimination. Conclusions: As a consequence, in these patients DNA sequencing or RFLP analysis is necessary to unequivocally assign the correct HFE genotype.

Carriers of the G20210A allele have high plasma prothrombin concentrations, which in turn are associated with an almost 3-fold increased risk of venous thrombosis (1). As it is the second most common genetic change associated with inherited thrombophilia, the variant site in the 3′ untranslated region is routinely examined in at-risk patients. We use the factor II (prothrombin) G20210A assay (Roche Diagnostics) for genotyping. In this assay, a 165-bp fragment of the prothrombin gene is amplified, and the different allelic variants are distinguished by melting-curve analysis using the fluorescence resonance energy transfer principle. The wild-type and variant alleles have melting peaks at ∼59 ± 2.5 °C and ∼49 ± 2.5 °C, respectively. Heterozygous samples exhibit a distinct combination of both melting peaks. We have genotyped genomic DNA from more than 2000 patients. Of these, 97% were homozygous wild type, ∼3% were heterozygous, and