Carmen G. Tag

Expression pattern of fibroblast growth factors (FGFs), their receptors and antagonists in primary endothelial cells and vascular smooth muscle cells.

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.

Bile Duct Ligation in Mice: Induction of Inflammatory Liver Injury and Fibrosis by Obstructive Cholestasis

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.

Hepatic stellate cells display a functional vascular smooth muscle cell phenotype in a three‐dimensional co‐culture model with endothelial cells

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.

The Marburg I variant (G534E) of the factor VII–activating protease determines liver fibrosis in hepatitis C infection by reduced proteolysis of platelet‐derived growth factor BB

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.)

Primary cultures of glomerular parietal epithelial cells or podocytes with proven origin.

Parietal epithelial cells (PECs) are crucially involved in the pathogenesis of rapidly progressive glomerulonephritis (RPGN) as well as in focal and segmental glomerulosclerosis (FSGS). In this study, transgenic mouse lines were used to isolate pure, genetically tagged primary cultures of PECs or podocytes using FACsorting. By this approach, the morphology of primary glomerular epithelial cells in culture could be resolved: Primary podocytes formed either large cells with intracytoplasmatic extensions or smaller spindle shaped cells, depending on specific culture conditions. Primary PECs were small and exhibited a spindle-shaped or polygonal morphology. In the very early phases of primary culture, rapid changes in gene expression (e.g. of WT-1 and Pax-2) were observed. However, after prolonged culture primary PECs and podocytes still segregated clearly in a transcriptome analysis - demonstrating that the origin of primary cell cultures is important. Of the classical markers, synaptopodin and podoplanin expression were differentially regulated the most in primary PEC and podocyte cultures. However, no expression of any endogenous gene allowed to differentiate between the two cell types in culture. Finally, we show that the transcription factor WT1 is also expressed by PECs. In summary, genetic tagging of PECs and podocytes is a novel and necessary tool to derive pure primary cultures with proven origin. These cultures will be a powerful tool for the emerging field of parietal epithelial cell biology.

Maternal factor V Leiden mutation is associated with HELLP syndrome in Caucasian women

Objective. 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 20210G >A mutation and the methylenetetrahydrofolate reductase (MTHFR) 677C >T polymorphism in women with HELLP syndrome and in their fetuses from the same index pregnancy. Design. The study was performed retrospectively in a case–control design. Sample. Seventy‐one mother–child pairs with HELLP syndrome and 79 control mother–child pairs with uncomplicated pregnancies were included in the study. Methods. 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. Results. 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. Conclusions. 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.

Evaluation of a novel reverse-hybridization StripAssay for typing DNA variants useful in diagnosis of adult-type hypolactasia

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.

Isolation and Time Lapse Microscopy of Highly Pure Hepatic Stellate Cells

Hepatic stellate cells (HSC) are the main effector cells for liver fibrosis. We aimed at optimizing HSC isolation by an additional step of fluorescence-activated cell sorting (FACS) via a UV laser. HSC were isolated from livers of healthy mice and animals subjected to experimental fibrosis. HSC isolation by iohexol- (Nycodenz) based density centrifugation was compared to a method with subsequent FACS-based sorting. We assessed cellular purity, viability, morphology, and functional properties like proliferation, migration, activation marker, and collagen expression. FACS-augmented isolation resulted in a significantly increased purity of stellate cells (>99%) compared to iohexol-based density centrifugation (60–95%), primarily by excluding doublets of HSC and Kupffer cells (KC). Importantly, this method is also applicable to young animals and mice with liver fibrosis. Viability, migratory properties, and HSC transdifferentiation in vitro were preserved upon FACS-based isolation, as assessed using time lapse microscopy. During maturation of HSC in culture, we did not observe HSC cell division using time lapse microscopy. Strikingly, FACS-isolated, differentiated HSC showed very limited molecular and functional responses to LPS stimulation. In conclusion, isolating HSC from mouse liver by additional FACS significantly increases cell purity by removing contaminations from other cell populations especially KC, without affecting HSC viability, migration, or differentiation.

An unusual melting curve profile in LightCycler multiplex genotyping of the hemochromatosis H63D/C282Y gene mutations

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.

Induction of experimental obstructive cholestasis in mice

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.