Vascularization and gene regulation of human endothelial cells growing on porous polyethersulfone (PES) hollow fiber membranes.

Open-cell hollow fibers made of polyethersulfone (PES) manufactured in the absence of solvents with pore diameters smaller than 100 microm were examined for vascularization by human endothelial cells. The goal of this study was to determine whether the 3-D porous character of the PES surface affected human endothelial cell morphology and functions. Freshly isolated human endothelial cells from the skin (HDMEC), from the lung (HPMEC) and from umbilical cords (HUVEC) and two human endothelial cell lines, HPMEC-ST1.6R and ISO-HAS.c1 were added to PES fibers and cell adherence and growth was followed by confocal laser scanning microscopy. Prior coating of PES with gelatin or fibronectin was necessary for adhesion and spreading of cells over the uneven porous surface with time. Confluent cells exhibited typical strong PECAM-1 expression at cell-cell borders. Little expression of the activation markers E-selectin, ICAM-1, and VCAM-1 was observed by RT-PCR of endothelial cells growing on PES. However, after stimulation for 4h by LPS, activation of these markers was observed and it was shown by immunofluorescent staining that induction occurred in most of the cells, thus confirming an intact functionality. Finally, cells growing as a monolayer on PES migrated to form microvessel-like structures when placed under conditions that stimulated angiogenesis. Thus, human endothelial cells grown on fibronectin-coated PES fibers retain important endothelial-cell specific morphological and functional properties and PES may serve as a useful biomaterial in tissue engineering and biotechnology applications.

Generation and characterization of transgenic mice expressing cobra venom factor.

Cobra venom factor (CVF), the anticomplementary protein in cobra venom, activates the alternative complement pathway, eventually leading to complement consumption. Here, we describe the development of a transgenic mouse model for CVF. We generated a DNA construct containing the full-length cDNA for single-chain pre-pro-CVF. Expression of CVF was controlled by the alpha(1)-antitrypsin promoter to achieve liver-specific expression. Linearized DNA was microinjected into murine ovary cells (strain CD(2)F(1) (BALB/cxDBA/2J)) and the newborn mice were analyzed for stable integration of CVF DNA. After establishing the transgene, mice were propagated in a BALB/c background. The CVF mRNA was detected in the liver and, in some animals, in the kidney. CVF protein was detected in small amounts in the serum. Serum complement hemolytic activity in CVF-transgenic mice was virtually absent. The concentration of plasma C3 was significantly reduced. The CVF-transgenic animals show no unusual phenotype. They provide an animal model to study the effect of long-term complement depletion by continued activation, as well as the role of complement in host immune response and pathogenesis of disease.

Cell cycle synchronization of porcine fetal fibroblasts by serum deprivation initiates a nonconventional form of apoptosis.

The success of somatic nuclear transfer depends upon the cell cycle stage of the donor nucleus and the recipient cytoplast. Recently, we established efficient cell cycle synchronization protocols for porcine fetal fibroblasts and found that serum withdrawal leads to cell death. Here, we examined whether the specific cell death induced by serum deprivation follows the conventional apoptotic pathway in porcine fibroblasts. Terminal deoxynucleotidyl transferase mediated dUTP nick end-labeling analysis revealed that serum deprivation induced DNA fragmentation in a concentration and time dependent manner. Semiquantitative RT-PCR and Western blotting revealed activation of cell death-related genes Bak and Bax of the Bcl-2 family. However, electrophoretic analysis of genomic DNA from serum deprived cells did not provide evidence for the internucleosomal DNA cleavage which is characteristic of conventional apoptosis. Thus, serum deprivation triggers initial steps in the apoptotic pathway, but does not lead to the typical oligonucleosome-sized DNA ladder. These findings contribute to a better understanding of apoptotic pathways and aid to define essential parameters of the donor nucleus for successful somatic cloning.

Liver-specific physiology of immortal, functionally differentiated hepatocytes and of deficient hepatocyte-like variants.

Five different immortalized transgenic hepatocyte cell lines derived from mice were investigated with respect to their potential to maintain the physiological properties of primary hepatocytes using chemically defined medium. This research completes a previous study by Klocke and coworkers in 2002, using gene expression analysis of the same cell lines by the respective physiological analysis for investigating the hepatocyte-like function. Three transgenic cell lines harboring a fusion gene derivative (construct 202) consisting of the complete SV40 early region, including the coding sequences for the transforming large and small tumor antigens, placed under the control of the murine metallothioneine 1-promotor/enhancer element, showed a hepatocyte-like function and physiology. They grew as a monolayer with a polygonal cell shape, consumed lactate, and secreted albumin at a cell-specific rate of 1.5 pg/h, which is in the range of primary hepatocytes. In addition, the potential of detoxifying ammonium could be maintained. Ammonium was metabolized and urea was produced and released into the medium. A complete urea cycle could be determined. A cell line established from neonatal transgenic mice and expressing a secretory variant of the human epidermal growth factor (IgEGF) under the control of the albumin promoter was characterized by an incomplete urea cycle. Another cell line isolated from the liver of homozygote neonatal p53-knockout mice showed no hepatocyte-specific functions but only properties of continuous cell lines. Specific nucleoside triphosphate (NTP) and uridine (U) ratios were used to characterize the differentiation status of the particular cell lines. A low NTP-U value was found for the three cell lines containing construct 202, which was identical to that observed for primary hepatocytes. In contrast, the cell line harvested from the liver of homozygote neonatal p53-knockout mice presented a NTP-U ratio characteristic for continuous cell lines. This study demonstrates that the four transgenic and the p53-knockout hepatocyte-derived cell lines can be used as models for investigating the conservation of tissue-specific functions in immortalized cells.

Remodeling of fibrinogen by endothelial cells in dependence on fibronectin matrix assembly. Effect of substratum wettability.

The endothelization of cardiovascular implants is desirable to improve their blood compatibility. The capacity of the endothelial cells to attach, migrate, proliferate and function on the implant surface depends on the presence of matrix proteins such as fibronectin (FN) and fibrinogen (FNG). In this study, we show that the deposition of fibrinogen into extracellular matrix-like structures by human umbilical vein endothelial cells (HUVEC) is dependent on FN matrix formation. We found further that the process of organization of both adsorbed and soluble FN and FNG is dependent on the wettability of materials since it was observed only on a hydrophilic and not on a hydrophobic model surface. beta(3) integrin was involved in the process of cell attachment to adsorbed FNG, while the mechanism of FNG fibrillogenesis required the activity of the beta(1) integrin. Studies of EC morphology showed the predominant peripheral organization of actin filaments and the formation of distinct leading and trailing cell edges suggesting a motile phenotype of cells when they are seeded on FNG. In summary, we concluded that adsorbed fibrinogen may enhance the motility of HUVEC and that soluble FNG requires FN matrix assembly to be organized in fibrilar structures.

Fibrinogen adsorption and platelet interactions on polymer membranes.

The hemocompatibility of four different wettable polymer membranes, namely Cuprophan (CE), polyether-polycarbonate (PC-PE), polysulfone (PSU), and polyetherimide (PEI), was investigated with respect to fibrinogen (Fng) adsorption and platelet adhesion/activation. In order to estimate the polar and dispersion components of the surface free energy, contact angles using water/vapor and water/n-hexadecane systems were measured. Adsorption of fibrinogen was studied using fluorescence-labeled protein. The adsorption isotherms showed that the amount and the affinity of adsorbed Fng increased with decreasing surface wettability of the membranes, which correlates with the dispersion and polar components of the surface free energy. The conformational changes of adsorbed Fng were detected by measuring the difference between monoclonal antibody binding to the conformation-sensitive epitope in the D-domain and the binding of polyclonal anti-Fng antibody. The anticipated conformational/orientational changes were greater for PEI and PSU membranes (the least wettable membranes) and negligible for the more wettable PC-PE and CE membranes. In addition, a possible relationship with the degree of platelet activation was found, showing negligible platelet adhesion on PC-PE and CE, but high platelet adhesion on PEI and PSU. Furthermore, platelets were spread to a large extent on PEI, while the formation of aggregates was observed on PSU. This may correspond to the anticipated differences in the conformational state of Fng on both membranes.

Progressive paralysis associated with diffuse astrocyte anaplasia in delta 202 mice homozygous for a transgene encoding the SV40 T antigen.

A convenient transgenic astrocytoma model in delta202 mice, homozygous for a construct encoding the early region of the SV40 virus genome, is described. In the offspring of crosses between delta202 mice heterozygous for the transgene nearly 60% were transgenic; one third of these developed progressive paralysis starting in the hindlimbs at approximately 35 days of age and died at 90 +/- 30 days of age. In affected mice proliferating-non-neuronal cells immunostained with antibodies to the GFAP, an astrocyte marker, whose number increased with age were found in the white matter of the brain, cerebellum and spinal cord, and progressive degeneration and necrosis of spinal motoneurons was observed that-may explain the paralysis. The early onset and reproducible time course of the neurological disease suggest that homozygous delta202 mice, whose proliferating astrocytes appear to damage spinal motoneurons, are a useful model to study astrocyte differentiation, function and tumorigenesis.

Interaction of human skin fibroblasts with moderate wettable polyacrylonitrile--copolymer membranes.

The development of a bioartificial skin is a step toward the treatment of patients with deep burns or nonhealing skin ulcers. One possible approach is based on growing dermal cells on membranes to obtain appropriate living cellular stroma (sheets) to cover the wound. New membrane-forming copolymers were synthesized, based on acrylonitrile (AN) copolymerization with hydrophilic N-vinylpyrrolidone (NVP) monomer, in different percentage ratios, such as 5, 20, and 30% w/w, and with two other relatively high polar comonomers--namely, sodium 2-methyl-2-propene-1-sulfonic acid (NaMAS) and aminoethylmethacrylate (AeMA). All these copolymers were characterized for their bulk composition and number average molecular weight, and used to prepare ultrafiltration membranes. Water contact angles and water uptake were estimated to characterize the wettability and scanning force microscopy to visualize the morphology of the resulting polymer surface. Cytotoxicity was estimated according to the international standard regulations, and the materials were found to be nontoxic. The interaction of the membranes with human skin fibroblasts was investigated considering that these cells are among the first to colonize membranes upon implantation or with prolonged external contact. The overall cell morphology, formation of focal adhesion contacts, and cell proliferation were estimated to characterize the cell material interactions. It was found that the pure polyacrylonitrile homopolymer (PAN) membrane provides excellent conditions for seeding with fibroblasts, comparable only to a copolymer containing AeMA. In contrast, the presence of NaMAS with acidic ionic groups decreased both the attachment and proliferation of fibroblasts. Low content of NVP in the copolymer, up to about 5%, still enabled good attachment and spreading of cells, as well as subsequent proliferation of fibroblasts, but higher ratios of 20 and 30% resulted in a significant decrease of these cellular activities.

entla, a novel epileptic and ataxic Cacna2d2 mutant of the mouse.

entla (ent) is a novel recessive phenotype of mice. The underlying mutation was mapped to chromosome 9 (60.1 centimorgans) and identified as an allele of the Cacna2d2 gene encoding the alpha2delta-2 subunit of voltage-gated calcium channels. The Cacna2d2entla allele harbors a 38-kb duplication comprising the 117 nucleotides of exon 3. The predicted duplication of 39 amino acid residues near the subunit's N terminus results in the expression of a full-length, membrane-associated protein. Western blot data were consistent with correct cleavage of the alpha2delta-2entla precursor into alpha2entla and delta2 proteins but indicated loss of the disulfide linkage between the two proteins. ent/ent mice develop ataxia by postnatal day 13-15, followed by paroxysmal dyskinesia a few days later. Two distinct types of cortical and hippocampal epileptic activity at 2 and 4 Hz were recorded, indicative of absence epilepsy. Homozygotes display reduced size and weight, increased mortality before weaning, and female infertility. No overt neuroanatomical abnormalities were detected. Ca2+ current densities recorded from acutely dissociated Purkinje cells of homozygous entla animals were reduced by 50% compared with wild type. Ligand binding assays using the antiepileptic drug [3H]gabapentin, a specific ligand of the alpha2delta-1 and alpha2delta-2 subunits, revealed a >60% reduced maximum binding to cerebellar membranes of ent/ent compared with unaffected littermates. entla is allelic to ducky and ducky2J, representing the third murine Cacna2d2 allele identified and so far the only one encoding an untruncated protein that is incorporated into membranes.

Establishment and characterization of immortal hepatocytes derived from various transgenic mouse lines.

The potential of three genetic changes introduced into mice by the transgenic or knockout technology aimed at immortalizing hepatocytes in vitro and concomitantly preserving their differentiated hepatic functions was analyzed. Six hepatocyte lines were isolated from neonatal and adult transgenic mice expressing either IgEGF (a secretable variant of hEGF) or SV40 T antigen in the liver and from neonatal and adult p53 knockout (KO) mice and have been subcultured >150 times in serum-free, arginine-deficient medium. Only in SV40 T antigen transgenic lines profiles of mRNAs encoding serum proteins, transcription factors, and liver-specific enzymes were similar to those found in livers and primary hepatocytes. Accordingly, these cells displayed basal and inducible expression of CYP proteins as well as testosterone metabolizing activities. Thus, either knockout of the p53 gene or expression of SV40 T antigen or of IgEGF imparts immortality to hepatocytes in vitro, but only SV40 T antigen expression is compatible with the concomitant long-term preservation of differentiated liver functions.

Determination of thiopurine methyltransferase phenotype in isolated human erythrocytes using a new simple nonradioactive HPLC method.

Genetic polymorphism of the S-methylation pathway catalyzed by thiopurine methyltransferase (TPMT) is responsible for variation in the metabolism, toxicity, and therapeutic efficacy of thiopurine drugs. This paper describe a new simple, nonradioactive HPLC method for determination of TPMT activity in isolated erythrocytes (Ery), based on the conversion of 6-mercaptopurine (pH 7.5, 37 degrees C) to 6-methylmercaptopurine (6-MMP) using S-adenosyl-l-methionine as methyl donor. The incubation step was stopped by a mixture of trichloroacetic acid/acetonitrile containing the internal standard 4-aminoacetophenone. 6-MMP was quantified by absorbance at 290 nm after chromatographic separation on a Zorbax SB-Phenyl column (5 microm, 4.6 x 250 mm) using mobile phases (flow rate 1.1 mL/min) consisting of acetonitrile, phosphate buffer pH 3.0, triethylamine, and dithiothreitol. The assay was linear up to 50 nmol/(mL Ery. h), and the detection limit was 0.3 nmol/(mL Ery. h). The extraction efficiency of 6-MMP was 95-103% (n = 3), and its analytic recovery ranged between 98.3% and 101.8% (n = 12). The within-day imprecision using pooled human erythrocytes (n = 12) was 4.4% at a TPMT activity of 14.3 nmol/(mL Ery.h) and 4.9% at 6.5 nmol/(mL Ery.h). The between-day imprecision (n = 12) was 6.8% and 7.5% nmol/(mL Ery.h), respectively. A very good agreement was found between TPMT activity determined with this method (y) and a widely used radiochemical procedure (x) (r = 0.94; n = 130; y = 0.502 + 0.946x; P < 0.05). Genotype analysis of all individuals with TPMT activity under 12.5 nmol/(mL Ery.h) revealed a genotype/phenotype concordance of 86%. The new HPLC method for determination of TPMT activity in Ery is a simple, rapid, and reliable nonradioactive procedure that can be successfully used for both research and routine clinical analysis.