Inhibition of neo-intimal hyperplasia in porcine coronary arteries utilizing a novel paclitaxel-coated scoring balloon catheter.

OBJECTIVE: Scoring balloons are particularly useful in the acute treatment of fibro-calcific, bifurcation and in-stent restenosis lesions but have not been shown to affect the restenosis rate. Conventional balloons coated with paclitaxel have recently been shown to reduce restenosis rates in certain lesion subsets, but are associated with suboptimal acute results. A novel paclitaxel-coated scoring balloon was developed to overcome these limitations. DESIGN: AngioSculpt(®) scoring balloons (SB) were coated with paclitaxel admixed with a specific excipient. SETTING AND INTERVENTIONS: Four in vitro and in vivo studies were performed: (a) loss of the drug during passage to the lesion, (b) transfer of the drug to the vessel wall; (c) inhibition of neo-intimal proliferation in porcine coronary arteries as compared to uncoated SB and the Paccocath™, and (d) evaluation of the dose-response to 1.5-12 µg of paclitaxel/mm(2) . MAIN OUTCOME MEASURES AND RESULTS: Drug loss during delivery to the lesion was 17% ± 8%, and transfer to the vessel wall was 9% ± 4% of dose on unused balloons. The paclitaxel-coated SB resulted in a lower late lumen loss of 0.27 ± 0.24 mm compared to 1.4 ± 0.7 mm with the uncoated SB (P = 0.001). Histomorphometry revealed larger luminal areas of 6.8 ± 1.6 mm(2) (paclitaxel-coated SB) and 5.8 ± 1.7 mm(2) (Paccocath) as compared to the uncoated SB (2.3 ± 1.5 mm(2) ; P = 0.001). No coating related adverse effects were observed on follow-up angiography or histologic examination at the treatment site or downstream myocardium. CONCLUSION: A novel paclitaxel-coated SB leads to a significant inhibition of neointimal proliferation in the porcine coronary model.

Functionalized self-assembling peptide hydrogel enhance maintenance of hepatocyte activity in vitro.

There is a major challenge in maintaining functional hepatocytes in vivo as these cells rapidly lose their metabolic properties in culture. In this work we have developed a bioengineered platform that replaces the use of the collagen I--in the traditional culture sandwich technique--by a defined extracellular matrix analogue, the self-assembling peptide hydrogel RAD16-I functionalized with biologically active motifs. Thus, after examining side by side the two culture systems we have found that in both cases hepatocytes acquired similar parenchymal morphology, presence of functional bile canaliculi structures, CYP3A2 induction by dexamethasone, urea production, secretion of proteins such as apolipoprotein (class A1, E, J), alpha(1)-microglobulin, alpha(1)-macroglobulin, retinol binding protein, fibronectin, alpha(1)-inhibitor III and biotin-dependent carboxylases. Interestingly, by assessing in more detail some other hepatic markers, one of the functionalized matrix analogues--carrying the 67 kD laminin receptor ligand--enhanced the gene expression of albumin, HNF4-alpha, MDR2 and tyrosine aminotransferase. We conclude that the use of a synthetic culture system with designed matrix functionalization has the advantage in controlling specific cellular responses.

Re-evaluation of tacrine hepatotoxicity using gel entrapped hepatocytes.

Controversial results about the involvement of CYP 1A2 and oxidative stress in tacrine-induced hepatotoxicity have been described by the different research groups. We suggested that different expression levels of CYP 1A2 in cell lines and primary hepatocytes in vitro may be the cause of the controversial results above. Therefore, this paper re-evaluated the toxicity of tacrine by using gel entrapment culture of rat hepatocytes. The toxic effect of tacrine on hepatocytes was assayed by the reduction of methyl thiazolyl tetrazolium (MTT) and intracellular glutathione (GSH), as well as by albumin synthesis. It was found that the detectable hepatotoxic dose of tacrine is much lower in hepatocytes entrapped in gel than in those in monolayer cultures. The fact that fluvoxamine, a potent cytochrome P450 (CYP) 1A2 inhibitor, reduced tacrine toxicity and the expression of the CYP 1A2 gene was maintained in gel entrapped hepatocytes, but not in hepatocyte monolayers, could illustrate a close association between CYP 1A2 expression levels and tacrine toxicity. Glycyrrhetinic acid (GA), a free radical scavenger, protected gel entrapped hepatocytes from tacrine toxicity, but was ineffective in hepatocyte monolayers. Hence, gel entrapped hepatocytes could reflect higher tacrine hepatotoxicity in vivo than hepatocyte monolayers.

Retargeting of adenoviral vector using basic fibroblast growth factor ligand for malignant glioma gene therapy.

OBJECT: Adenovirus vector (AdV)-mediated gene delivery has been recently demonstrated in clinical trials as a novel potential treatment for malignant gliomas. Combined coxsackievirus B and adenovirus receptor (CAR) has been shown to function as an attachment receptor for multiple adenovirus serotypes, whereas the vitronectin integrins (alphavbeta3 and alphavbeta5) are involved in AdV internalization. In resected glioma specimens, the authors demonstrated that malignant gliomas have varying levels of CAR, alphavbeta3, and alphavbeta5 expression. METHODS: A correlation between CAR expression and the transduction efficiency of AdV carrying the green fluorescent protein in various human glioblastoma multiforme (GBM) cell lines and GBM primary cell lines was observed. To increase transgene activity in in vitro glioma cells with low or deficient levels of CAR, the authors used basic fibroblast growth factor (FGF2) as a targeting ligand to redirect adenoviral infection through its cognate receptor, FGF receptor 1 (FGFR1), which was expressed at high levels by all glioma cells. These findings were confirmed by in vivo study data demonstrating enhanced transduction efficiency of FGF2-retargeted AdV in CAR-negative intracranial gliomas compared with AdV alone, without evidence of increased angiogenesis. CONCLUSIONS: Altogether, the results demonstrated that AdV-mediated gene transfer using the FGF2/FGFR system is effective in gliomas with low or deficient levels of CAR and suggested that FGF2-retargeting of AdV may be a promising approach in glioma gene therapy.

The role of contortrostatin, a snake venom disintegrin, in the inhibition of tumor progression and prolongation of survival in a rodent glioma model.

OBJECT: Malignant gliomas are not curable because of diffuse brain invasion. The tumor cells invade the surrounding brain tissue without a clear tumor-brain demarcation line, making complete resection impossible. Therapy aimed at inhibition of invasion is crucial not only for prevention of tumor spread, but also for selectively blocking migrating cells that may be more resistant to chemotherapy and radiation. Recently, investigations have shown that the snake venom disintegrin contortrostatin specifically binds to certain integrins on the surface of glioma cells and thereby inhibits their interaction with the extracellular matrix (ECM), resulting in a blockage of cell motility and invasiveness. To translate these in vitro findings into clinical settings, the authors examined the effect of contortrostatin on glioma progression in a rodent model. METHODS: Athymic mice were intracranially or subcutaneously injected with U87 glioma cells, and the effect of intratumorally administered contortrostatin on tumor progression and animal survival was then studied. In addition, the authors evaluated the pharmacological safety of contortrostatin use in the brains of tumor-free animals. CONCLUSIONS: The results demonstrate that contortrostatin is able to inhibit tumor growth and angiogenesis and to prolong survival in a rodent glioma model. Moreover, contortrostatin appears to be well tolerated by the animal and lacks obvious neurotoxic side effects. Thus, contortrostatin may have potential as a novel therapeutic agent for the treatment of malignant gliomas.

Functional effect of contortrostatin, a snake venom disintegrin, on human glioma cell invasion in vitro.

The metastatic spread of cancer is a complex process that involves the combination of different cellular actions including cell adhesion to the extracellular matrix (ECM), breakdown of the ECM by specific matrix-degrading proteinases, and active cell locomotion. Contortrostatin (CN), a homodimeric snake venom disintegrin, has previously been demonstrated to be effective in blocking vitronectin/fibronectin-dependent adhesion and invasion of T98G human glioblastoma cells through Matrigel using in vitro studies. However, it is not known at what step of the invasion process CN exerts its inhibitory effect. In the present report, CN is shown to decrease invasion of various glioma cell lines through Matrigel affecting neither cell adhesion, nor cell viability. While CN had no effect on cell binding to laminin and type IV collagen, it blocked adhesion of alphav beta3-positive, but not alphav beta3-negative cells, to vitronectin and fibronectin. Furthermore, members of the matrix metalloproteinase (MMP) family and their physiological inhibitors, and of the plasminogen activator (PA)/plasmin system were demonstrated not to be involved in CN-induced loss of glioma cell invasiveness. Instead, CN inhibited active locomotion of cells on Matrigel. These data suggest that CN-mediated inhibition of glioma cell invasion through Matrigel is a direct result of impaired cell motility. Moreover, use of several glioma cell lines and integrin antibodies strongly indicates the versatility of CN in inhibiting the invasion process based on the ability of CN to interact with different integrins, including alphav beta3, alphav beta5, and alpha5beta1.

Impact of human autoantibodies on ß1-adrenergic receptor conformation, activity, and internalization.

AIMS: Autoantibodies against second extracellular loops of ß(1)-adrenergic receptors frequent in dilated cardiomyopathy confer myocardial dysfunction presumably via cAMP stimulation. Here, we investigate the autoantibody impact on receptor conformation and function. METHODS AND RESULTS: IgG was prepared from patients with dilated cardiomyopathy, matched healthy donors (10 each) or commercial IgG preparations (2). IgG binding to ß(1)-adrenergic receptor peptides was detected in 5 of 10 patients and 2 of 10 controls. IgG colocalization with the native receptor was detected in 8 of 10 patients and 1 of 10 controls (10 of 10 patients and 7 of 10 controls at >30 mg IgG/L). All IgGs exhibiting receptor colocalization triggered changes in receptor conformation (determined with fluorescent sensors) not stringently correlated to cAMP stimulation, suggesting the induction of more or less active receptor conformations. Receptor-activating IgG was detected in 8 of 10 patients but only 1 of 10 controls. In addition, IgG from 8 of 10 patients and 3 of 10 controls attenuated receptor internalization (measured by total internal reflection fluorescence microscopy). IgG-inducing inactive receptor conformations had no effect on subsequent cAMP stimulation by isoproterenol. IgG-inducing active receptor conformations dampened or augmented subsequent cAMP stimulation by isoproterenol, depending on whether receptor internalization was attenuated or not. Corresponding IgG effects on the basal beating rate and chronotropic isoproterenol response of embryonic human cardiomyocytes were observed. CONCLUSIONS: (i) Autoantibodies trigger conformation changes in the ß(1)-adrenergic receptor molecule. (ii) Some also attenuate receptor internalization. (iii) Combinations thereof increase the basal beating rate of cardiomyocytes and optionally entail dampening of their chronotropic catecholamine responses. (iv) The latter effects seem specific for patient autoantibodies, which also have higher levels.

Effect of native and NH3 plasma-functionalized polymeric membranes on the gene expression profiles of primary hepatocytes.

Little is known about how cells respond to different biomaterials at the molecular level. Biomaterials could stimulate specific cellular responses at the molecular level, such as activation of signalling pathways that control gene activity involved in the maintenance, growth and functional regeneration of liver tissue in vitro. This aspect is an important step in liver tissue engineering. Currently, there are no data available concerning the modulation of cellular genomic response by using synthetic membranes in a bioartificial system. For the first time we investigated gene expression profiles of primary hepatocytes cultured on different substrates: collagen sandwich, native and NH(3) plasma-grafted PEEK-WC-PU membranes. Gene expression in cell suspension prepared after cell isolation was used as a control. Generally, microarray data revealed that the expression of the majority of genes remained unchanged compared to the control. Among 31 000 genes, 52 were significantly changed: 20 were upregulated and 32 downregulated. There were similar changes in gene expression of hepatocytes cultured in the membranes and collagen sandwich. However, some genes involved in the cell proliferation and functional metabolic pathways are more expressed in cells cultured on the membranes and especially on the functionalized ones. Both membranes sustained liver functions at the molecular level, demonstrating their suitability for the reconstruction of liver and as a toxicogenomic tool to predict the liver response to novel drugs.

Glutathione Peroxidase 2 Inhibits Cyclooxygenase-2-Mediated Migration and Invasion of HT-29 Adenocarcinoma Cells but Supports Their Growth as Tumors in Nude Mice.

The selenoprotein gastrointestinal glutathione peroxidase 2 (GPx2) is up-regulated in a variety of cancer cells with thus far unknown consequences. Therefore, two clones of a human colon cancer cell line (HT-29) in which GPx2 was stably knocked down by small interfering RNA (siRNA; siGPx2) were used to test whether cancer-relevant processes are affected by GPx2. The capacity to grow anchorage independently in soft agar was significantly reduced in siGPx2 cells when compared with controls (i.e., HT-29 cells stably transfected with a scramble siRNA). The weight of tumors derived from siGPx2 cells injected into nude mice was lower in 9 of 10 animals. In contrast, in a wound-healing assay, wound closure was around 50% in controls and 80% in siGPx2 cells, indicating an enhanced capacity of the knockdown cells to migrate. Similarly, invasion of siGPx2 cells in a Transwell assay was significantly increased. Migration and invasion of siGPx2 cells were inhibited by celecoxib, a cyclooxygenase-2 (COX-2)-specific inhibitor, but not by alpha-tocopherol. Selenium supplementation of cell culture medium did not influence the results obtained with siGPx2 cells, showing that none of the other selenoproteins could replace GPx2 regarding the described effects. The data show that GPx2 inhibits malignant characteristics of tumor cells, such as migration and invasion, obviously by counteracting COX-2 expression but is required for the growth of transformed intestinal cells and may, therefore, facilitate tumor cell growth. The data also shed new light on the use of selenium as a chemopreventive trace element: a beneficial effect may depend on the stage of tumor development.

Present and Future Developments in Hepatic Tissue Engineering for Liver Support Systems : State of the art and future developments of hepatic cell culture techniques for the use in liver support systems.

The liver is the most important organ for the biotransformation of xenobiotics, and the failure to treat acute or acute-on-chronic liver failure causes high mortality rates in affected patients. Due to the lack of donor livers and the limited possibility of the clinical management there has been growing interest in the development of extracorporeal liver support systems as a bridge to liver transplantation or to support recovery during hepatic failure. Earlier attempts to provide liver support comprised non-biological therapies based on the use of conventional detoxification procedures, such as filtration and dialysis. These techniques, however, failed to meet the expected efficacy in terms of the overall survival rate due to the inadequate support of several essential liver-specific functions. For this reason, several bioartificial liver support systems using isolated viable hepatocytes have been constructed to improve the outcome of treatment for patients with fulminant liver failure by delivering essential hepatic functions. However, controlled trials (phase I/II) with these systems have shown no significant survival benefits despite the systems' contribution to improvements in clinical and biochemical parameters. For the development of improved liver support systems, critical issues, such as the cell source and culture conditions for the long-term maintenance of liver-specific functions in vitro, are reviewed in this article. We also discuss aspects concerning the performance, biotolerance and logistics of the selected bioartificial liver support systems that have been or are currently being preclinically and clinically evaluated.

Development and characterization of a small-scale bioreactor based on a bioartificial hepatic culture model for predictive pharmacological in vitro screenings.

A vast majority of pharmacons are beset by possible interactions and side effects which have usually been tested in laboratory animals. However, better methods are needed to reduce the number of animal experiments and interspecies differences with respect to drug metabolism, as well as to provide a faster and more cost-effective way of analysis. These facts have led to the development of in vitro models based on isolated primary hepatocytes to better assess drug metabolism, interactions, and toxicity. A new small-scale bioreactor with the hepatic sandwich model and a gas-permeable membrane at the bottom allowing a definable oxygen exchange, has been constructed and compared with the conventional well plates. Compared to hepatocytes cultured in conventional systems, the cells exhibited a stronger liver-specific capacity and remained in a differentiated state in the small-scale bioreactor over a cultivation period of 17 days. This in vitro model could serve as a tool to predict the liver response to newly developed drugs.

Potent mimicry of fibronectin-induced intracellular signaling in glioma cells by the homodimeric snake venom disintegrin contortrostatin.

OBJECTIVE: The snake venom disintegrin contortrostatin (CN) is able to inhibit tumor progression and angiogenesis in vivo and therefore is of considerable interest as a potential antitumor drug. CN specifically binds to certain integrins on the tumor cell and angiogenic endothelial cell surface and inhibits their interaction with the extracellular matrix, resulting in blockage of cell motility and invasiveness. To understand the molecular consequences of CN binding to integrins, we set out to investigate and compare the effects of CN and fibronectin (FN) on integrin-induced signaling and the resulting alteration in cellular cytoskeletal morphology. METHODS: Two different malignant glioma cell lines were exposed to soluble or immobilized CN, FN, or both, and the consequences for intracellular signaling and cellular adhesion to matrix were investigated. RESULTS: CN binding to integrins can mimic the intracellular signaling cascade evoked by FN, because the phosphorylation of the key signaling proteins focal adhesion kinase, paxillin, and p130 Crk-associated substrate and the association of Src with focal adhesion kinase are similar. However, CN is at least one order of magnitude more potent than FN. When soluble CN is added to cells that are already attached to an FN-coated matrix, it effectively disrupts the binding of integrin to FN, leading to a decrease in integrin signaling, which, in turn, results in the disruption of the cytoskeleton and cellular detachment. CONCLUSION: Our results provide a mechanistic explanation of how soluble CN might block cellular migration and invasion, namely, by disrupting and preventing the binding of integrins to the extracellular matrix. We envision that this property of CN could be used in the treatment of gliomas, namely, by intratumoral infusion of CN to prevent glioma and endothelial cell interactions with the extracellular matrix, leading to inhibition of cell invasion.

Purification and characterization of the thyrotropin-releasing hormone (TRH)-degrading serum enzyme and its identification as a product of liver origin.

Previous biochemical studies have indicated that the membrane-bound thyrotropin-releasing hormone (TRH)-degrading enzyme (TRH-DE) from brain and liver and the serum TRH-DE are derived from the same gene. These studies also suggested that the serum enzyme is of liver origin. The present study was undertaken to verify these hypotheses. In different species, a close relationship between the activities of the serum enzyme and the particulate liver enzyme was noticed. The activity of the serum enzyme decreased when rats were treated with thioacetamide, a known hepatotoxin. With hepatocytes cultured in a sandwich configuration, release of the TRH-DE into the culture medium could also be demonstrated. The trypsin-solubilized particulate liver TRH-DE and the serum TRH-DE were purified to electrophoretic homogeneity. Both enzymes and the brain TRH-DE were recognized by a monoclonal antibody generated with the purified brain enzyme as antigen. Lectin blot analysis indicated that the serum enzyme and the liver enzyme are glycoproteins containing a sugar structure of the complex type, whereas the brain enzyme exhibits an oligomannose/hybrid glycostructure. A molecular mass of 97 000 Da could be estimated for all three enzymes after deglycosylation and SDS/PAGE followed by Western blotting. Fragment analysis of the serum TRH-DE revealed that the peptide sequences correspond to the cDNA deduced amino-acid sequences of the membrane-bound brain TRH-DE, whereby two peptides were identified that are encoded by exon 1. These data strongly support the hypothesis that the TRH-DEs are all derived from the same gene, whereby the serum enzyme is generated by proteolytic cleavage of the particulate liver enzyme.