Human Cell Line ECV304 Research Articles

Classification of the mechanism of toxicity as applied to human cell line ECV304

The objective of this study was to identify the pattern of cytotoxicity testing of the human cell line ECV304 using three techniques of an ensemble learning algorithm (bagging, boosting and stacking). The study of cell morphology of ECV304 cell line was conducted using impedimetric measurement. Three types of toxins were applied to the ECV304 cell line namely 1 mM hydrogen peroxide (H2O2), 5% dimethyl sulfoxide and 10 μg Saponin. The measurement was conducted using electrodes and lock-in amplifier to detect impedance changes during cytotoxicity testing within a frequency range 200 and 830 kHz. The results were analysed, processed and extracted using detrended fluctuation analysis to obtain characteristics and features of the cells when exposed to the each of the toxins. Three ensemble algorithms applied showed slightly different results on the performance for classifying the data set from the feature extraction that was performed. However, the results show that the cell reaction to the toxins could be classified.

The application of detrended fluctuation analysis to assess physical characteristics of the human cell line ECV304 following toxic challenges

In this paper we present a non-contact, impedance-based sensor system capable of characterizing the toxic response of cells to three different types of toxin. ECV304 cells were treated with 1 mM Hydrogen peroxide, 5% Dimethyl Sulfoxide, and 10 μg/ml saponin. Impedance spectroscopy was performed over a 2 h period on the cells within a commercial cell growth chamber, positioned on a pair of measurement electrodes, at frequencies between 200 and 830 kHz at 10 kHz intervals. Analysis of the impedance data was undertaken using the feature-extraction technique, Detrended Fluctuation Analysis (DFA). DFA scales the autocorrelation of a non-stationary signal, such as those generated using impedance spectroscopy for cytotoxicity testing. The correlation between the average fluctuation of the signal, F(n) (and scaling exponent, α) and a measurement of the cell size from image analysis was evaluated. The results showed that F(n) and α were strongly related to the changes of the morphological size of the cells. The results demonstrated that non-contact impedance spectroscopy, coupled with DFA can be used to monitor cell size in real time.

Effects of NMDA receptor modulators on a blood–brain barrier in vitro model

Changes of the functionality of the blood–brain barrier (BBB) have been reported in the context of several brain related diseases such as multiple sclerosis, epilepsy, Alzheimer's disease and stroke. Several publications indicated the presence and functionality of the NMDA receptor (NMDAR) at the brain endothelium and a possible involvement of the NMDAR in the above-mentioned diseases. Recently, it was shown that the application of the NMDAR antagonist MK801 can block several adverse effects at the BBB in vitro, but also that MK801 can significantly change the proteome of brain endothelial cells without simultaneous stimulation of NMDAR by glutamate. Based on these reports we investigated if NMDAR antagonists MK801 and D-APV can affect the intracellular calcium level (Ca2+i) of an in vitro BBB model based on human cell line ECV304 on their own and compared these results to effects mediated by NMDAR agonists glutamate and NMDA. Treatment of ECV304 cells for 30min with glutamate resulted in no significant change of Ca2+i. On the contrary, application of NMDA and NMDAR antagonists D-APV and MK801 led to a significant and concentration dependent decrease of Ca2+i. Further studies revealed that glutamate was able to decrease the transendothelial electrical resistance (TEER) of the BBB in vitro model, whereas NMDA and D-APV were able to increase TEER. Analysis of the protein expression levels of tight junctional molecules ZO-1 and occludin showed a complex regulation after application of NMDAR modulators. In summary, it was shown that NMDAR antagonists can alter BBB key properties in vitro on their own. Moreover, although qPCR results confirmed the presence of NMDA receptor subunits NR1, NR2A, NR2B and NR2C, membrane binding studies failed to prove the typical plasma membrane localization and functionality in human BBB cell line ECV304.

Functionability of NMDA-Receptors in an in vitro model of the Blood-Brain-Barrier

A diversity of disease processes and conditions such as Alzheimer´s disease, schizophrenia, epilepsy and stroke – although they are caused by different mechanism – share one common target: the NMDA-receptor [1]. Recent studies reported the presence and the participation of this glutamate-gated ion-channel in major regulatory mechanisms at the blood-brain barrier (BBB). In order to show the functionability of this receptor and his effects on the BBB, we used an in vitro model based on human cell line ECV304 [2].

Expression of Claudin-1, Claudin-3 and Claudin-5 in human blood–brain barrier mimicking cell line ECV304 is inducible by glioma-conditioned media

Up to now no standard cell culture model of the blood–brain barrier is available. However, several models based on primary cells or continuous cell lines have been characterized and described in respect of different applications. One of the most important characteristics of the blood–brain barrier is the restriction of paracellular transport, respectively its tightness. Human cell line ECV304 is one of the promising continuous cell lines for blood–brain barrier modelling due to two reasons: on the one hand the cells are able to form significant tighter layers than most of the other cell lines used and on the other hand several properties of the blood–brain barrier are inducible by using glioma-conditioned medium. Claudins are transmembranal proteins which form the backbone of the tight junctions at the blood–brain barrier. We have investigated the presence and inducibility of the expression of Claudin-1, Claudin-3 and Claudin-5 using immunofluorescence microscopy. For the first time this study proves the presence of Claudin-1, Claudin-3 and Claudin-5 in ECV304 (obtained from ECACC) cell layers and the inducibility of their expression by glioma-conditioned media.

Transport of a GABAAReceptor Modulator and Its Derivatives fromValeriana officinalisL. s. l.Across anin VitroCell Culture Model of the Blood-Brain Barrier

The roots and rhizome of Valeriana officinalis L . s. l. are therapeutically used for their sedative and sleep-enhancing effects. Some of the active compounds found in commonly used extracts are the sesquiterpenic acids, especially valerenic acid, which was recently identified as a GABA (A) receptor modulator. To interact with this receptor in the brain, substances such as valerenic acid and its derivatives acetoxyvalerenic acid and hydroxyvalerenic acid have to cross the blood-brain barrier (BBB). The aim of our study was to obtain BBB permeability data of these compounds for the first time and to elucidate possible transport pathways across our BBB in vitro model. Transport of valerenic acid, acetoxyvalerenic acid and hydroxyvalerenic acid was compared with the permeability of the GABA (A) modulator diazepam, which is known to penetrate into the central nervous system transcellularly by passive diffusion. Experiments were carried out with an established Transwell in vitro model based on the human cell line ECV304. Results indicated clearly that all three acids permeated significantly slower than diazepam. The ranking was confirmed in group studies as well as in single-substance studies after normalization to diazepam. Valerenic acid (1.06 +/- 0.29 microm/min, factor 0.03 related to diazepam) was the slowest to permeate in the group study, followed by hydroxyvalerenic acid (2.72 +/- 0.63 microm/min, factor 0.07 related to diazepam) and acetoxyvalerenic acid (3.54 +/- 0.58 microm/min, factor 0.09 related to diazepam). To elucidate the contribution of the paracellular transport, studies were performed at different tightness status of the cell layers reflected by different transendothelial electrical resistance (TEER) values. Results showed an exponential correlation between transport and TEER for all three acids, whereas diazepam permeated TEER independently. In summary, it is hypothesized that the investigated compounds from Valeriana officinalis L. S. L. can probably only pass through the BBB by a still unknown transport system and not transcellularly by passive diffusion.

Microparticles of Human Atherosclerotic Plaques Enhance the Shedding of the Tumor Necrosis Factor-α Converting Enzyme/ADAM17 Substrates, Tumor Necrosis Factor and Tumor Necrosis Factor Receptor-1

Human atherosclerotic plaques express the metalloprotease tumor necrosis factor (TNF)-alpha converting enzyme (TACE/ADAM-17), which cleaves several transmembrane proteins including TNF and its receptors (TNFR-1 and TNFR-2). Plaques also harbor submicron vesicles (microparticles, MPs) released from plasma membranes after cell activation or apoptosis. We sought to examine whether TACE/ADAM17 is present on human plaque MPs and whether these MPs would affect TNF and TNFR-1 cellular shedding. Flow cytometry analysis detected 12,867 +/- 2007 TACE/ADAM17(+) MPs/mg of plaques isolated from 25 patients undergoing endarterectomy but none in healthy human internal mammary arteries. Plaque MPs harbored mainly mature active TACE/ADAM17 and dose dependently cleaved a pro-TNF mimetic peptide, whereas a preferential TACE/ADAM17 inhibitor (TMI-2) and recombinant TIMP-3 prevented this cleavage. Plaque MPs increased TNF shedding from the human cell line ECV-304 overexpressing TNF (ECV-304(TNF)), as well as TNFR-1 shedding from activated human umbilical vein endothelial cells or ECV-304(TNF) cells, without affecting TNF or TNFR-1 synthesis. MPs also activated the shedding of the endothelial protein C receptor from human umbilical vein endothelial cells. All these effects were inhibited by TMI-2. The present study shows that human plaque MPs carry catalytically active TACE/ADAM17 and significantly enhance the cell surface processing of the TACE/ADAM17 substrates TNF, TNFR-1, and endothelial protein C receptor, suggesting that TACE/ADAM17(+) MPs could regulate the inflammatory balance in the culprit lesion.

Overexpression of glycosyl phosphatidylinositol-anchored tissue factor pathway inhibitor-1 inhibits tissue factor activity.

The cellular initiation of coagulation by the tissue factor (TF)-activated factor VII complex is transiently inhibited by endogenous tissue factor pathway inhibitor-1 (TFPI-1), whereas exogenously added TFPI-1 is targeted to a degradation pathway. This study investigates the relevance of glycosyl phosphatidylinositol (GPI) anchoring for the anticoagulant properties of TFPI-1. Experiments were performed with the human cell line ECV304 using liposomal gene transfer. For GPI anchoring of TFPI-1 we used a fusion protein of TFPI-1 and the GPI attachment sequence of decay-accelerating factor (GPI-TFPI-1), and compared it with wild-type TFPI-1. We measured TF and TFPI-1 surface expression by flow cytometry and TF proteolytic activity by a chromogenic assay for activated factor X generation. After transfection of GPI-TFPI-1, surface expression of TFPI-1 increased to 134 +/- 9% of mock transfected cells (mean +/- SEM, P = 0.004), and transfection with wild-type TFPI-1 did not significantly alter TFPI-1 surface expression. After transfection with GPI-TFPI-1, TF activity was reduced by 18 +/- 9% compared with mock transfections (P = 0.003), whereas after transfection with TFPI-1 wild type no significant inhibition was observed. This effect was not due to altered TF expression. GPI anchoring is an essential prerequisite for surface expression of TFPI-1 and inhibition of TF activity. Gene transfer of GPI-anchored TFPI, therefore, may be an efficient tool to inhibit local TF-induced coagulation.