Impact of di-ethylhexylphthalate exposure on metabolic programming in P19 ECC-derived cardiomyocytes.

Di(2-ethylhexyl)phthalate (DEHP) is the most common plasticizer in plastic devices of everyday use. It is a ubiquitous environmental contaminant and primarily known to impair male gonadal development and fertility. Studies concerning the long-term effects of prenatal DEHP exposure on certain diseases [The Developmental Origins of Health and Disease paradigm (DOHaD) hypothesis] are scarce although it is proven that DEHP crosses the placenta. Rising environmental pollution during the last centuries coincides with an increasing prevalence of cardiovascular and metabolic diseases. We have investigated the effects of an early embryonic DEHP exposure at different developmental stages on cardiomyogenesis. We used an in-vitro model, the murine P19 embryonic carcinoma cell line (P19 ECC), mimicking early embryonic stages up to differentiated beating cardiomyocytes. P19 ECC were exposed to DEHP (5, 50, 100 µg ml(-1)) at the undifferentiated stage for 5 days and subsequently differentiated to beating cardiomyocytes. We analyzed the expression of metabolic (Pparg1, Fabp4 and Glut4), cardiac (Myh6, Gja1) and methylation (Dnmt1, Dnmt3a) marker genes by quantitative real-time PCR (qRT-PCR), beating rate and the differentiation velocity of the cells. The methylation status of Pparg1, Ppara and Glut4 was investigated by pyrosequencing. DEHP significantly altered the expression of all investigated genes. The beating rate and differentiation velocity were accelerated. Exposure to DEHP led to small but statistically significant increases in methylation of specific CpGs within Ppara and Pparg1, which otherwise were generally hypomethylated, but methylation of Glut4 was unaltered. Early DEHP exposure of P19 ECC alters the expression of genes associated with cellular metabolism and the functional features of cardiomyocytes.

Short-time glucose exposure of embryonic carcinoma cells impairs their function as terminally differentiated cardiomyocytes.

The fetal and postnatal phenotype is influenced by developmental conditions experienced prenatally. Among prenatal development metabolic factors are of particular importance as they are supposed to predispose for pathophysiological alterations later in life and to pioneer functional impairment in senescence (metabolic programming). Till now the mechanisms of metabolic programming are not well understood. We have investigated various concentrations of glucose during differentiation of pluripotent P19 embryonic carcinoma cells (ECC) into cardiomyocytes. Undifferentiated P19 cells were exposed to 5mM (low), 25 mM (control), 40 mM or 100mM (high) glucose for 48 h during embryoid body (EB) formation, followed by plating and differentiation into cardiomyocytes in vitro with standard glucose supplementation (25 mM) for 10-15 days. The amount of cardiac clusters, the frequency of spontaneous beatings as well as the expression of metabolic and cardiac marker genes and their promoter methylation were measured. We observed a metabolic programming effect of glucose during cardiac differentiation. Whereas the number of beating clusters and the expression of the cardiac marker alpha myosin heavy chain (α-MHC) were comparable in all groups, the frequencies of beating clusters were significantly higher in the high glucose group compared to low glucose. However, neither the insulin receptor (IR) or insulin like growth factor 1 receptor (IGF1R) nor the metabolic gene glucose transporter 4 (GLUT4) were influenced in RNA expression or in promoter methylation. Our data indicate that a short time glucose stress during embryonic cell determination leads to lasting effects in terminally differentiated cell function.

Biosensing and drug delivery at the microscale : novel devices for controlled and responsive drug delivery.

An overall objective of pharmaceutical research is the controlled release or delivery of drugs at the biological target site in a therapeutically and pharmacodynamically optimal amount. In relation to "intelligent" drug delivery, several basic aspects are important, i.e., release of active pharmaceutical ingredients from the formulation, transport to and penetration across biological barriers, and subsequent biotransformation depending on a controlled release process. Future development of advanced and/or controlled drug releasing systems, e.g. polymeric or particulate drug targeting systems, nano-carbon tube related and/or nano-pillar based drug release, or electronically mediated molecule delivery, is expected to take advantage of progress in molecular cell biology, cell and tissue engineering, membrane nano-biophysics, and bioelectronic properties (Bramstedt et al. 2005; Gardner et al. 2006). In this chapter novel aspects of the development of innovative drug delivery systems described and are categorized into polymeric, lipid-based or electronically mediated delivery systems (De la Heras et al. 2004).

An impedimetric microelectrode-based array sensor for label-free detection of tau hyperphosphorylation in human cells.

Tauopathies such as Alzheimer's disease (AD) belong to the group of neurodegenerative diseases that are characterised by hyperphosphorylation of the protein tau. Hyperphosphorylation of tau is one of the salient events leading to neuronal cytotoxicity and cognitive impairments. In this context, inhibition of tau hyperphosphorylation by specific tau kinase inhibitors can provide an excellent drug target for the treatment of AD and other tau-related neurodegenerative diseases. To improve the identification, optimisation and validation during the high-cost hit-to-lead cycle of AD drugs, we established a fast and sensitive label-free technique for testing the efficacy of tau kinase inhibitors in vitro. Here, we report for the first time that microelectrode-based impedance spectroscopy can be used to detect the pathological risk potential of hyperphosphorylated tau in the human neuroblastoma cell line SH-SY5Y. Our findings provide a novel real-time recording technique for testing the efficiency of tau kinase inhibitors or other lead structures directed to tau hyperphosphorylation on differentiated SH-SY5Y cells.

Microcavity array (MCA)-based biosensor chip for functional drug screening of 3D tissue models.

Multicellular tumour spheroids that mimic a native cellular environment are widely used as model systems for drug testing. To study drug effects on three-dimensional cultures in real-time we designed and fabricated a novel type of sensor chip for fast, non-destructive impedance spectroscopy and extracellular recording. Precultured spheroids are trapped between four gold electrodes. Fifteen individual 100microm deep square microcavities with sizes from 200 to 400microm allow an optimised positioning during the measurement. Although apoptosis was induced in human melanoma spheroids by Camptothecin (CTT), treated cultures did not show disintegration but displayed increased impedance magnitudes compared to controls after 8h resulting from an altered morphology of the outer cells. Contractions in cardiomyocyte spheroids were monitored when the innovative chip was used for recording of extracellular potentials. The silicon-based electrode array is used as an acute test system for the monitoring of any kind of 3D cell cultures. Since no adherence of cells or labelling is necessary the multifunctional sensor chip provides a basis for improved drug development by high content screenings with reduced costs and assay times. Additional improvements for parallel testing of different substances on one chip are presented.

Artificial design of three-dimensional retina-like tissue from dissociated cells of the mammalian retina by rotation-mediated cell aggregation.

The goal of this study was to establish a reliable three-dimensional culture system for the mammalian retina that allows the analysis of retinal function and dysfunction. To produce three-dimensional retinal tissues in vitro, dissociated retinal cells of neonatal rats were maintained in culture dishes on a self-made orbital shaker. On the basis of well-defined rotation conditions, dissociated free-floating cells reaggregate in the center of the culture dish to form a multicellular cluster. Subsequently, cells begin to proliferate, whereby they form spherelike retinal tissues that grow to a size of 180-210 microm. Immunohistochemical characterization of mature retinal spheres revealed the presence of ganglion cells, amacrine cells, Müller cells, and rod photoreceptors, which are arranged in different retina-like layers. Although a small number of cells undergo programmed cell death, retinal spheres remain viable for at least 35 days in culture as revealed by fluorescein diacetate and TUNEL staining. Because most biological processes involved in tissue organization such as proliferation, differentiation, apoptosis, and survival are also observable in retinal spheres, the presented novel mammalian three-dimensional culture system is not only an outstanding model for basic research but may also be of great benefit for stem cell tissue engineering and the pharmaceutical industry.

Ethyl Pyruvate Emerges as a Safe and Fast Acting Agent against Trypanosoma brucei by Targeting Pyruvate Kinase Activity.

BACKGROUND: Human African Trypanosomiasis (HAT) also called sleeping sickness is an infectious disease in humans caused by an extracellular protozoan parasite. The disease, if left untreated, results in 100% mortality. Currently available drugs are full of severe drawbacks and fail to escape the fast development of trypanosoma resistance. Due to similarities in cell metabolism between cancerous tumors and trypanosoma cells, some of the current registered drugs against HAT have also been tested in cancer chemotherapy. Here we demonstrate for the first time that the simple ester, ethyl pyruvate, comprises such properties. RESULTS: The current study covers the efficacy and corresponding target evaluation of ethyl pyruvate on T. brucei cell lines using a combination of biochemical techniques including cell proliferation assays, enzyme kinetics, phasecontrast microscopic video imaging and ex vivo toxicity tests. We have shown that ethyl pyruvate effectively kills trypanosomes most probably by net ATP depletion through inhibition of pyruvate kinase (Ki = 3.0±0.29 mM). The potential of ethyl pyruvate as a trypanocidal compound is also strengthened by its fast acting property, killing cells within three hours post exposure. This has been demonstrated using video imaging of live cells as well as concentration and time dependency experiments. Most importantly, ethyl pyruvate produces minimal side effects in human red cells and is known to easily cross the blood-brain-barrier. This makes it a promising candidate for effective treatment of the two clinical stages of sleeping sickness. Trypanosome drug-resistance tests indicate irreversible cell death and a low incidence of resistance development under experimental conditions. CONCLUSION: Our results present ethyl pyruvate as a safe and fast acting trypanocidal compound and show that it inhibits the enzyme pyruvate kinase. Competitive inhibition of this enzyme was found to cause ATP depletion and cell death. Due to its ability to easily cross the blood-brain-barrier, ethyl pyruvate could be considered as new candidate agent to treat the hemolymphatic as well as neurological stages of sleeping sickness.

A novel 3D label-free monitoring system of hES-derived cardiomyocyte clusters: a step forward to in vitro cardiotoxicity testing.

Unexpected adverse effects on the cardiovascular system remain a major challenge in the development of novel active pharmaceutical ingredients (API). To overcome the current limitations of animal-based in vitro and in vivo test systems, stem cell derived human cardiomyocyte clusters (hCMC) offer the opportunity for highly predictable pre-clinical testing. The three-dimensional structure of hCMC appears more representative of tissue milieu than traditional monolayer cell culture. However, there is a lack of long-term, real time monitoring systems for tissue-like cardiac material. To address this issue, we have developed a microcavity array (MCA)-based label-free monitoring system that eliminates the need for critical hCMC adhesion and outgrowth steps. In contrast, feasible field potential derived action potential recording is possible immediately after positioning within the microcavity. Moreover, this approach allows extended observation of adverse effects on hCMC. For the first time, we describe herein the monitoring of hCMC over 35 days while preserving the hCMC structure and electrophysiological characteristics. Furthermore, we demonstrated the sensitive detection and quantification of adverse API effects using E4031, doxorubicin, and noradrenaline directly on unaltered 3D cultures. The MCA system provides multi-parameter analysis capabilities incorporating field potential recording, impedance spectroscopy, and optical read-outs on individual clusters giving a comprehensive insight into induced cellular alterations within a complex cardiac culture over days or even weeks.

Cells on a chip--the use of electric properties for highly sensitive monitoring of blood-derived factors involved in angiotensin II type 1 receptor signalling.

BACKGROUND: We developed a highly sensitive cardiomyocyte based screening system for the non-destructive electronic detection of chronotropic drugs and tissue-secreted factors involved in AT1 receptor-mediated cardiovascular diseases. METHODS: For this purpose we cultured spontaneously beating neonatal rat cardiomyocytes on microelectrode arrays (MEAs), and tested the optimised, stable culture parameters for a reproducible real-time recording of alterations in contraction frequency. After the evaluation of culture parameters, computer-based electronic measurement systems were used for counting of contractions by recording of the field potential of cardiomyocytes. RESULTS: Using the biosensor, angiotensin II, the predominant ligand of the AT1 receptor, was detected at very low concentrations of 10(-11) M via altered contractions of cardiomyocytes. Moreover, we demonstrated that cardiomyocyte coupled microarrays allow the detection of blood-derived low concentrated anti-AT1 receptor autoimmune antibodies of pregnant women suffering from preeclampsia. CONCLUSION: This study demonstrates the first well-suited electrophysiological recording of cardiomyocytes on multielectrode arrays as a benefit for functional biomonitoring for the detection of AT1 receptor/ligand interactions and other marker proteins in sera directed to cardiovascular diseases.