Functional implications of Cav 2.3 R-type voltage-gated calcium channels in the murine auditory system - novel vistas from brainstem-evoked response audiometry.

Voltage-gated Ca2+ channels (VGCCs) are considered to play a key role in auditory perception and information processing within the murine inner ear and brainstem. In the past, Cav 1.3 L-type VGCCs gathered most attention as their ablation causes congenital deafness. However, isolated patch-clamp investigation and localization studies repetitively suggested that Cav 2.3 R-type VGCCs are also expressed in the cochlea and further components of the ascending auditory tract, pointing to a potential functional role of Cav 2.3 in hearing physiology. Thus, we performed auditory profiling of Cav 2.3+/+ controls, heterozygous Cav 2.3+/- mice and Cav 2.3 null mutants (Cav 2.3-/- ) using brainstem-evoked response audiometry. Interestingly, click-evoked auditory brainstem responses (ABRs) revealed increased hearing thresholds in Cav 2.3+/- mice from both genders, whereas no alterations were observed in Cav 2.3-/- mice. Similar observations were made for tone burst-related ABRs in both genders. However, Cav 2.3 ablation seemed to prevent mutant mice from total hearing loss particularly in the higher frequency range (36-42 kHz). Amplitude growth function analysis revealed, i.a., significant reduction in ABR wave WI and WIII amplitude in mutant animals. In addition, alterations in WI -WIV interwave interval were observed in female Cav 2.3+/- mice whereas absolute latencies remained unchanged. In summary, our results demonstrate that Cav 2.3 VGCCs are mandatory for physiological auditory information processing in the ascending auditory tract.

Data Acquisition and Analysis In Brainstem Evoked Response Audiometry In Mice.

Brainstem evoked response audiometry (BERA) is of central relevance in the clinical neurophysiology. As other evoked potential (EP) techniques, such as visually evoked potentials (VEPs) or somatosensory evoked potentials (SEPs), the auditory evoked potentials (AEPs) are triggered by the repetitive presentation of identical stimuli, the electroencephalographic (EEG) response of which is subsequently averaged resulting in distinct positive (p) and negative (n) deflections. In humans, both the amplitude and the latency of individual peaks can be used to characterize alterations in synchronization and conduction velocity in the underlying neuronal circuitries. Importantly, AEPs are also applied in basic and preclinical science to identify and characterize the auditory function in pharmacological and genetic animal models. Even more, animal models in combination with pharmacological testing are utilized to investigate for potential benefits in the treatment of sensorineural hearing loss (e.g., age- or noise-induced hearing deficits). Here we provide a detailed and integrative description of how to record auditory brainstem-evoked responses (ABRs) in mice using click and tone-burst application. A specific focus of this protocol is on pre-experimental animal housing, anesthesia, ABR recording, ABR filtering processes, automated wavelet-based amplitude growth function analysis, and latency detection.

Road Map for Development of Stem Cell-Based Alternative Test Methods.

Much progress has been made in establishing strategies for differentiation of induced human pluripotent stem cells (hiPSCs). However, differentiated hiPSCs are not yet routinely used for prediction of toxicity. Here, limiting factors are summarised and possibilities for improvement are discussed, with a focus on hepatocytes, cardiomyocytes, tubular epithelial cells, and developmental toxicity. Moreover, we make recommendations for further fine-tuning of differentiation protocols for hiPSCs to hepatocyte-like cells by comparing individual steps of currently available protocols to the mechanisms occurring during embryonic development. A road map is proposed to facilitate test system development, including a description of the most useful performance metrics.

yylncT Defines a Class of Divergently Transcribed lncRNAs and Safeguards the T-mediated Mesodermal Commitment of Human PSCs.

Human protein-coding genes are often accompanied by divergently transcribed non-coding RNAs whose functions, especially in cell fate decisions, are poorly understood. Using an hESC-based cardiac differentiation model, we define a class of divergent lncRNAs, termed yin yang lncRNAs (yylncRNAs), that mirror the cell-type-specific expression pattern of their protein-coding counterparts. yylncRNAs are preferentially encoded from the genomic loci of key developmental cell fate regulators. Most yylncRNAs are spliced polyadenylated transcripts showing comparable expression patterns in vivo in mouse and in human embryos. Signifying their developmental function, the key mesoderm specifier BRACHYURY (T) is accompanied by yylncT, which localizes to the active T locus during mesoderm commitment. yylncT binds the de novo DNA methyltransferase DNMT3B, and its transcript is required for activation of the T locus, with yylncT depletion specifically abolishing mesodermal commitment. Collectively, we report a lncRNA-mediated regulatory layer safeguarding embryonic cell fate transitions.

Cell death mechanisms of the anti-cancer drug etoposide on human cardiomyocytes isolated from pluripotent stem cells.

Etoposide (ETP) and anthracyclines are applied for wide anti-cancer treatments. However, the ETP-induced cardiotoxicity remains to be a major safety issue and the underlying cardiotoxic mechanisms are not well understood. This study is aiming to unravel the cardiotoxicity profile of ETP in comparison to anthracyclines using physiologically relevant human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs). Using xCELLigence real-time cell analyser (RTCA), we found that single high dose of ETP induces irreversible increase in hPSC-CMs beating rate and decrease in beating amplitude. We also identified 58 deregulated genes consisting of 33 upregulated and 25 downregulated genes in hPSC-CMs after ETP treatment. Gene ontology (GO) and pathway analysis showed that most upregulated genes are enriched in GO categories like positive regulation of apoptotic process, regulation of cell death, and mitochondria organization, whereas most downregulated genes were enriched in GO categories like cytoskeletal organization, muscle contraction, and Ca2+ ion homeostasis. Moreover, we also found upregulation in 5 miRNAs (has-miR-486-3p, has-miR-34c-5p, has-miR-4423-3p, has-miR-182-5p, and has-miR-139-5p) which play role in muscle contraction, arginine and proline metabolism, and hypertrophic cardiomyopathy (HCM). Immunostaining and transmission electron microscopy also confirmed the cytoskeletal and mitochondrial damage in hPSC-CMs treated with ETP, as well as noticeable alterations in intracellular calcium handling and mitochondrial membrane potential were also observed. The apoptosis inhibitor, Pifithrin-α, found to protect hPSC-CMs from ETP-induced cardiotoxicity, whereas hPSC-CMs treated with ferroptosis inhibitor, Liproxstatin-1, showed significant recovery in hPSC-CMs functional properties like beating rate and amplitude after ETP treatment. We suggest that the damage to mitochondria is a major contributing factor involved in ETP-induced cardiotoxicity and the activation of the p53-mediated ferroptosis pathway by ETP is likely the critical pathway in ETP-induced cardiotoxicity. We also conclude that the genomic biomarkers identified in this study will significantly contribute to develop and predict potential cardiotoxic effects of novel anti-cancer drugs in vitro.

Fingerprinting of neurotoxic compounds using a mouse embryonic stem cell dual luminescence reporter assay.

Identification of neurotoxic drugs and environmental chemicals is an important challenge. However, only few tools to address this topic are available. The aim of this study was to develop a neurotoxicity/developmental neurotoxicity (DNT) test system, using the pluripotent mouse embryonic stem cell line CGR8 (ESCs). The test system uses ESCs at two differentiation stages: undifferentiated ESCs and ESC-derived neurons. Under each condition, concentration-response curves were obtained for three parameters: activity of the tubulin alpha 1 promoter (typically activated in early neurons), activity of the elongation factor 1 alpha promoter (active in all cells), and total DNA content (proportional to the number of surviving cells). We tested 37 compounds from the ESNATS test battery, which includes polypeptide hormones, environmental pollutants (including methylmercury), and clinically used drugs (including valproic acid and tyrosine kinase inhibitors). Different classes of compounds showed distinct concentration-response profiles. Plotting of the lowest observed adverse effect concentrations (LOAEL) of the neuronal promoter activity against the general promoter activity or against cytotoxicity, allowed the differentiation between neurotoxic/DNT substances and non-neurotoxic controls. Reporter activity responses in neurons were more susceptible to neurotoxic compounds than the reporter activities in ESCs from which they were derived. To relate the effective/toxic concentrations found in our study to relevant in vivo concentrations, we used a reverse pharmacokinetic modeling approach for three exemplary compounds (teriflunomide, geldanamycin, abiraterone). The dual luminescence reporter assay described in this study allows high-throughput, and should be particularly useful for the prioritization of the neurotoxic potential of a large number of compounds.

Human Embryonic and Induced Pluripotent Stem Cell Based Toxicity Testing Models: Future Applications in New Drug Discovery.

New drug discovery (NDD) is a fascinating discipline encompassing different facets of medicine, pharmacology, biotechnology and chemistry. NDD is very often restricted by efficacy or safety problems of the new clinical candidate in human patients. Drug regulatory authorities have provided various guidelines for advancement of safe new chemical entities (NCEs) in clinical trials which must be strictly followed. In spite of this, various drugs have failed in clinical trials or withdrawn from market because of human safety issues related to cardiotoxicity, hepatotoxicity, neurotoxicity and teratogenicity. The failure of safety prediction was pointed to species specificity issues, lack of mechanistic toxicity data and inadequate clinical trials. These drugs not only affect human health but also cause loss of resources and time. The species specificity issues are partially addressed by use of primary human cells but their availability is very limited. Human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) offer sources for generation of an unlimited number of human somatic cells. The emergence of mechanistic models for toxicity testing with transcriptomics, proteomics along with toxicokinetics readouts based on hESCs and hiPSCs is paving the way to design new human relevant testing strategies. Introduction of these models at the timeframe of lead selection and optimization in parallel with in vitro pharmacokinetic studies will significantly reduce compound attrition rate by selection of safer lead molecules. We focused on upcoming hESCs and hiPSCs based toxicity testing models and their future role to address safety gaps of present drug discovery and development.

MicroRNAs as early toxicity signatures of doxorubicin in human-induced pluripotent stem cell-derived cardiomyocytes.

An in depth investigation at the genomic level is needed to identify early human-relevant cardiotoxicity biomarkers that are induced by drugs and environmental toxicants. The main objective of this study was to investigate the role of microRNAs (miRNAs) as cardiotoxicity biomarkers using human-induced pluripotent stem cell (hiPSC)-derived cardiomyocytes (CMs) that were exposed to doxorubicin (DOX) as a "gold standard" cardiotoxicant. hiPSC-CMs were exposed to 156 nM DOX for 2 days or for 6 days of repeated exposure, followed by drug washout and incubation in drug-free culture medium up to day 14 after the onset of exposure. The induced miRNAs were profiled using miRNA microarrays, and the analysis of the data was performed using the miRWalk 2.0 and DAVID bioinformatics tools. DOX induced early deregulation of 14 miRNAs (10 up-regulated and 4 down-regulated) and persistent up-regulation of 5 miRNAs during drug washout. Computational miRNA gene target predictions suggested that several DOX-responsive miRNAs might regulate the mRNA expression of genes involved in cardiac contractile function. The hiPSC-CMs exposed to DOX in a range from 39 to 156 nM did not show a significant release of the cytotoxicity marker lactate dehydrogenase (LDH) compared to controls. Quantitative real-time PCR analyses confirmed the early deregulation of miR-187-3p, miR-182-5p, miR-486-3p, miR-486-5p, miR-34a-3p, miR-4423-3p, miR-34c-3p, miR-34c-5p and miR-1303, and also the prolonged up-regulation of miR-182-5p, miR-4423-3p and miR-34c-5p. Thus, we identified and validated miRNAs showing differential DOX-responsive expression before the occurrence of cytotoxicity markers such as LDH, and these miRNAs also demonstrated the significant involvement in heart failure in patients and animal models. These results suggest that the DOX-induced deregulated miRNAs in human CMs may be used as early sensitive cardiotoxicity biomarkers for screening potential drugs and environmental cardiotoxicants with a similar mechanism of action.

Identification of genomic biomarkers for anthracycline-induced cardiotoxicity in human iPSC-derived cardiomyocytes: an in vitro repeated exposure toxicity approach for safety assessment.

The currently available techniques for the safety evaluation of candidate drugs are usually cost-intensive and time-consuming and are often insufficient to predict human relevant cardiotoxicity. The purpose of this study was to develop an in vitro repeated exposure toxicity methodology allowing the identification of predictive genomics biomarkers of functional relevance for drug-induced cardiotoxicity in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). The hiPSC-CMs were incubated with 156 nM doxorubicin, which is a well-characterized cardiotoxicant, for 2 or 6 days followed by washout of the test compound and further incubation in compound-free culture medium until day 14 after the onset of exposure. An xCELLigence Real-Time Cell Analyser was used to monitor doxorubicin-induced cytotoxicity while also monitoring functional alterations of cardiomyocytes by counting of the beating frequency of cardiomyocytes. Unlike single exposure, repeated doxorubicin exposure resulted in long-term arrhythmic beating in hiPSC-CMs accompanied by significant cytotoxicity. Global gene expression changes were studied using microarrays and bioinformatics tools. Analysis of the transcriptomic data revealed early expression signatures of genes involved in formation of sarcomeric structures, regulation of ion homeostasis and induction of apoptosis. Eighty-four significantly deregulated genes related to cardiac functions, stress and apoptosis were validated using real-time PCR. The expression of the 84 genes was further studied by real-time PCR in hiPSC-CMs incubated with daunorubicin and mitoxantrone, further anthracycline family members that are also known to induce cardiotoxicity. A panel of 35 genes was deregulated by all three anthracycline family members and can therefore be expected to predict the cardiotoxicity of compounds acting by similar mechanisms as doxorubicin, daunorubicin or mitoxantrone. The identified gene panel can be applied in the safety assessment of novel drug candidates as well as available therapeutics to identify compounds that may cause cardiotoxicity.

Human skin-derived stem cells as a novel cell source for in vitro hepatotoxicity screening of pharmaceuticals.

Human skin-derived precursors (hSKP) are postnatal stem cells with neural crest properties that reside in the dermis of human skin. These cells can be easily isolated from small (fore) skin segments and have the capacity to differentiate into multiple cell types. In this study, we show that upon exposure to hepatogenic growth factors and cytokines, hSKP acquire sufficient hepatic features that could make these cells suitable in vitro tools for hepatotoxicity screening of new chemical entities and already existing pharmaceutical compounds. Indeed, hepatic differentiated hSKP [hSKP-derived hepatic progenitor cells (hSKP-HPC)] express hepatic progenitor cell markers (EPCAM, NCAM2, PROM1) and adult hepatocyte markers (ALB), as well as key biotransformation enzymes (CYP1B1, FMO1, GSTA4, GSTM3) and influx and efflux drug transporters (ABCC4, ABCA1, SLC2A5). Using a toxicogenomics approach, we could demonstrate that hSKP-HPC respond to acetaminophen exposure in a comparable way to primary human hepatocytes in culture. The toxicological responses "liver damage", "liver proliferation", "liver necrosis" and "liver steatosis" were found to be significantly enriched in both in vitro models. Also genes associated with either cytotoxic responses or induction of apoptosis (BCL2L11, FOS, HMOX1, TIMP3, and AHR) were commonly upregulated and might represent future molecular biomarkers for hepatotoxicity. In conclusion, our data gives a first indication that hSKP-HPC might represent a suitable preclinical model for in vitro screening of hepatotoxicity. To the best of our knowledge, this is the first report in which human postnatal stem cells derived from skin are described as a potentially relevant cell source for in vitro hepatotoxicity testing of pharmaceutical compounds.

Identification of specific pluripotent stem cell death--inducing small molecules by chemical screening.

A potential application of embryonic and inducible pluripotent stem cells for the therapy of degenerative diseases involves pure somatic cells, free of tumorigenic undifferentiated embryonic and inducible pluripotent stem cells. In complex collections of chemicals with pharmacological potential we expect to find molecules able to induce specific pluripotent stem cell death, which could be used in some cell therapy settings to eliminate undifferentiated cells. Therefore, we have screened a chemical library of 1120 small chemicals to identify compounds that induce specifically apoptotic cell death in undifferentiated mouse embryonic stem cells (ESCs). Interestingly, three compounds currently used as clinically approved drugs, nortriptyline, benzethonium chloride and methylbenzethonium chloride, induced differential effects in cell viability in ESCs versus mouse embryonic fibroblasts (MEFs). Nortriptyline induced apoptotic cell death in MEFs but not in ESCs, whereas benzethonium and methylbenzethonium chloride showed the opposite effect. Nortriptyline, a tricyclic antidepressant, has also been described as a potent inhibitor of mitochondrial permeability transition, one of two major mechanisms involved in mitochondrial membrane permeabilization during apoptosis. Benzethonium chloride and methylbenzethonium chloride are quaternary ammonium salts used as antimicrobial agents with broad spectrum and have also been described as anticancer agents. A similar effect of benzethonium chloride was observed in human induced pluripotent stem cells (hiPSCs) when compared to both primary human skin fibroblasts and an established human fibroblast cell line. Human fibroblasts and hiPSCs were similarly resistant to nortriptyline, although with a different behavior. Our results indicate differential sensitivity of ESCs, hiPSCs and fibroblasts to certain chemical compounds, which might have important applications in the stem cell-based therapy by eliminating undifferentiated pluripotent stem cells from stem cell-derived somatic cells to prevent tumor formation after transplantation for therapy of degenerative diseases.

Dietary small molecules and large-scale gene expression studies: an experimental approach for understanding their beneficial effects on the development of malignant and non-malignant proliferative diseases.

Epidemiological studies have repeatedly demonstrated a correlation between nutrition, development and the severity of malignant and non-malignant proliferative diseases such as cancer and atherosclerosis. Therefore, the prevention of chronic proliferative diseases through dietary intervention is currently receiving considerable attention. Until now, much of the research is being focused on the cellular and molecular action mechanisms of dietary small molecules explaining their beneficial effects. Dietary chemicals may affect gene expression in several human diseases. However, significant progress has been made and several molecular action mechanisms have been proposed. Alteration of genetical pathways by nutrition, also called "Nutrigenomics", may offer a new approach for understanding the beneficial effects of dietary compounds on the development of severe polygenic diseases, such as cardiovascular disease, diabetes and hypertension. This review focuses on the nutritional genomics of dietary chemicals with a special emphasis on catechins. Catechins belong to the flavonoid family, which are polyphenolic compounds available in foods of plant origin. Several epidemiological studies have reported that consumption of flavonoids, and especially catechins might function as chemopreventive agents against cancer and cardiovascular diseases.

Trapping of growth factors by catechins: a possible therapeutical target for prevention of proliferative diseases.

The prevention of cancer through dietary intervention is currently receiving considerable attention. Several epidemiological studies substantiate that green tea has a protective effect against a variety of malignant proliferative disorders such as lung cancer, breast cancer and prostate cancer. This preventive potential of green tea against cancer is attributed to the biologically active flavonoids called catechins. Epigallocatechin 3-o-gallate, the major catechin found in green tea, mediates diverse physiological and pharmacological actions in bringing about the regression of the tumors and also lowers the risk of nonmalignant cardiovascular proliferative diseases. Much of the current research is being focused on how these catechins specifically bring about the regression of the experimentally induced tumors both in vitro and in vivo. These catechins exert diverse physiological effects against proliferative diseases by several mechanisms, most of which are not completely characterized. This review summarizes the mechanisms by which these catechins play an essential role in regulating the process of carcinogenesis, with a special emphasis on how these catechins antagonize the growth factor-induced proliferative disorders.

Local delivery of green tea catechins inhibits neointimal formation in the rat carotid artery injury model.

It has been shown that green tea catechins (GTC) suppress proliferation of vascular smooth muscle cells (VSMCs) and that epigallocatechin-3-gallate (EGCG), which is a major constituent of GTC, selectively inhibits the platelet-derived growth factor-BB (PDGF-BB)-induced intracellular signaling transduction pathway. Vascular smooth muscle cell proliferation is one of major mechanisms of restenosis following percutaneous coronary intervention. This study tested whether GTC can inhibit VSMC proliferation and prevent neointimal formation in a rat carotid artery injury model. Vascular smooth muscle cell proliferation inhibition was analyzed with [3H]thymidine incorporation. Green tea catechins were applied to the endothelium-denuded carotid arteries of rats for 20 min. Angiography and morphometric analysis was performed after 2 weeks. Green tea catechins decreased [3H]thymidine incorporation stimulated with PDGF-BB dose dependently. In the absence of PDGF-BB, the decrement of [3H]thymidine incorporation was evident above a concentration of 10 micro g/ml of GTC. Carotid arteriographic evaluation showed that the minimum luminal diameter in the GTC-treated group (n=12) was 5.9 +/- 1.6 arbitrary units (a.u.) and was significantly larger than in the control group (4.3 +/- 1.4 a.u., n=10) ( P <0.05). The GTC-treated group also showed a significant reduction in neointimal formation compared with the control group (0.29 +/- 0.11 vs 0.42 +/- 0.10 mm2, P < 0.05). To identify the active ingredients, we performed a similar experiment using EGCG. The effects of EGCG were similar to those of GTC. Green tea catechins effectively inhibited VSMC proliferation. Neointimal formation was prevented in the rat carotid artery injury model by local delivery of GTC. As EGCG showed similar effects, it may be one of the major constituents of GTC having these effects.

Inhibitory effect of epigallocatechin 3-O-gallate on vascular smooth muscle cell hypertrophy induced by angiotensin II.

Recent evidence indicates that epigallocatechin 3-O-gallate (EGCG), the major catechin derived from green tea leaves, lowers the risk of cardiovascular diseases such as atherosclerosis and hypertension. However, a precise mechanism for this biologic function has not yet been clearly delineated. Angiotensin II (Ang II) stimulates vascular smooth muscle cell (VSMC) hypertrophy, which is a critical event in the development of atherosclerosis, hypertension, and angioplasty-induced restenosis. In the present study, we show that EGCG inhibits Ang II-stimulated VSMC hypertrophy, as determined by [3H]leucine incorporation into VSMC. Since mitogen-activated protein kinase (MAPK) families are involved in cell growth, we determined whether EGCG affects them. EGCG pretreatment did not exert any significant changes in Ang II-stimulated activation of extracellular signal-regulated kinase (ERK) and p38 MAPK. EGCG only inhibited Ang II-stimulated activation of c-Jun N-terminal kinase (JNK). Moreover, EGCG suppressed Ang II-induced c-jun mRNA expression. In contrast, EGC, a structural analogue of EGCG, did not inhibit the JNK activity or c-jun mRNA expression. In addition, a specific JNK inhibitor, SP600125, dose-dependently suppressed Ang II-stimulated VSMC hypertrophy. These results suggest that the effect of EGCG on Ang II-induced VSMC hypertrophy is due to specific inhibition of the JNK signaling pathway at both transcriptional and posttranslational levels, which may underlie its beneficial effect on the cardiovascular diseases.