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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, Ca(v)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 Ca(v)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 Ca(v)2.3 in hearing physiology. Thus, we performed auditory profiling of Ca(v)2.3(+/+) controls, heterozygous Ca(v)2.3(+/-) mice and Ca(v)2.3 null mutants (Ca(v)2.3(-/-)) using brainstem-evoked response audiometry. Interestingly, click-evoked auditory brainstem responses (ABRs) revealed increased hearing thresholds in Ca(v)2.3(+/-) mice from both genders, whereas no alterations were observed in Ca(v)2.3(-/-) mice. Similar observations were made for tone burst-related ABRs in both genders. However, Ca(v)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 W-I and W-III amplitude in mutant animals. In addition, alterations in W-I-W-IV interwave interval were observed in female Ca(v)2.3(+/-) mice whereas absolute latencies remained unchanged. In summary, our results demonstrate that Ca(v)2.3 VGCCs are mandatory for physiological auditory information processing in the ascending auditory tract.

The generation of germline competent rat embryonic stem cells (rESCs) allows the study of their lineage commitment. Here, we developed a highly efficient system for rESC‐derived cardiomyocytes, and even the formation of three‐dimensional (3D)‐like cell clusters with cTNT and α‐Actinin. We have validated that laminin can interact with membrane integrin to promote the phosphorylation of both phosphatidylinositol 3‐kinase (PI3K) p85 and the focal adhesion kinase (FAK). In parallel, GATA4 was up‐regulated. Upon inhibiting the integrin, laminin loses the effect on cardiomyocyte differentiation, accompanied with a down‐regulation of phosphorylation level of PI3K p85 and FAK. Meanwhile, the expression of Gata4 was inhibited as well. Taken together, laminin is a crucial component in the differentiation of rESCs into cardiomyocytes through increasing their proliferation via interacting with integrin pathway. These results provide new insights into the pathways mediated by extracellular laminin involved in the fate of rESC‐derived cardiomyocytes.

Availability of homogeneous astrocyte populations would facilitate research concerning cell plasticity (metabolic and transcriptional adaptations; innate immune responses) and cell cycle reactivation. Current protocols to prepare astrocyte cultures differ in their final content of immature precursor cells, preactivated cells or entirely different cell types. A new method taking care of all these issues would improve research on astrocyte functions. We found here that the exposure of a defined population of pluripotent stem cell-derived neural stem cells (NSC) to BMP4 results in pure, nonproliferating astrocyte cultures within 24-48 h. These murine astrocytes generated from embryonic stem cells (mAGES) expressed the positive markers GFAP, aquaporin 4 and GLT-1, supported neuronal function, and acquired innate immune functions such as the response to tumor necrosis factor and interleukin 1. The protocol was applicable to several normal or disease-prone pluripotent cell lines, and the corresponding mAGES all exited the cell cycle and lost most of their nestin expression, in contrast to astrocytes generated by serum-addition or obtained as primary cultures. Comparative gene expression analysis of mAGES and NSC allowed quantification of differences between the two cell types and a definition of an improved marker set to define astrocytes. Inclusion of several published data sets in this transcriptome comparison revealed the similarity of mAGES with cortical astrocytes in vivo. Metabolic analysis of homogeneous NSC and astrocyte populations revealed distinct neurochemical features: both cell types synthesized glutamine and citrate, but only mature astrocytes released these metabolites. Thus, the homogeneous cultures allowed an improved definition of NSC and astrocyte features.

Aims Heart failure (HF) is associated with development of AF and life-threatening ventricular tachycardia and fibrillation (VT/VF). Vulnerability to development of AF and VT/VF at different stages of HF and the underlying pathophysiological mechanisms are poorly defined. The present study was designed to determine the time-course of development of electrical and structural remodelling of the atria and ventricles, and their contribution to induction of AF and VT/VF in a canine model of HF. Methods and results Dogs were ventricular tachypaced (VTP) for 2–3 weeks or 5–6 weeks (‘early’ and ‘late’ HF, respectively). Electrophysiological studies were performed in isolated atrial and ventricular preparations and correlated with cardiac dimensions and haemodynamic parameters recorded in vivo. Vulnerability to programmed electrical stimulation-induced AF was greater in early vs. late stages of HF (78% vs. 38%). In contrast, VT/VF was inducible in late but not in early stages of HF (38% vs. 0%). The temporal distinction in atrial and ventricular arrhythmia susceptibility was associated with a much more rapid development of electrical and structural remodelling in atria. Vulnerability to AF developed following moderate electro-structural remodelling and waned with further progression to severe remodelling, which averted rapid atrial activation. Conclusions A temporal window of vulnerability for AF appears relatively early during development of VTP-induced HF in dogs, whereas VT/VF vulnerability is observed at more advanced stages of HF. These findings, if confirmed in humans, may have clinical implications with regard to prognosis and approach to therapy of patients with HF.

BACKGROUND AND PURPOSE Teratogenic substances induce adverse effects during the development of the embryo. Multilineage differentiation of human embryonic stem cells (hESCs) mimics the development of the embryo in vitro. Here, we propose a transcriptomic approach in hESCs for monitoring specific toxic effects of compounds as an alternative to traditional time-consuming and cost-intensive in vivo tests requiring large numbers of animals. This study was undertaken to explore the adverse effects of cytosine arabinoside (Ara-C) on randomly differentiated hESCs. EXPERIMENTAL APPROACH Human embryonic stem cells were used to investigate the effects of a developmental toxicant Ara-C. Sublethal concentrations of Ara-C were given for two time points, day 7 and day 14 during the differentiation. Gene expression was assessed with microarrays to determine the dysregulated transcripts in presence of Ara-C. KEY RESULTS Randomly differentiated hESCs were able to generate the multilineage markers. The low concentration of Ara-C (1 nM) induced the ectoderm and inhibited the mesoderm at day 14. The induction of ectodermal markers such as MAP2, TUBB III, PAX6, TH and NESTIN was observed with an inhibition of mesodermal markers such as HAND2, PITX2, GATA5, MYL4, TNNT2, COL1A1 and COL1A2. In addition, no induction of apoptosis was observed. Gene ontology revealed unique dysregulated biological process related to neuronal differentiation and mesoderm development. Pathway analysis showed the axon guidance pathway to be dysregulated. CONCLUSIONS AND IMPLICATIONS Our results suggest that hESCs in combination with toxicogenomics offer a sensitive in vitro developmental toxicity model as an alternative to traditional animal experiments.

Establishment of fetal bovine serum (FBS)-free cell culture conditions is essential for transplantation therapies. Blood-derived endothelial colony-forming cells (ECFCs) are potential candidates for regenerative medicine applications. ECFCs were isolated from term umbilical cord blood units and characterized by flow cytometry, capillary formation and responsiveness to cytokines. ECFCs were expanded under standard, FBS-containing endothelial medium, or transferred to chemically defined endothelial media without FBS. Microarray expression profiling was applied to compare the transcriptome profiles in FBS-containing versus FBS-free culture. ECFC outgrowth in standard medium was successful in 92% of cord blood units. The karyotype of expanded ECFCs remained normal. Without FBS, ECFC initiation and expansion failed. Modest proliferation, changes in cell morphology and organization and cell death have been observed after passaging. Gene ontology analysis revealed a broad down-regulation of genes involved in cell cycle progression and up-regulation of genes involved in stress response and apoptosis. Interestingly, genes participating in lipid biosynthesis were markedly up-regulated. Detection of several endothelial cell-specific marker genes showed the maintenance of the endothelial cell characteristics during serum-free culture. Although ECFCs maintain their endothelial characteristics during serum-free culturing, they could not be expanded. Additional supply of FBS-free media with lipid concentrates might increase the ECFC survival.

A transgenic murine embryonic stem (ES) cell lineage expressing enhanced green fluorescent protein (EGFP) under the control of alpha-myosine heavy chain (alpha-MHC) promoter (palpha-MHC-EGFP) was used to investigate the effects of (thio)urea and cinchona alkaloid derivatives on cardiomyogenesis. The screening of the compounds yielded cardiomyogenesis inducing substances with good (IV-5, V-4) to very good activities (II-16, IV-8), as determined by a 50 to 80 % increase in the EGFP fluorescence compared to untreated cells. Time-dependent screening approaches in which compounds were added at different developmental stages of the ES cells appeared to be of limited suitability for the identification of potential cellular targets.

Embryonic stem (ES) cells are revolutionizing the field of developmental biology as a potential tool to understand the molecular mechanisms occurring during the process of differentiation from the embryonic stage to the adult phenotype. ES cells harvested from the inner cell mass (ICM) of the early embryo can proliferate indefinitely in vitro while retaining the ability to differentiate into all somatic cells. Emerging results from mice models with ES cells are promising and raising tremendous hope among the scientific community for the ES‐cell based cell replacement therapy (CRT) of various severe diseases. ES cells could potentially revolutionize medicine by providing an unlimited renewable source of cells capable of replacing or repairing tissues that have been damaged in almost all degenerative diseases such as diabetes, myocardial infarction and Parkinson's disease. This review updates the progress of ES cell research in CRT, discusses about the problems encountered in the practical utility of ES cells in CRT and evaluates how far this approach is successful experimentally.

Catechins have been reported to possess anti-cancer activity in vitro and in vivo. To identify target genes that may be involved in the anti-tumorigenic effect of catechins, gene expression profiles in adherent human HT 29 colon carcinoma cells, in HT 29 spheroids and in epigallocatechin-3 gallate (EGCG)-treated HT 29 cells have been analysed by high-density oligonucleotide microarrays. Treatment of HT 29 cells with EGCG (2.5-50 microm) resulted in a dose-dependent inhibition of spheroid formation of HT 29 cells. Forty transcripts were induced at least twofold in 3-day-old spheroids relative to normal adherent cells using three replicates. Oncogenes like c-fos and c-jun are significantly up-regulated in spheroids. We identified several signal transduction and proliferation genes which are down-regulated in response to EGCG treatment. Increase in the mRNA expression profile of c-Fos correlated well with protein levels in HT 29 spheroids whereas EGCG did not affect protein formation. In agreement with the DNA chip data, IQGAP2 protein was not increased in spheroids but protein formation was totally blocked in EGCG-treated cells. Interestingly, no change in expression of cytotoxic or apoptotic related genes has been observed in EGCG-treated cells. Our findings suggest that EGCG may exert its anti-cancer activity through modulation of expression of a number of genes that are involved in cell proliferation, cell-cell contacts and cell-matrix interactions.

A contributing factor to the failure of trials of neuroprotectants in acute ischemic stroke may be the differing vulnerability to ischemia of white compared with gray matter. To address this issue, we determined to establish the existence of potentially viable tissue in white matter and its evolution to infarction or salvage in both gray and white matter compartments in patients with ischemic stroke. Twenty-seven patients (mean age, 73.4 years) at a median of 16.5 hours after symptom onset were studied using the hypoxic marker 18F-misonidazole with positron emission tomography (PET). Tissue was segmented using an magnetic resonance probabilistic map. Although there was a greater volume of initially "at-risk tissue" in gray matter (58.3 cm3, 29.9-93.0 cm3 than white matter (42.0 cm3, 15.8-74.0 cm3; p

Stromal cell-derived factor-1 (SDF-1), mainly known as a chemotactic factor for haematopoietic progenitor cells, also provides angiogenetic potency. Since the intracellular signalling of SDF-1-induced neovascularization remains unclear, we studied in human umbilical arterial endothelial cells (HUAEC) the influence of SDF-1alpha on induction of the genes of early growth response-1 (Egr-1) and VEGF, as well as the activation of extracellular regulated kinases (ERK) 1/2, which are all known to be involved in endothelial cell proliferation. We found a time-dependent induction of Egr-1 and VEGF mRNA expression and phosphorylation of ERK1/2 by SDF-1alpha. Furthermore, we demonstrated that Egr-1 expression is dependent on ERK 1/2 activation. Finally, we tried to confirm the relevance of the induced gene expression by detecting the [3H]thymidine incorporation as a marker for cell proliferation in HUAEC after stimulation with SDF-1alpha alone or together with VEGF. This particular test showed, that SDF-1alpha alone has no effect, but is able to significantly enhance VEGF induced DNA synthesis. In summary, SDF-1alpha is involved in different steps of endothelial cell proliferation, but, since Egr-1 and VEGF offer different functions, it may also play a so far undefined role on other conditions of the endothelium.

Chronic myeloid leukemia (CML) is a cancer of one of the two main types of white blood cells, the neutrophiles. The Philadelphia (Ph) chromosome is the most prominent cytogenic abnormality in CML. The existence of this chromosome is due to a translocation, involving the bcr-gene on chromosome 22 and the abl-gene on chromosomes 9. The expression of the tyrosine kinase p210bcr-abl, a 210kDa fusion protein, is the result of this translocation. Tyrphostins (1, 2) are inhibitors of this enzyme and have been synthesised as potential drugs. The tyrphostin AG957 is of particular interest because of its small size, which enables the molecule to permeate cells (3), and because of its selectivity for p210bcr-abl over the normally expressed p140c-abl. In this study, we have radiolabelled AG957 (1) with the radioisotope C-11 in order to be evaluated as a potential agent for clinical PET studies of chronic myeloid leukemia.

Pyrimidine-specific ribonucleases are a superfamily of structurally related enzymes with distinct catalytic and biological properties. We used a combination of enzymatic and non-enzymatic assays to investigate the release of such enzymes by isolated cells in serum-free and serum-containing media. We found that human endothelial cells typically expressed large amounts of a pancreatic-type RNase that is related to, if not identical to, human pancreatic RNase. This enzyme exhibits pyrimidine-specific catalytic activity, with a marked preference for poly(C) substrate over poly(U) substrate. It was potently inhibited by placental RNase inhibitor, the selective pancreatic-type RNase inhibitor Inhibit-Ace, and a polyclonal antibody against human pancreatic RNase. The enzyme isolated from medium conditioned by immortalized umbilical vein endothelial cells (EA.hy926) possesses an amino-terminal sequence identical to that of pancreatic RNase, and shows molecular heterogeneity (molecular weights 18,000-26,000) due to different degrees of N-glycosylation. Endothelial cells from arteries, veins, and capillaries secreted up to 100 ng of this RNase daily per million cells, whereas levels were low or undetectable in media conditioned by other cell types examined. The corresponding messenger RNA was detected by RT-PCR in most cell types tested so far, and level of its expression was in keeping with the amounts of protein. The selective strong release of pancreatic-type RNase by endothelial cells suggests that it is endowed with non-digestive functions and involved in vascular homeostasis.

The important peptide labelling reagent succinimidyl 4-[18F]fluorobenzoate ([18F]SFB) has been synthesised in 75-85% decay corrected radiochemical yield using the IBA Synthera platform (IBA Cyclotron Solutions, Louvain-la-neuve, Belgium) with the fluorodeoxyglucose-integrated fluidic processor nucleophilic and only four reagent vials in a single reactor. (4-ethoxycarbonylphenyl) trimethylammonium triflate was used as the labelling precursor and 1 M aqueous tetramethylammonium hydroxide for the hydrolysis of the intermediate ethyl 4-[1eF]fluorobenzoate. N,N,N',N'-tetramethyl-O-(N-succinimidyl)uronium tetrafluoroborate (TSTU) was then used to form [18F]SFB from 4-[18F]fluorobenzoate. By omitting the addition of acetic acid and introducing a combined hydrolysis/water removal step, the synthesis time was shortened to 58 minutes. After SepPak purification, the radiochemical purity of [18F]SFB was 95.8-98.2%. These simplifications might be of significance to users of other automated synthesis modules.

There are contradictory findings regarding the effects of free fatty acids on vascular smooth muscle cell (VSMC) growth. In the present study we investigated the effects of fatty acids released from hydrolysis of human VLDL triglycerides by lipoprotein lipase and of the fatty acids most abundant in the hydrolysed VLDL, namely oleic, linoleic, palmitic and myristic acid, all non albumin-bound, on VSMC growth. The effect of fatty acids on VSMC growth was assessed by [3H]-thymidine incorporation, colourimetrically, by cell counting, by determination of the cytoplasmic histone-associated DNA fragments and the caspase 3 activity. The fatty acid concentrations were determined by gas chromatography-mass spectrometry. Stimulation of ERK1/2 and p38 was determined by the chemiluminescence Western blotting method. Incubation of VSMC with purified VLDL (100 μg ml−1) and lipoprotein lipase (35 u ml−1) led to almost complete cell death although the ERK1/2 and the p38 MAP kinases were stimulated. The EC50 of oleic, linoleic, myristic and palmitic acid were 4.6±1.3, 2.4±0.2, 116±10 and 287±30 μM, respectively. The estimated EC50 of myristic and palmitic acid when derived from hydrolysed VLDL were 10 and 8 times, respectively, lower than when used alone. Apoptosis was not involved in the fatty acid-induced VSMC growth suppression/death. We conclude that (a) non albumin-bound fatty acids cause VSMC necrosis in a dose-dependent manner with a parallel ERK1/2 and p38 stimulation, (b) unsaturated fatty acids are more toxic to VSMC than saturated, and (c) saturated fatty acids are more toxic to VSMC in the hydrolysed VLDL than when used individually. British Journal of Pharmacology (2001) 132, 1725–1734; doi:10.1038/sj.bjp.0704026

ERKs belong to MAP kinase family and are activated by several growth and stress factors. Although ethanol has been shown to modulate ERK1 and ERK2 (p44mapk and p42mapk) activity, it can also act as an antiproliferative agent in various mammalian cells. Since the nature of the antiproliferative effect of ethanol in VSMCs has not been defined, we examined its effects on growth and on early intracellular events normally induced by growth factors in VSMCs. Measurement of cytosolic Ca2+ and pH in cell monolayers was performed using fura-2/AM and BCECF/AM, respectively. The effect of ethanol on VSMCs growth was assessed by [3H]-thymidine incorporation, by cell counting and by determination of the caspase 3 activity. Stimulation of ERK1 and ERK2 was examined by the chemiluminescence Western blotting method. The expression of c-fos was quantitated by Northern blotting. Determination of inositolphosphates was performed after labelling of VSMCs with myo-[2-3H]-inositol and separation of inositolphosphates by HPLC. Ethanol (0.3–1.0% v v−1, 17–170 mM) induced a dose-dependent maximal stimulation of p44mapk/p42mapk at 30 min and expression of c-fos mRNA with a maximum at 120 min. Intracellular events upstream to MAP kinase, like an increase in [Ca2+]i, activation of the Na+/H+ exchanger and formation of phosphoinositol metabolites were also markedly activated by ethanol. Treatment of VSMCs with ethanol for 3–5 min induced an increase in DNA synthesis whereas treatment of the cells for more than 30 min was toxic. Caspase 3 activity was not modulated by ethanol treatment of VSMCs. We may postulate that the activation of these mitogenic signals including the elevation of DNA synthesis reflects a cell effort to protect itself against the toxic effects of ethanol. British Journal of Pharmacology (1999) 128, 1761–1771; doi:10.1038/sj.bjp.0702969

1. The composition of glycosphingolipids is altered in atherosclerotic tissue. In order to study the possible modulation of interleukin-1beta (IL-1beta)-induced expression of inducible nitric oxide synthase (iNOS) by endogenously synthesized glycosphingolipids, we investigated rat aortic vascular smooth muscle cells (VSMC) grown in the presence of the inhibitor of glycosphingolipid synthesis, threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP). 2. Depletion of glycosphingolipids by PDMP (20-30 microM) was demonstrated by thin-layer chromatography of D-[1-(14)C]-galactose- or L-[-U14C]-serine-labelled glycosphingolipids. Nitrite generation was measured by the diaminonaphthalene assay, nitric oxide was determined by the oxyhaemoglobin technique and iNOS protein was detected by immunocytochemistry. 3. In VSMC grown in the presence of PDMP, the glycosphingolipid content was reduced by 30-50%. In PDMP-treated VSMC, IL-1beta (3 micro ml[-1])-stimulated release of nitrite (135 +/- 4 nmol mg(-1) protein 48 h[-1]) was significantly increased as compared to IL-1beta-stimulated control cells (40 +/- 3 nmol mg(-1) protein 48 h(-1); n = 6, P < 0.001). Similarly, IL-1beta (3 micro ml(-1), 36 h)-stimulated release of nitric oxide was higher in PDMP-treated VSMC (6.1 +/- 0.5 nmol mg(-1) protein h[-1]) as compared to untreated cells (2.0 +/- 0.6 nmol mg(-1) protein h(-1); n = 3, P < 0.01). These findings were confirmed by the demonstration of increased expression of iNOS protein (14.9 +/- 1.2% vs 6.4 +/- 0.2%; n = 4, P < 0.001), as shown by immunocytochemistry. 4. Evidence is presented that endogenous glycosphingolipids are important modulators of cytokine-induced iNOS expression. In view of an altered glycosphingolipid profile in atherosclerotic arteries, these mechanisms might be of relevance for the pathogenesis of atherosclerosis and restenosis subsequent to vessel injury.

1. Hoe 694 (3-methylsulphonyl-4-piperidinobenzoyl)guanidine methanesulphonate) was characterized as a new, potent, non-amiloride inhibitor of the Na+/H+ exchanger. In order to elucidate the role of the Na+/H+ exchanger isoform 1 (NHE-1) in the regulation of vascular smooth muscle cell growth, we investigated the effects of different amiloride analogues and of Hoe 694 on angiotensin II-induced cell growth. Since intracellular pH, the intracellular free Ca2+ concentration and the expression of the transcription factor c-fos seem to be involved in the regulation of cell growth, the effects of the amiloride analogues and Hoe 694 on the angiotensin II-induced changes in these three parameters were examined. 2. Measurement of cytosolic Ca2+ and pH in cell monolayers was performed using fura-2/AM and BCECF/AM, respectively. The effect of angiotensin II on cell growth was examined using (1) [3H]-thymidine incorporation, (2) the bromo-2-deoxyuridine (BrdU) immunfluorescence assay, (3) the colorimetric determination of cell mitochondrial dehydrogenase activity and (4) determination of cell number. Total RNA was extracted from cells by the guanidinium isothiocyanate/CsCl procedure. The expression of c-fos was quantitated by Northern blotting. 3. Various amiloride analogues inhibited the angiotensin II-induced stimulation of the Na+/H+ exchanger, the increase in cytosolic Ca2+ and cell growth but not the induction of c-fos mRNA. Hoe 694 (1-25 microM) dose-dependently inhibited the angiotensin II-induced stimulation of the Na+/H+ exchanger but had no significant effects on cytosolic Ca2+, c-fos mRNA levels or cell growth. 4. Our findings support the concept that activation of the Na+/H+ exchanger is not essential for angiotensin II-induced vascular smooth muscle cell growth.

SPECIFIC AIMSAbnormal vascular smooth muscle cell (VSMC) proliferation and the platelet-derived growth factor (PDGF) play an important role in the development and progression of proliferative cardiovascular diseases. In the present study, we examined the effects of natural plant-derived catechins (isolated from green tea leafs) with different chemical structure on cell growth and on the intracellular signal transduction pathway of PDGF-BB in rat and human aortic VSMC.PRINCIPAL FINDINGS1. Treatment of rat aortic VSMC for 24 h with 20 μM epigallocatechin-3 gallate (EGCG), catechin-3 gallate (CG), and epicatechin-3 gallate (ECG), which possess a galloyl group in the 3 position of the catechin structure, caused an ∼70% inhibition as well as complete inhibition of the (50 ng/ml) PDGF-BB-induced DNA synthesis, respectively.Epigallocatechin (EGC) that possess a galloyl group in the 2 position also caused a 70% inhibition of the DNA synthesis. Treatment of VSMC with 50 μM CG, EGCG, ECG, and EGC resulted in comple...