Molecular signatures of angiogenesis inhibitors: a single-embryo untargeted metabolomics approach in zebrafish.

Angiogenesis is a key process in embryonic development, a disruption of this process can lead to severe developmental defects, such as limb malformations. The identification of molecular perturbations representative of antiangiogenesis in zebrafish embryo (ZFE) may guide the assessment of developmental toxicity from an endpoint- to a mechanism-based approach, thereby improving the extrapolation of findings to humans. Thus, the aim of the study was to discover molecular changes characteristic of antiangiogenesis and developmental toxicity. We exposed ZFEs to two antiangiogenic drugs (SU4312, sorafenib) and two developmental toxicants (methotrexate, rotenone) with putative antiangiogenic action. Molecular changes were measured by performing untargeted metabolomics in single embryos. The metabolome response was accompanied by the occurrence of morphological alterations. Two distinct metabolic effect patterns were observed. The first pattern comprised common effects of two specific angiogenesis inhibitors and the known teratogen methotrexate, strongly suggesting a shared mode of action of antiangiogenesis and developmental toxicity. The second pattern involved joint effects of methotrexate and rotenone, likely related to disturbances in energy metabolism. The metabolites of the first pattern, such as phosphatidylserines, pterines, retinol, or coenzyme Q precursors, represented potential links to antiangiogenesis and related developmental toxicity. The metabolic effect pattern can contribute to biomarker identification for a mechanism-based toxicological testing.

The short-term toxicity and metabolome of dicyclopentadiene.

Dicyclopentadiene (DCPD) was investigated in a 14-day oral rat toxicity study based on the OECD 407 guideline in combination with plasma metabolomics. Wistar rats received the compound daily via gavage at dose levels of 0, 50 and 150 mg/kg bw. The high dose induced transient clinical signs of toxicity and in males only reduced body weight gain. High dose liver changes were characterized by altered clinical chemistry parameters in both sexes and pathological changes in females. In high dose males an accumulation of alpha-2 u-globulin in the kidney was noted. Comparing the DCPD metabolome with previously established specific metabolome patterns in the MetaMap® Tox data base suggested that the high dose would result in liver enzyme induction leading to increased breakdown of thyroid hormones for males and females. An indication for liver toxicity in males was also noted. Metabolomics also suggested an effect on the functionality of the adrenals in high dose males, which together with published data, is suggestive of a stress related effect in this organ. The results of the present 14-day combined toxicity and metabolome investigations were qualitatively in line with literature data from subchronic oral studies in rats with DCPD. Importantly no other types of organ toxicity, or hormone dysregulation beyond the ones associated with liver enzyme induction and stress were indicated, again in line with results of published 90-day studies. It is therefore suggested that short term "smart" studies, combining classical toxicity with 'omics technologies, could be a 2 R (refine and reduce) new approach method allowing for the reduction of in vivo toxicity testing.

A metabolomics approach to reveal the mechanism of developmental toxicity in zebrafish embryos exposed to 6-propyl-2-thiouracil.

A crucial component of a substance registration and regulation is the evaluation of human prenatal developmental toxicity. Current toxicological tests are based on mammalian models, but these are costly, time consuming and may pose ethical concerns. The zebrafish embryo has evolved as a promising alternative model to study developmental toxicity. However, the implementation of the zebrafish embryotoxicity test is challenged by lacking information on the relevance of observed morphological alterations in fish for human developmental toxicity. Elucidating the mechanism of toxicity could help to overcome this limitation. Through LC-MS/MS and GC-MS metabolomics, we investigated whether changes to the endogenous metabolites can indicate pathways associated with developmental toxicity. To this aim, zebrafish embryos were exposed to different concentrations of 6-propyl-2-thiouracil (PTU), a compound known to induce developmental toxicity. The reproducibility and the concentration-dependence of the metabolome response and its association with morphological alterations were studied. Major morphological findings were reduced eye size, and other craniofacial anomalies; major metabolic changes included increased tyrosine, pipecolic acid and lysophosphatidylcholine levels, decreased methionine levels, and disturbance of the 'Phenylalanine, tyrosine and tryptophan biosynthesis' pathway. This pathway, and the changes in tyrosine and pipecolic acid levels could be linked to the mode of action of PTU, i.e., inhibition of thyroid peroxidase (TPO). The other findings suggested neurodevelopmental impairments. This proof-of-concept study demonstrated that metabolite changes in zebrafish embryos are robust and provide mechanistic information associated with the mode of action of PTU.

Protocols for the Evaluation of Neurodevelopmental Alterations in Rabbit Models In Vitro and In Vivo.

The rabbit model is gaining importance in the field of neurodevelopmental evaluation due to its higher similarity to humans in terms of brain development and maturation than rodents. In this publication, we detailed 14 protocols covering toxicological relevant endpoints for the assessment of neurodevelopmental adverse effects in the rabbit species. These protocols include both in vitro and in vivo techniques, which also cover different evaluation time-points, the neonatal period, and long-term examinations at postnatal days (PNDs) 50-70. Specifically, the protocols (P) included are as follows: neurosphere preparation (GD30/PND0; P2) and neurosphere assay (P3), behavioral ontogeny (PND1; P4), brain obtaining and brain weight measurement at two different ages: PND1 (P5) and PND70 (P12), neurohistopathological evaluations after immersion fixation for neurons, astrocytes, oligodendrocytes and microglia (PND1; P6-9) or perfusion fixation (PND70; P12), motor activity (P11, open field), memory and sensory function (P11, object recognition test), learning (P10, Skinner box), and histological evaluation of plasticity (P13 and P14) through dendritic spines and perineuronal nets. The expected control values and their variabilities are presented together with the information on how to troubleshoot the most common issues related to each protocol. To sum up, this publication offers a comprehensive compilation of reliable protocols adapted to the rabbit model for neurodevelopmental assessment in toxicology.

Pluripotent stem cell assays: Modalities and applications for predictive developmental toxicity.

This manuscript provides a review focused on embryonic stem cell-based models and their place within the landscape of alternative developmental toxicity assays. Against the background of the principles of developmental toxicology, the wide diversity of alternative methods using pluripotent stem cells developed in this area over the past half century is reviewed. In order to provide an overview of available models, a systematic scoping review was conducted following a published protocol with inclusion criteria, which were applied to select the assays. Critical aspects including biological domain, readout endpoint, availability of standardized protocols, chemical domain, reproducibility and predictive power of each assay are described in detail, in order to review the applicability and limitations of the platform in general and progress moving forward to implementation. The horizon of innovative routes of promoting regulatory implementation of alternative methods is scanned, and recommendations for further work are given.

A Systematic Review to Compare Chemical Hazard Predictions of the Zebrafish Embryotoxicity Test With Mammalian Prenatal Developmental Toxicity.

Originally developed to inform the acute toxicity of chemicals on fish, the zebrafish embryotoxicity test (ZET) has also been proposed for assessing the prenatal developmental toxicity of chemicals, potentially replacing mammalian studies. Although extensively evaluated in primary studies, a comprehensive review summarizing the available evidence for the ZET's capacity is lacking. Therefore, we conducted a systematic review of how well the presence or absence of exposure-related findings in the ZET predicts prenatal development toxicity in studies with rats and rabbits. A two-tiered systematic review of the developmental toxicity literature was performed, a review of the ZET literature was followed by one of the mammalian literature. Data were extracted using DistillerSR, and study validity was assessed with an amended SYRCLE's risk-of-bias tool. Extracted data were analyzed for each species and substance, which provided the basis for comparing the 2 test methods. Although limited by the number of 24 included chemicals, our results suggest that the ZET has potential to identify chemicals that are mammalian prenatal developmental toxicants, with a tendency for overprediction. Furthermore, our analysis confirmed the need for further standardization of the ZET. In addition, we identified contextual and methodological challenges in the application of systematic review approaches to toxicological questions. One key to overcoming these challenges is a transition to more comprehensive and transparent planning, conduct and reporting of toxicological studies. The first step toward bringing about this change is to create broad awareness in the toxicological community of the need for and benefits of more evidence-based approaches.

Implementation of a functional endpoint to the zebrafish embryotoxicity test to evaluate craniofacial abnormalities.

The inclusion of a read-out to detect functional consequences of craniofacial alterations in the zebrafish embryotoxicity test will allow to evaluate these alterations which are difficult to assess morphologically, and to detect alterations in cranial nerves functions leading to impairment of jaw movements. In this study we have established an ingestion test in zebrafish larvae younger than 120 hpf. To overcome the challenge of evaluating larvae which still do not present independent feeding behaviour, we have tested the ability of 72, 96 or 102 hpf larvae to ingest food mixed with fluorescent microspheres under several conditions (dark/light, with/without shaking) to find the best experimental set-up for the test. We have included the investigation of two substances as potential positive controls: ketoconazole and tricaine. Ketoconazole 10 µM exposure during development produced significant embryotoxic effects including a characteristic craniofacial alteration pattern consisting in impaired development of brain, nasal cavity, mouth opening and jaw, as well as a significant decrease in food intake. Tricaine exposure at 380 µM during the food availability period significantly decreased the food intake. The method proposed will be a useful alternative tool to animal testing to detect compounds inducing adverse effects on craniofacial development.

Adaptation of the Systematic Review Framework to the Assessment of Toxicological Test Methods: Challenges and Lessons Learned With the Zebrafish Embryotoxicity Test.

Systematic review methodology is a means of addressing specific questions through structured, consistent, and transparent examinations of the relevant scientific evidence. This methodology has been used to advantage in clinical medicine, and is being adapted for use in other disciplines. Although some applications to toxicology have been explored, especially for hazard identification, the present preparatory study is, to our knowledge, the first attempt to adapt it to the assessment of toxicological test methods. As our test case, we chose the zebrafish embryotoxicity test (ZET) for developmental toxicity and its mammalian counterpart, the standard mammalian prenatal development toxicity study, focusing the review on how well the ZET predicts the presence or absence of chemical-induced prenatal developmental toxicity observed in mammalian studies. An interdisciplinary team prepared a systematic review protocol and adjusted it throughout this piloting phase, where needed. The final protocol was registered and will guide the main study (systematic review), which will execute the protocol to comprehensively answer the review question. The goal of this preparatory study was to translate systematic review methodology to the assessment of toxicological test method performance. Consequently, it focused on the methodological issues encountered, whereas the main study will report substantive findings. These relate to numerous systematic review steps, but primarily to searching and selecting the evidence. Applying the lessons learned to these challenges can improve not only our main study, but may also be helpful to others seeking to use systematic review methodology to compare toxicological test methods. We conclude with a series of recommendations that, if adopted, would help improve the quality of the published literature, and make conducting systematic reviews of toxicological studies faster and easier over time.

Uterine adenocarcinoma in the rat induced by afidopyropen. An analysis of the lesion's induction, progression and its relevance to humans.

Afidopyropen is a novel insecticide that acts as a TRPV channel modulator in chordotonal organs of target insects. In two carcinogenicity studies with Fischer rats, an increased incidence of uterine adenocarcinomas was observed at 1000 and 3000 ppm. This finding prompted an investigation of the mechanism of the tumor formation as well as the relevance of this mechanism to humans. The mechanistic work took parallel paths: one path investigated the pharmacokinetic properties of the test substance at the doses where the tumors were found; while the second path examined the key mechanistic events that culminated in uterine adenocarcinomas. The results of the investigation indicated that the tumors only occurred at doses where excretion of test substance was saturated - indicating that homeostatic biological and/or physiological processes were overwhelmed. At the doses where these processes were overwhelmed, the test substance acted through a mechanism of dopamine agonism, triggering a cascade key events that resulted in uterine adenocarcinomas. An analysis of these mechanisms observed in rat showed that they are both quantitatively (pharmacokinetic mechanism) and qualitatively (dopamine agonism mechanism) not relevant to humans. Therefore the uterine adenocarcinomas observed in the rat associated with high doses of Afidopyropen are not expected to pose a carcinogenic risk to humans.

Investigations of putative reproductive toxicity of low-dose exposures to vinclozolin in Wistar rats.

The current investigation examines whether the fungicide vinclozolin, which has an anti-androgenic mode of action, is capable of disrupting endocrine homeostasis at very low doses. The data generated clarify whether a non-monotonic dose-response relationship exists to enhance the current debate about the regulation of endocrine disruptors. Moreover, it is part of a series of investigations assessing the dose-response relationship of single and combined administration of anti-androgenic substances. A pre-postnatal in vivo study design was chosen which was compliant with regulatory testing protocols. The test design was improved by additional endpoints addressing hormone levels, morphology and histopathological examinations. Doses were chosen to represent an effect level (20 mg/kg bw/d), the current NOAEL (4 mg/kg bw/d), and a dose close to the "ADI" (0.005 mg/kg bw/d) for the detection of a possible non-monotonic dose-response curve. Anti-androgenic changes were observable at the effect level but not at lower exposures. Nipple/areola counts appeared to be the most sensitive measure of effect, followed by male sex organ weights at sexual maturation, and finally gross and histopathological findings. The results indicate the absence of evidence for effects at low or very low dose levels. A non-monotonic dose-response relationship was not evident.

Extended evaluation on the ES-D3 cell differentiation assay combined with the BeWo transport model, to predict relative developmental toxicity of triazole compounds.

The mouse embryonic stem D3 (ES-D3) cell differentiation assay is based on the morphometric measurement of cardiomyocyte differentiation and is a promising tool to detect developmental toxicity of compounds. The BeWo transport model, consisting of BeWo b30 cells grown on transwell inserts and mimicking the placental barrier, is useful to determine relative placental transport velocities of compounds. We have previously demonstrated the usefulness of the ES-D3 cell differentiation assay in combination with the in vitro BeWo transport model to predict the relative in vivo developmental toxicity potencies of a set of reference azole compounds. To further evaluate this combined in vitro toxicokinetic and toxicodynamic approach, we combined ES-D3 cell differentiation data of six novel triazoles with relative transport rates obtained from the BeWo model and compared the obtained ranking to the developmental toxicity ranking as derived from in vivo data. The data show that the combined in vitro approach provided a correct prediction for in vivo developmental toxicity, whereas the ES-D3 cell differentiation assay as stand-alone did not. In conclusion, we have validated the combined in vitro approach for developmental toxicity, which we have previously developed with a set of reference azoles, for a set of six novel triazoles. We suggest that this combined model, which takes both toxicodynamic and toxicokinetic aspects into account, should be further validated for other chemical classes of developmental toxicants.

Stem cell-derived systems in toxicology assessment.

Industrial sectors perform toxicological assessments of their potential products to ensure human safety and to fulfill regulatory requirements. These assessments often involve animal testing, but ethical, cost, and time concerns, together with a ban on it in specific sectors, make appropriate in vitro systems indispensable in toxicology. In this study, we summarize the outcome of an EPAA (European Partnership of Alternatives to Animal Testing)-organized workshop on the use of stem cell-derived (SCD) systems in toxicology, with a focus on industrial applications. SCD systems, in particular, induced pluripotent stem cell-derived, provide physiological cell culture systems of easy access and amenable to a variety of assays. They also present the opportunity to apply the vast repository of existing nonclinical data for the understanding of in vitro to in vivo translation. SCD systems from several toxicologically relevant tissues exist; they generally recapitulate many aspects of physiology and respond to toxicological and pharmacological interventions. However, focused research is necessary to accelerate implementation of SCD systems in an industrial setting and subsequent use of such systems by regulatory authorities. Research is required into the phenotypic characterization of the systems, since methods and protocols for generating terminally differentiated SCD cells are still lacking. Organotypical 3D culture systems in bioreactors and microscale tissue engineering technologies should be fostered, as they promote and maintain differentiation and support coculture systems. They need further development and validation for their successful implementation in toxicity testing in industry. Analytical measures also need to be implemented to enable compound exposure and metabolism measurements for in vitro to in vivo extrapolation. The future of SCD toxicological tests will combine advanced cell culture technologies and biokinetic measurements to support regulatory and research applications. However, scientific and technical hurdles must be overcome before SCD in vitro methods undergo appropriate validation and become accepted in the regulatory arena.

Identification of a potent endothelium-derived angiogenic factor.

The secretion of angiogenic factors by vascular endothelial cells is one of the key mechanisms of angiogenesis. Here we report on the isolation of a new potent angiogenic factor, diuridine tetraphosphate (Up4U) from the secretome of human endothelial cells. The angiogenic effect of the endothelial secretome was partially reduced after incubation with alkaline phosphatase and abolished in the presence of suramin. In one fraction, purified to homogeneity by reversed phase and affinity chromatography, Up4U was identified by MALDI-LIFT-fragment-mass-spectrometry, enzymatic cleavage analysis and retention-time comparison. Beside a strong angiogenic effect on the yolk sac membrane and the developing rat embryo itself, Up4U increased the proliferation rate of endothelial cells and, in the presence of PDGF, of vascular smooth muscle cells. Up4U stimulated the migration rate of endothelial cells via P2Y2-receptors, increased the ability of endothelial cells to form capillary-like tubes and acts as a potent inducer of sprouting angiogenesis originating from gel-embedded EC spheroids. Endothelial cells released Up4U after stimulation with shear stress. Mean total plasma Up4U concentrations of healthy subjects (N=6) were sufficient to induce angiogenic and proliferative effects (1.34 ± 0.26 nmol L(-1)). In conclusion, Up4U is a novel strong human endothelium-derived angiogenic factor.

A testing strategy for the identification of mammalian, systemic endocrine disruptors with particular focus on steroids.

Most endocrine disruptors interact with hormone receptors or steroid biosynthesis and metabolism, thereby modifying the physiological function of endogenous hormones. Here, we present an alternative testing paradigm for detection of endocrine modes of action that replace and reduce animal testing through refinement. Receptor mediated endocrine effects were assessed using the yeast-based receptor-mediated transcriptional activation YES/YAS assays and effects on steroid hormone biosynthesis were assessed using the human cell line H295R in the steroidogenesis assay. In our testing paradigm we propose to complement the in vitro assays with a single in vivo repeated dose study in which plasma samples are analyzed for their metabolome profile in addition to classical parameters such as histopathology. The combination of these methods does not only contribute to refinement and reduction of animal testing, but also has significantly increased the efficient allocation of resources and allows for a sound assessment of the endocrine disruption potential of compounds. Thus, this proposal constitutes a potentially attractive alternative to EPA's Endocrine Disruptor Screening Program to identify mammalian, systemic endocrine modes of action. Data on 14 reference substances for which the in vitro YES/YAS and steroidogenesis assays and the in vivo metabolome analysis were performed to assess their putative endocrine modes of action are presented here.

The impact of vascular endothelial growth factor and basic fibroblast growth factor on cardiac fibroblasts grown under altered gravity conditions.

BACKGROUND: Myocardium is very sensitive to gravitational changes. During a spaceflight cardiovascular atrophy paired with rhythm problems and orthostatic intolerance can occur. The aim of this study was to investigate the impact of basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) on cardiac fibroblasts (CF) grown under altered gravity conditions. METHODS: We examined the influence of exposure to a Random Positioning Machine (RPM) on CF, derived from porcine hearts. We focused on growth, extracellular matrix protein (ECMP) synthesis and apoptosis. RESULTS: When cultured on a RPM, CF began to form 3D spheroids within 24h, irrespective of growth factor treatment. Exposure to RPM induced an increased synthesis of ECMP and also resulted in elevated apoptosis in adherent CF as measured by terminal deoxynucleotidyl transferase-mediated dUTP digoxigenin nick end labeling (TUNEL) analysis, 4',6-diamidino-2-phenylindole (DAPI) staining, and caspase-3 detection. bFGF and VEGF significantly decreased the amount of ECMP (collagen type I, III, chondroitin sulfate) in 1g and RPM cultures, and also significantly reduced the amount of apoptotic CF as well as caspase-3. CONCLUSIONS: Altered gravity conditions on a RPM induced 3D growth, elevated ECMP synthesis and apoptosis in cardiac fibroblasts. Growth factor treatment attenuated programmed cell death and ECMP secretion.

Embryotoxic potential of N-methyl-pyrrolidone (NMP) and three of its metabolites using the rat whole embryo culture system.

N-methyl-2-pyrrolidone (NMP), which undergoes extensive biotransformation, has been shown in vivo to cause developmental toxicity and, especially after oral treatment, malformations in rats and rabbits. Data are lacking as to whether the original compound or one of its main metabolites is responsible for the toxic effects observed. Therefore, the relative embryotoxicity of the parent compound and its metabolites was evaluated using rat whole embryo culture (WEC) and the balb/c 3T3 cytotoxicity test. The resulting data were evaluated using two strategies; namely, one based on using all endpoints determined in the WEC and the other including endpoints from both the WEC and the cytotoxicity test. On basis of the first analysis, the substance with the highest embryotoxic potential is NMP, followed by 5-hydroxy-N-methyl-pyrrolidone (5-HNMP), 2-hydroxy-N-methylsuccinimide (2-HMSI) and N-methylsuccinimide (MSI). Specific dysmorphogeneses induced by NMP and 5-HNMP were aberrations in the head region of the embryos, abnormal development of the second visceral arches and open neural pores. The second evaluation strategy used only two endpoints of the WEC, i.e. the no observed adverse effect concentration (NOAEC(WEC)) and the lowest concentration leading to dysmorphogenesis in 100% of the cultured embryos (IC(Max WEC)). In addition to these WEC endpoints the IC(50 3T3) from the cytotoxicity test (balb/c 3T3 fibroblasts) was included in the evaluation scheme. These three endpoints were applied to a prediction model developed during a validation study of the European Centre for the Validation of Alternative Methods (ECVAM) allowing the classification of the embryotoxic potential of each compound into three classes (non-, weakly- and strongly embryotoxic). Consistent results from both evaluation strategies were observed, whereby NMP and its metabolites revealed a direct embryotoxic potential. Hereby, only NMP and 5-HNMP induced specific embryotoxic effects and were classified as weakly embryotoxic, whereas the other two metabolites, 2-HMSI and MSI, were determined to be non-embryotoxic.

A delayed type of three-dimensional growth of human endothelial cells under simulated weightlessness.

Endothelial cells (ECs) form three-dimensional (3D) aggregates without any scaffold when they are exposed to microgravity simulated by a random positioning machine (RPM) but not under static conditions at gravity. Here we describe a delayed type of formation of 3D structures of ECs that was initiated when ECs cultured on a desktop RPM remained adherent for the first 5 days but spread over neighboring adherent cells, forming little colonies. After 2 weeks, tube-like structures (TSs) became visible in these cultures. They included a lumen, and they elongated during another 2 weeks of culturing. The walls of these TSs consisted mainly of single-layered ECs, which had produced significantly more beta(1)-integrin, laminin, fibronectin, and alpha-tubulin than ECs simultaneously grown adhering to the culture dishes under microgravity or normal gravity. The amount of actin protein was similar in ECs incorporated in TSs and in ECs growing at gravity. The ratio of tissue inhibitor of metalloproteinases-1 to matrix metalloproteinase-2 found in the supernatants was lower at the seventh than at the 28th day of culturing. These results suggest that culturing ECs under conditions of modeled gravitational unloading represents a new technique for studying the formation of tubes that resemble vascular intimas.

Effects of macrolides on proinflammatory epitopes on endothelial cells in vitro.

An inflammatory reaction at the site of infusion is a common clinical problem that is observed after the intravenous application of antibiotics and other drugs. The pathomechanism of this infusion-related phlebitis is not fully understood. We analyzed the effects of the three macrolide antibiotics erythromycin, clarithromycin and azithromycin on human endothelial cells in vitro. As a positive control quinupristin/dalfopristin was studied. The cytotoxicity of all substances was analyzed by a modified MTT cytotoxicity assay with 3T3-fibroblasts and EA.hy 926 endothelial cells. Cells were incubated for 10 days with the antibiotics. After adding MTT the optical density was measured which correlates with cell death. Clarithromycin exhibited the strongest cytotoxic effect on EA.hy 926 cells (EC(50) 30 mg/L), followed by azithromycin (EC(50) 40 mg/L), a cytotoxic effect of erythromycin could only be observed at much higher concentrations (EC(50) 310 mg/L). The reaction of the endothelial cells was further analyzed in detail by means of flow cytometry. For these experiments the endothelial cell line EA.hy 926 as well as primary cells (HUVEC) were used. The antigens were stained with fluoresceinisothiocyanat- or phycoerythrin-conjugated monoclonal antibodies for the following surface antigens: CD34, E-selectin (CD62E), ICAM-1 (CD54) and VCAM-1 (CD106). Cells were incubated with the antibiotics at concentrations ranging from 100 to 800 mg/L (clarithromycin and azithromycin) and from 200 to 1,200 mg/L (erythromycin). These concentrations occur under therapeutic conditions at the site of infusion. Cells were incubated for 2 h and analysis was carried out after an additional culture period of 22 h without test compounds. A significantly enhanced expression of all four antigens was observed which was most pronounced at 800 mg/L (erythromycin), 600 mg/L (azithromycin) and 400 mg/L (clarithromycin). A concentration of 800 mg/L erythromycin medium caused an increase of the expression of CD34 (+6%), E-selectin (+5%), ICAM-1 (+14%) and VCAM-1 (+5%). At lower concentrations (600 mg/L) azithromycin provokes a stronger upregulation of the proinflammatory antigens: CD34 (+17%), E-selectin (+18%), ICAM-1 (+27%) and VCAM-1 (+17%). At a concentration of 400 mg/L medium clarithromycin induced a similar effect as erythromycin at twice this concentration: CD34 (+5%), E-selectin (+7%), ICAM-1 (+23%) and VCAM-1 (+4%). Reactions of the HUVECs were less pronounced than those of the EA.hy 926 cells. Cell surface markers involved in interactions between endothelial cells and leukocytes proved to be useful markers to study differences in the proinflammatory potential of the three macrolides. By analysing the upregulation of these antigens on EA.hy 926 cells in vitro the risk of phlebitis could be predictable for other drugs as well.

Effects of the vasodilating beta-blocker nebivolol on smoking-induced endothelial dysfunction in young healthy volunteers.

OBJECTIVE: To assess the effect of nebivolol, a highly selective third generation beta1-adrenoceptor antagonist with an endothelium-dependent vasodilatory action, on smoking-induced endothelial dysfunction. RESEARCH DESIGN AND METHODS: This open-label study examined the effect of 14 daily doses of 5 mg nebivolol on forearm blood flow in 21 healthy, young, male, light smokers (< or =5 cigarettes/day), measured by plethysmography on Days 1, 7, and 14. The primary endpoint was the difference in forearm blood flow after smoking one standard cigarette from baseline (Day 1) until treatment end on Day 14. Secondary outcomes included the difference in forearm blood flow between Day 1 and Day 7 compared with Day 14 before and after smoking, the effect of nebivolol on blood coagulation parameters, high-sensitive-C-reactive protein (hs-CRP), and the safety and tolerability of nebivolol. RESULTS: Nebivolol for 14 days did not significantly affect forearm blood flow after smoking. On Day 7 of nebivolol treatment, forearm blood flow after smoking was significantly greater than blood flow before smoking (increase of 0.44 mL/min; p = 0.00656). Serum level ofhs-CRP showed a marked decrease from Day 1 to Day 14. No changes in coagulation parameters were observed over the course of nebivolol treatment. Nebivolol was well tolerated throughout the study. CONCLUSIONS: The increase in forearm blood flow and the marked decrease in hs-CRP over 14 days of treatment suggest that nebivolol has a positive effect on endothelial function in light smokers, but larger studies are required to confirm these observations.

Effects of basic fibroblast growth factor on endothelial cells under conditions of simulated microgravity.

Fibroblast growth factors interact with appropriate endothelial cell (EC) surface receptors and initiate intracellular signal cascades, which participate in modulating blood vessel growth. EC, upon exposure to basic fibroblast growth factors (bFGFs) undergo profound functional alterations, which depend on their actual sensitivity and involve gene expression and de novo protein synthesis. We investigated the effects of bFGF on signaling pathways of EA.hy926 cells in different environments. EC were cultured under normal gravity (1 g) and simulated microgravity (micro g) using a three-dimensional (3D) clinostat. Microgravity induced early and late apoptosis, extracellular matrix proteins, endothelin-1 (ET-1) and TGF-beta(1) expression. Microgravity reduced eNOS mRNA within 24 h. Moreover, a six- to eightfold higher amount of IL-6 and IL-8 was secreted within 24 h micro g. In addition, microgravity induced a duplication of NF-kappaB p50, while p65 was quadrupled. At 1 g, bFGF application (4 h) reduced ET-1, TGF-beta(1) and eNOS gene expression. After 24 h, bFGF enhanced fibronectin, VEGF, Flk-1, Flt-1, the release of IL-6, IL-8, and TGF-beta(1). Furthermore, bFGF promoted apoptosis, reduced NFkB p50, but enhanced NFkB p65. After 4 h micro g, bFGF decreased TGF-beta(1), eNOS, and ET-1 gene expression. After 24 h micro g, bFGF elevated fibronectin, Flk-1 and Flt-1 protein, and reduced IL-6 and IL-8 compared with vehicle treated micro g cultures. In micro g, bFGF enhanced NF-KappaB p50 by 50%, Bax by 25% and attenuated p65, activation of caspase-3 and annexin V-positive cells. bFGF differently changes intracellular signals in ECs depending whether it is applied under microgravity or normal gravity conditions. In microgravity, bFGF contributes to protect the EC from apoptosis.