Validation of the hen's egg test for micronucleus induction (HET-MN): detailed protocol including scoring atlas, historical control data and statistical analysis.

A validation exercise of the hen's egg test for micronucleus induction was finalised with a very good predictivity based on the analysis of micronuclei in peripheral erythrocytes of fertilised chicken eggs (Reisinger et al. The hen's egg test for micronucleus-induction (HET-MN): validation data set. Mutagenesis, this issue). For transparency reasons this complementary publication provides further details on the assay especially as it was the first validation study in the field of genotoxicity testing involving the use of chicken eggs. Thus, the experimental protocol is described in detail and is complemented by a scoring atlas for microscopic analysis in blood cells. In addition, general characteristics of the test system, which is able to mirror the systemic availability of test compounds, are delineated: the test compound passes the egg membrane and is taken up by the blood vessels of the underlying chorioallantoic membrane. Subsequently, it is distributed by the circulating blood, metabolised by the developing liver and the yolk sac membrane and finally excreted into the allantois, a bladder equivalent. In specific, the suitability of the test system for genotoxicity testing is shown by, inter alia, a low background DNA damage in a comprehensive historical control database. In addition, the state-of-the-art statistical method used to evaluate obtained data is delineated. It combines laboratory-specific effect threshold with the Umbrella-Williams test, a statistical model also of interest for other genotoxicity test methods.

The hen's egg test for micronucleus induction (HET-MN): validation data set.

The classical in vitro genotoxicity test battery is known to be sensitive for indicating genotoxicity. However, a high rate of 'misleading positives' was reported when three assays were combined as required by several legislations. Despite the recent optimisations of the standard in vitro tests, two gaps could hardly be addressed with assays based on 2D monolayer cell cultures: the route of exposure and a relevant intrinsic metabolic capacity to transform pro-mutagens into reactive metabolites. Following these considerations, fertilised chicken eggs have been introduced into genotoxicity testing and were combined with a classical read-out parameter, the micronucleus frequency in circulating erythrocytes, to develop the hen's egg test for micronucleus induction (HET-MN). As a major advantage, the test mirrors the systemic availability of compounds after oral exposure by reflecting certain steps of Absorption, Distribution, Metabolism, Excretion (ADME) without being considered as an animal experiment. The assay is supposed to add to a toolbox of assays to follow up on positive findings from initial testing with classical in vitro assays. We here report on a validation exercise, in which >30 chemicals were tested double-blinded in three laboratories. The specificity and sensitivity of the HET-MN were calculated to be 98 and 84%, respectively, corresponding to an overall accuracy of 91%. A detailed protocol, which includes a picture atlas detailing the cell and micronuclei analysis, is published in parallel (Maul et al. Validation of the hen's egg test for micronucleus induction (HET-MN): detailed protocol including scoring atlas, historical control data and statistical analysis).

Landscape of T Follicular Helper Cell Dynamics in Human Germinal Centers.

T follicular helper (Tfh) cells play a very important role in mounting a humoral response. Studies conducted in mouse models have revealed with good kinetic and spatial resolution the dynamics of these cells in germinal centers (GC) and their cross-talk with B cells upon an immune response. However, whether a similar migratory behavior is performed by human Tfh cells is unclear, as technology to track them in situ has been lacking. In this study, we combined traditional immunohistochemistry and real-time fluorescent imaging approaches on fresh human adenoid slices to provide static and dynamic information on Tfh cells. Our data indicate that GC light zones are composed of two distinct areas in terms of Tfh cell distribution and migration. In the outer GC light zones, Tfh cells migrate actively and with a high ability to form dynamic clusters showing intense and rapid reorganization. In these outer regions, Tfh cells demonstrate multiple interactions between each other. Conversely, in central regions of GC light zones, Tfh cells are much more static, forming long-lasting conjugates. These findings reveal for the first time, to our knowledge, the dynamic behavior whereby Tfh cells migrate in human GC and highlight the heterogeneity of GC for Tfh cell motility.

Validation of the 3D reconstructed human skin Comet assay, an animal-free alternative for following-up positive results from standard in vitro genotoxicity assays.

As part of the safety assessment process, all industrial sectors employ genotoxicity test batteries, starting with well-established in vitro assays. However, these batteries have limited predictive capacity for the in vivo situation, which may result in unnecessary follow-up in vivo testing or the loss of promising substances where animal tests are prohibited or not desired. To address this, a project involving regulators, academia and industry was established to develop and validate in vitro human skin-based genotoxicity assays for topically exposed substances, such as cosmetics ingredients. Here, we describe the validation of the 3D reconstructed skin (RS) Comet assay. In this multicenter study, chemicals were applied topically three times to the skin over 48 h. Isolated keratinocytes and fibroblasts were transferred to slides before electrophoresis and the resulting comet formation was recorded as % tail DNA. Before decoding, results of the validation exercise for 32 substances were evaluated by an independent statistician. There was a high predictive capacity of this assay when compared to in vivo outcomes, with a sensitivity of 77 (80)%, a specificity of 88 (97)% and an overall accuracy of 83 (92)%. The numbers reflect the calls of the performing laboratories in the coded phase, whereas those in parenthesis reflect calls according to the agreed evaluation criteria. Intra- and inter-laboratory reproducibility was also very good, with a concordance of 93 and 88%, respectively. These results generated with the Phenion® Full-Thickness skin model demonstrate its suitability for this assay, with reproducibly low background DNA damage and sufficient metabolic capacity to activate pro-mutagens. The validation outcome supports the use of the RS Comet assay to follow up positive results from standard in vitro genotoxicity assays when the expected route of exposure is dermal. Based on the available data, the assay was accepted recently into the OECD test guideline development program.

Knowledge sharing to facilitate regulatory decision-making in regard to alternatives to animal testing: Report of an EPAA workshop.

The European Partnership for Alternative Approaches to Animal Testing (EPAA) convened a workshop Knowledge sharing to facilitate regulatory decision-making. Fifty invited participants from the European Commission, national and European agencies and bodies, different industry sectors (chemicals, cosmetics, fragrances, pharmaceuticals, vaccines), and animal protection organizations attended the workshop. Four case studies exemplarily revealed which procedures are in place to obtain regulatory acceptance of new test methods in different sectors. Breakout groups discussed the status quo identifying the following facilitators for regulatory acceptance of alternatives to animal testing: Networking and communication (including cross-sector collaboration, international cooperation and harmonization); involvement of regulatory agencies from the initial stages of test method development on; certainty on prerequisites for test method acceptance including the establishment of specific criteria for regulatory acceptance. Data sharing and intellectual property issues affect many aspects of test method development, validation and regulatory acceptance. In principle, all activities should address replacement, reduction and refinement methods (albeit animal testing is generally prohibited in the cosmetics sector). Provision of financial resources and education support all activities aiming at facilitating the acceptance and use of alternatives to animal testing. Overall, workshop participants recommended building confidence in new methodologies by applying and gaining experience with them.

Dermal xenobiotic metabolism: a comparison between native human skin, four in vitro skin test systems and a liver system.

The xenobiotic metabolism of 4 in vitro human skin test systems (2D and 3D) was compared with that of the native human skin samples from which the skin test systems had been produced. In total 3 skin samples were investigated, each from a different donor to exclude variability due to gender, donor or tissue supplier. In addition, the skin cultures were compared with a surrogate of the liver. Basal and induced phase I and phase II enzymes were analyzed regarding gene/protein expression as well as enzyme activity. The distinctions between the different test systems and the two dermal compartments (epidermis and dermis) were more noticeable than any donor variability. The 3D models of skin and liver mirrored the in vivo situation more realistically than did the monolayer cultures. Phase I metabolism was more pronounced in the hepatic model, whereas phase II metabolism was more prominent in the reconstructed skin. These results show that reconstructed skin models are a valuable tool for organ-specific safety assessment with regard to xenobiotic metabolism.

Comet assay in reconstructed 3D human epidermal skin models--investigation of intra- and inter-laboratory reproducibility with coded chemicals.

Reconstructed 3D human epidermal skin models are being used increasingly for safety testing of chemicals. Based on EpiDerm™ tissues, an assay was developed in which the tissues were topically exposed to test chemicals for 3h followed by cell isolation and assessment of DNA damage using the comet assay. Inter-laboratory reproducibility of the 3D skin comet assay was initially demonstrated using two model genotoxic carcinogens, methyl methane sulfonate (MMS) and 4-nitroquinoline-n-oxide, and the results showed good concordance among three different laboratories and with in vivo data. In Phase 2 of the project, intra- and inter-laboratory reproducibility was investigated with five coded compounds with different genotoxicity liability tested at three different laboratories. For the genotoxic carcinogens MMS and N-ethyl-N-nitrosourea, all laboratories reported a dose-related and statistically significant increase (P < 0.05) in DNA damage in every experiment. For the genotoxic carcinogen, 2,4-diaminotoluene, the overall result from all laboratories showed a smaller, but significant genotoxic response (P < 0.05). For cyclohexanone (CHN) (non-genotoxic in vitro and in vivo, and non-carcinogenic), an increase compared to the solvent control acetone was observed only in one laboratory. However, the response was not dose related and CHN was judged negative overall, as was p-nitrophenol (p-NP) (genotoxic in vitro but not in vivo and non-carcinogenic), which was the only compound showing clear cytotoxic effects. For p-NP, significant DNA damage generally occurred only at doses that were substantially cytotoxic (>30% cell loss), and the overall response was comparable in all laboratories despite some differences in doses tested. The results of the collaborative study for the coded compounds were generally reproducible among the laboratories involved and intra-laboratory reproducibility was also good. These data indicate that the comet assay in EpiDerm™ skin models is a promising model for the safety assessment of compounds with a dermal route of exposure.

Statistical analysis of the hen's egg test for micronucleus induction (HET-MN assay).

The HET-MN assay (hen's egg test for micronucleus induction) is different from other in vitro genotoxicity assays in that it includes toxicologically important features such as absorption, distribution, metabolic activation, and excretion of the test compound. As a promising follow-up to complement existing in vitro test batteries for genotoxicity, the HET-MN is currently undergoing a formal validation. To optimize the validation, the present study describes a critical analysis of previously obtained HET-MN data to check the experimental design and to identify the most appropriate statistical procedure to evaluate treatment effects. Six statistical challenges (I-VI) of general relevance were identified, and remedies were provided which can be transferred to similarly designed test methods: a Williams-type trend test is proposed for overdispersed counts (II) by means of a square-root transformation which is robust for small sample sizes (I), variance heterogeneity (III), and possible downturn effects at high doses (IV). Due to near-to-zero or even zero-count data occurring in the negative control (V), a conditional comparison of the treatment groups against the mean of the historical controls (VI) instead of the concurrent control was proposed, which is in accordance with US-FDA recommendations. For the modified Williams-type tests, the power can be estimated depending on the magnitude and shape of the trend, the number of dose groups, and the magnitude of the MN counts in the negative control. The experimental design used previously (i.e. six eggs per dose group, scoring of 1000 cells per egg) was confirmed. The proposed approaches are easily available in the statistical computing environment R, and the corresponding R-codes are provided.

Applicability and robustness of the hen's egg test for analysis of micronucleus induction (HET-MN): results from an inter-laboratory trial.

The hen's egg test for analysis of micronucleus formation (HET-MN) was developed several years ago to provide an alternative test system to the in vivo micronucleus test. In order to assess its applicability and robustness, a study was carried out at the University of Osnabrueck (lab A) and at the laboratories of Henkel AG & Co. KGaA (lab B). Following transfer of the method to lab B, a range of test substances that had been pre-tested at lab A, were tested at Henkel: the genotoxins cyclophosphamide, dimethylbenz(a)anthracene, methotrexate, acrylamide, azorubin, N-nitroso-dimethylamine and the non-genotoxins, orange G and isopropyl myristate. In a second phase, additional compounds with known in vivo properties were examined in both labs: the non-genotoxin, ampicillin, the "irrelevant" positives, isophorone and 2,4-dichlorophenol ("irrelevant" means positive in standard in vitro tests, but negative in vivo), the clastogen p-chloroaniline, and the aneugens carbendazim and vinorelbine. All substances were correctly predicted in both labs with respect to their in vivo genotoxic properties, indicating that the HET-MN may have an improved predictivity compared with current standard in vitro test systems. The results support the promising role of the HET-MN assay as a supplement to existing test batteries.

The reconstructed skin micronucleus assay (RSMN) in EpiDerm™: detailed protocol and harmonized scoring atlas.

The European Cosmetic Toiletry and Perfumery Association (COLIPA), along with contributions from the European Centre for the Validation of Alternative Methods (ECVAM), initiated a multi-lab international prevalidation project on the reconstructed skin micronucleus (RSMN) assay in EpiDerm™ for the assessment of the genotoxicity of dermally applied chemicals. The first step of this project was to standardize the protocol and transfer it to laboratories that had not performed the assay before. Here we describe in detail the protocol for the RSMN assay in EpiDerm™ and the harmonized guidelines for scoring, with an atlas of cell images. We also describe factors that can influence the performance of the assay. Use of these methods will help new laboratories to conduct the assay, thereby further increasing the database for this promising new in vitro genotoxicity test.

Eye irritation potential: usefulness of the HET-CAM under the Globally Harmonized System of classification and labeling of chemicals (GHS).

Extensive research has been conducted over the past decades to develop alternatives to the rabbit eye irritation test (Draize test) used in a regulatory context to assess eye irritation potentials. Although no single in vitro test has emerged as being completely acceptable for full replacement, various tests are considered to be suitable and are regularly used to assess certain aspects. Amongst these, the Hen's Egg Test Chorioallantoic Membrane (HET-CAM) has gained regulatory acceptance in various countries to classify severe eye irritants. In this retrospective study, historical eye irritation data (in vivo and in vitro) from 137 samples (approx. 75% non-irritants; 25% (severe) irritants) tested both in the HET-CAM and Draize eye test was compared with regard to the predicted eye irritation classes under the GHS and the traditional EU classification system (DSD).The overall concordance was in the range of 80-90%. A high specificity (96-98%, depending on the classification system and the chosen discrimination) but rather low sensitivity (48-65%) was observed. The study indicates that HET-CAM results are useful as part of weight-of-evidence assessments or in tiered approaches to assess eye irritation potentials rather than as stand-alone classification method.

International prevalidation studies of the EpiDerm 3D human reconstructed skin micronucleus (RSMN) assay: transferability and reproducibility.

Recently, a novel in vitro reconstructed skin micronucleus (RSMN) assay incorporating the EpiDerm 3D human skin model (Curren et al., Mutat. Res. 607 (2006) 192-204; Mun et al., Mutat. Res. 673 (2009) 92-99) has been shown to produce comparable data when utilized in three different laboratories in the United States (Hu et al., Mutat. Res. 673 (2009) 100-108). As part of a project sponsored by the European cosmetics companies trade association (COLIPA), with a contribution from the European Center for the Validation of Alternative Methods (ECVAM), international prevalidation studies of the RSMN assay have been initiated. The assay was transferred and optimized in two laboratories in Europe, where dose-dependent, reproducibly positive results for mitomycin C and vinblastine sulfate were obtained. Further intra- and inter-laboratory reproducibility of the RSMN assay was established by testing three coded chemicals, N-ethyl-N-nitrosourea, cyclohexanone, and mitomycin C. All chemicals were correctly identified by all laboratories as either positive or negative. These results support the international inter-laboratory and inter-experimental reproducibility of the assay and reinforce the conclusion that the RSMN assay in the EpiDerm 3D human skin model is a valuable in vitro method for assessment of genotoxicity of dermally applied chemicals.

A tiered approach to the use of alternatives to animal testing for the safety assessment of cosmetics: genotoxicity. A COLIPA analysis.

For the assessment of genotoxic effects of cosmetic ingredients, a number of well-established and regulatory accepted in vitro assays are in place. A caveat to the use of these assays is their relatively low specificity and high rate of false or misleading positive results. Due to the 7th amendment to the EU Cosmetics Directive ban on in vivo genotoxicity testing for cosmetics that was enacted March 2009, it is no longer possible to conduct follow-up in vivo genotoxicity tests for cosmetic ingredients positive in in vitro genotoxicity tests to further assess the relevance of the in vitro findings. COLIPA, the European Cosmetics Association, has initiated a research programme to improve existing and develop new in vitro methods. A COLIPA workshop was held in Brussels in April 2008 to analyse the best possible use of available methods and approaches to enable a sound assessment of the genotoxic hazard of cosmetic ingredients. Common approaches of cosmetic companies are described, with recommendations for evaluating in vitro genotoxins using non-animal approaches. A weight of evidence approach was employed to set up a decision-tree for the integration of alternative methods into tiered testing strategies.

Use of in vitro 3D tissue models in genotoxicity testing: Strategic fit, validation status and way forward. Report of the working group from the 7th International Workshop on Genotoxicity Testing (IWGT).

Use of three-dimensional (3D) tissue equivalents in toxicology has been increasing over the last decade as novel preclinical test systems and as alternatives to animal testing. In the area of genetic toxicology, progress has been made with establishing robust protocols for skin, airway (lung) and liver tissue equivalents. In light of these advancements, a "Use of 3D Tissues in Genotoxicity Testing" working group (WG) met at the 7th IWGT meeting in Tokyo in November 2017 to discuss progress with these models and how they may fit into a genotoxicity testing strategy. The workshop demonstrated that skin models have reached an advanced state of validation following over 10 years of development, while liver and airway model-based genotoxicity assays show promise but are at an early stage of development. Further effort in liver and airway model-based assays is needed to address the lack of coverage of the three main endpoints of genotoxicity (mutagenicity, clastogenicity and aneugenicity), and information on metabolic competence. The IWGT WG believes that the 3D skin comet and micronucleus assays are now sufficiently validated to undergo an independent peer review of the validation study, followed by development of individual OECD Test Guidelines.

Hen's Egg Test for Micronucleus Induction (HET-MN).

The classical in vitro genotoxicity test battery is known to be sensitive for indicating genotoxicity. However, a high rate of "misleading" positives was reported when three assays were combined as required by several legislations. Despite the recent optimizations of the standard in vitro tests, two gaps could merely be addressed with assays based on monolayer cell cultures, that is, the route of exposure and a relevant intrinsic metabolic capacity to transform chemicals into reactive metabolites. Following these considerations, fertilized chicken eggs have been introduced into genotoxicity testing and were combined with a classical readout parameter, i.e., the analysis of micronucleus frequency in erythrocytes, to develop the hen's egg test for micronucleus induction, the HET-MN. As a major advantage the test mirrors the systemic availability of compounds after oral exposure reflecting certain steps of ADME without being considered as an animal experiment. After a successful validation exercise the detailed protocol is given here.

3D image analysis reveals differences of CD30 positive cells and network formation in reactive and malignant human lymphoid tissue (classical Hodgkin Lymphoma).

AIMS: The examination of histological sections is still the gold standard in diagnostic pathology. Important histopathological diagnostic criteria are nuclear shapes and chromatin distribution as well as nucleus-cytoplasm relation and immunohistochemical properties of surface and intracellular proteins. The aim of this investigation was to evaluate the benefits and drawbacks of three-dimensional imaging of CD30+ cells in classical Hodgkin Lymphoma (cHL) in comparison to CD30+ lymphoid cells in reactive lymphoid tissues. MATERIALS AND RESULTS: Using immunoflourescence confocal microscopy and computer-based analysis, we compared CD30+ neoplastic cells in Nodular Sclerosis cHL (NScCHL), Mixed Cellularity cHL (MCcHL), with reactive CD30+ cells in Adenoids (AD) and Lymphadenitis (LAD). We confirmed that the percentage of CD30+ cell volume can be calculated. The amount in lymphadenitis was approx. 1.5%, in adenoids around 2%, in MCcHL up to 4,5% whereas the values for NScHL rose to more than 8% of the total cell cytoplasm. In addition, CD30+ tumour cells (HRS-cells) in cHL had larger volumes, and more protrusions compared to CD30+ reactive cells. Furthermore, the formation of large cell networks turned out to be a typical characteristic of NScHL. CONCLUSION: In contrast to 2D histology, 3D laser scanning offers a visualisation of complete cells, their network interaction and spatial distribution in the tissue. The possibility to differentiate cells in regards to volume, surface, shape, and cluster formation enables a new view on further diagnostic and biological questions. 3D includes an increased amount of information as a basis of bioinformatical calculations.

Validation of the 3D Skin Comet assay using full thickness skin models: Transferability and reproducibility.

Recently revised OECD Testing Guidelines highlight the importance of considering the first site-of-contact when investigating the genotoxic hazard. Thus far, only in vivo approaches are available to address the dermal route of exposure. The 3D Skin Comet and Reconstructed Skin Micronucleus (RSMN) assays intend to close this gap in the in vitro genotoxicity toolbox by investigating DNA damage after topical application. This represents the most relevant route of exposure for a variety of compounds found in household products, cosmetics, and industrial chemicals. The comet assay methodology is able to detect both chromosomal damage and DNA lesions that may give rise to gene mutations, thereby complementing the RSMN which detects only chromosomal damage. Here, the comet assay was adapted to two reconstructed full thickness human skin models: the EpiDerm™- and Phenion® Full-Thickness Skin Models. First, tissue-specific protocols for the isolation of single cells and the general comet assay were transferred to European and US-American laboratories. After establishment of the assay, the protocol was then further optimized with appropriate cytotoxicity measurements and the use of aphidicolin, a DNA repair inhibitor, to improve the assay's sensitivity. In the first phase of an ongoing validation study eight chemicals were tested in three laboratories each using the Phenion® Full-Thickness Skin Model, informing several validation modules. Ultimately, the 3D Skin Comet assay demonstrated a high predictive capacity and good intra- and inter-laboratory reproducibility with four laboratories reaching a 100% predictivity and the fifth yielding 70%. The data are intended to demonstrate the use of the 3D Skin Comet assay as a new in vitro tool for following up on positive findings from the standard in vitro genotoxicity test battery for dermally applied chemicals, ultimately helping to drive the regulatory acceptance of the assay. To expand the database, the validation will continue by testing an additional 22 chemicals.

PPARalpha activators inhibit vascular endothelial growth factor receptor-2 expression by repressing Sp1-dependent DNA binding and transactivation.

Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors, originally implicated in the regulation of lipid and glucose homeostasis. In addition, natural and synthetic PPAR activators may control inflammatory processes by inhibition of distinct proinflammatory genes. As signaling via the vascular endothelial growth factor receptor-2 (VEGFR2) pathway is critical for angiogenic responses during chronic inflammation, we explored whether known antiinflammatory effects of PPAR ligands are mediated in part through diminished VEGFR2 expression. In this study, PPARalpha agonists are found to inhibit endothelial VEGFR2 expression, whereas predominant PPARgamma ligands remained without discernible effects. Time- and concentration-dependent inhibition is demonstrated both at the level of protein and mRNA VEGFR2 expression. Inhibitory effects of PPARalpha agonists on transcriptional activity of the VEGFR2 promoter are conveyed by an element located between base pairs -60 and -37 that contains two adjacent consensus Sp1 transcription factor binding sites. Constitutive Sp1-containing complex formation to this sequence is decreased by PPARalpha treatment, indicating that VEGFR2 gene expression is inhibited by repressing Sp1 site-dependent DNA binding and transactivation. Our coimmunoprecipitation experiments revealed enhanced protein interactions between PPARalpha and Sp1 on PPARalpha activation, thus constituting a probable mechanism by which PPARalpha activators decrease Sp-dependent binding activity to the VEGFR2 promoter. Hence, molecular mechanisms by which PPARs modulate the rate of gene transcription may include direct interactions between specific transcription factors and PPARs that ultimately result in reduced DNA binding to their respective response elements.

Systematic evaluation of non-animal test methods for skin sensitisation safety assessment.

The need for non-animal data to assess skin sensitisation properties of substances, especially cosmetics ingredients, has spawned the development of many in vitro methods. As it is widely believed that no single method can provide a solution, the Cosmetics Europe Skin Tolerance Task Force has defined a three-phase framework for the development of a non-animal testing strategy for skin sensitization potency prediction. The results of the first phase ­ systematic evaluation of 16 test methods ­ are presented here. This evaluation involved generation of data on a common set of ten substances in all methods and systematic collation of information including the level of standardisation, existing test data,potential for throughput, transferability and accessibility in cooperation with the test method developers.A workshop was held with the test method developers to review the outcome of this evaluation and to discuss the results. The evaluation informed the prioritisation of test methods for the next phase of the non-animal testing strategy development framework. Ultimately, the testing strategy ­ combined with bioavailability and skin metabolism data and exposure consideration ­ is envisaged to allow establishment of a data integration approach for skin sensitisation safety assessment of cosmetic ingredients.

Fluid shear stress-induced transcriptional activation of the vascular endothelial growth factor receptor-2 gene requires Sp1-dependent DNA binding.

Hemodynamic forces play a fundamental role in the regulation of endothelial cell survival. As signaling via the vascular endothelial growth factor (VEGF) receptor-2 pathway has been previously demonstrated to impact endothelial cell survival, we hypothesized that laminar shear stress may facilitate survival in part by inducing VEGF receptor-2 expression. This study shows a time- and dose-dependent upregulation of endothelial VEGF receptor-2 expression by fluid shear stress in microvascular and large-vessel derived endothelial cells. A functional analysis of the 5'-regulatory region of the VEGF receptor-2 promoter localized the shear stress-response element to a sequence between bp -60 and -37 that encompasses two adjacent consensus Sp1 transcription factor binding sites. Constitutive and shear stress-inducible Sp1-dependent complexes are bound to this element, indicating that fluid shear stress-induced transcriptional activation of the VEGF receptor-2 gene requires Sp1-dependent DNA binding. Together, these results suggest that biomechanical stimulation may lead to endothelial cell survival by upregulating VEGF receptor-2 expression.