endothelial cell tube formation assay

Within the best responses in terms of treated/control ratio, Junction Number/Sphere (JN/S), Total Length/Sphere (TL/S) and Total Segment Length/Sphere (TSL/S) VEGF-A/Ctrl ratios were respectively 2.9 (p<0.001), 1.8 (p<0.0001) and 2.9 (p<0.001) for the maximal activation at 25ng/ml (Fig. 2E). PubMed Central Goodwin, A. M. In vitro assays of angiogenesis for assessment of angiogenic and anti-angiogenic agents. J. Nat. By using this new approach, we dramatically increased the repeatability and precision of angiogenesis measurements. Effect of living cellular sheets on the angiogenic potential of human microvascular endothelial cells. FBA: HUVEC were cultured in EBM-2 with 1% penicillin/streptomycin supplemented with 2% Supplement Mix, thus constituting the EGM-2 as previously described14. Tissues were then blocked for 30 min at room temperature with normal goat serum and incubated with primary antibodies including Although the disruption leads to a higher decrease in Total Segment Length (TSL) by a length value that corresponds to the double head blue arrow (Fig. Cybern. Meng, H. et al. WebEndothelial Cell Tube Formation is an in vitro assay system composed of a BD Falcon 96-well black plate with clear bottom uniformly coated with BD Matrigel Matrix. 0000038088 00000 n WebEndothelial cell tube formation assay is one of the most widely used and reliable methods for studying in vitro angiogenesis. Briefly, 3.0 10 5 HUVECs were seeded into each well of a 6-well plate containing 10 g/ml exosomes and 5% FBS and incubated for 72 h. After 50 l of Matrigel (Corning, NY, USA, Cat. Junctions were formed by the group of dots associated to a bifurcation (Fig. x[e7^J. This can be explained as the early meshing structure of HUVEC constrained differences in amplitude in ETFA. This quick, quantitative, reliable, and easy assay has been used for more than 40 years. Microvasc. https://doi.org/10.1038/s41598-020-67289-8, DOI: https://doi.org/10.1038/s41598-020-67289-8. Bookshelf The .gov means its official. Cells were suspended in EGM2-MV medium without VEGF-A (control condition), or complemented with 5, 10, 25 or 50ng/ml VEGF-A. Madu, C., Li, L. & Lu, Y. Glasbey, C. A. labels correspond to steps described in the Results section. The VEGF-A and NF-B p65 protein expressions in the quercetin-treated HT-29 cells were determined by fluorescence assay and Western blot analysis. (A to D) Represent four pixels (in red) each linked to three neighborning pixels (green). Ferguson, F. M. & Gray, N. S. Kinase inhibitors: the road ahead. Ann. Cancer Biol. All reagents were of culture grade. 2022 Nov 1;132(21):e153430. 108 0 obj <>stream ETFA and FBA) in their efficiency, accuracy and statistical relevance to model angiogenesis patterns of Human Umbilical Vein EC (HUVEC). Even with a higher cultured area for ETFA compared to FBA (about 2-fold more), a significant difference compared to the control was only found with one VEGF-A treatment (VEGF-A 10ng/ml, p<0.05), and with TMA parameter only using ETFA. Nat. These assays were performed in parallel on HUVEC cells, using both activating (VEGF-A) and inhibiting (sunitinib) conditions of angiogenesis. Actually, WimTube is focused on quantifying Tube Formation Assay and it is designed with flexibility to adapt to images covering the whole range of microscopy imaging techniques. Queiroz, M. M. F. et al. Initial clonogenic potential of human endothelial progenitor cells is predictive of their further properties and establishes a functional hierarchy related to immaturity. <> Rep. 5, 11652, https://doi.org/10.1038/srep11652 (2015). Zhou H, Hong Y, Scoto M, Thomson A, Pead E, MacGillivray T, Hernandez-Gerez E, Catapano F, Meng J, Zhang Q, Hunter G, Shorrock HK, Ng TK, Hamida A, Sanson M, Baranello G, Howell K, Gillingwater TH, Brogan P, Thompson DA, Parson SH, Muntoni F. J Clin Invest. Tree structure analysis started with Junctions and Extremities detection. Sci Rep 10, 11568 (2020). Publishers note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Scale bar: 200 m. 1), (2) tree detection (Fig. Bergers, G. & Benjamin, L. E. Tumorigenesis and the angiogenic switch. G iE @Z- eCollection 2022. Samarelli, A. V. et al. The analysis can be divided into three main steps: (1) sphere detection (Fig. 0000029010 00000 n 2021 May;146:115883. doi: 10.1016/j.bone.2021.115883. Model. MMS increased as TMA, segment disruptions and mesh fusion leading to an increase in residual meshes (Fig. 0 An official website of the United States government. The https:// ensures that you are connecting to the This site needs JavaScript to work properly. Bio-inspired green light crosslinked alginate-heparin hydrogels support HUVEC tube formation. Part. Discov. MeSH This method has the advantage of being closely related, in terms of 3D sprouting freedom with a solid anchorage point, to an in vivo assay known as the Rat Aortic Sprouting Assay. Nihon Yakurigaku Zasshi. Article 3): 10. A critical analysis of current in vitro and in vivo angiogenesis assays. Med. Using the same principle, the Junctions that intercepted Circles corresponding to sphere limits were defined as Anchorage Junctions (zoom inset 4 Fig. sharing sensitive information, make sure youre on a federal 5E). Issa, M. E., Berndt, S., Carpentier, G., Pezzuto, J. M. & Cuendet, M. Bruceantin inhibits multiple myeloma cancer stem cell proliferation. Taylor BE, Lee CA, Zapadka TE, Zhou AY, Barber KG, Taylor ZRR, Howell SJ, Taylor PR. Disclaimer. labels correspond to steps described in the Results section. Characterization of large extracellular vesicles (L-EV) derived from human regulatory macrophages (Mreg): novel mediators in wound healing and angiogenesis? 4B). 0000004269 00000 n Tube formation assays were performed to evaluate the EC tube-forming ability, as described befores [17, 18]. Afterwards, researchers can analyze different aspects. 2022. To ensure successful results and limit variability, proper selection of basement membrane extracts and endothelial cells is necessary, and conditions must be optimized. Evid Based Complement Alternat Med. Careers. 5 0 obj Lett. Angiogenesis, the growth of new blood vessels from pre-existing ones, is a complex and critical process that takes place during vertebrate development, in specific physiological conditions in adult individuals and during different pathologies1,2. government site. ETFA (left column images) and FBA, (right column images). This image analysis software was successfully used to characterize meshed and/or branched structures in more than 150 different studies (http://image.bio.methods.free.fr/ImageJ/?Angiogenesis-Analyzer-for-ImageJ&artpage=6-6#outil_sommaire_6), which include endothelial in vitro ETFA cell differentiation in phase contrast20,21, fluorescence microscopy22, and in vivo studies using the mouse retina angiogenesis model23,24. Comparing the angiogenic potency of nave marrow stromal cells and Notch-transfected marrow stromal cells. 2013 Sep 21;13:231. doi: 10.1186/1472-6882-13-231. Clipboard, Search History, and several other advanced features are temporarily unavailable. Article WebIn Vitro Angiogenesis cell based assays provide a convenient system for the evaluation of endothelial cell tube formation in a convenient 96-well format using either ECM Gel or Fibrin ECM gels. Tree detection in FBA. 2A), v1 is the minimum size of structures to keep and v2 is a value corresponding to the size of the shadows to be removed. 37, 2, https://doi.org/10.1186/s41232-016-0033-2 (2017). HUVECs were seeded at a density of 25,000 cells/cm 2 on Matrigel-coated plates, Tube formation assay of control and QD 2 In this work, we developed and implemented a new algorithm for AA able to recognize microspheres and to analyze the attached capillary-like structures from the FBA model. National Library of Medicine 2C). Anti-Angiogenic Treatments Interact with Steroid Secretion in Inflammatory Breast Cancer Triple Negative Cell Lines. stream WebThe tube formation assay is a simple in vitro angiogenesis assay which is routinely used to screen the pro-angiogenic or anti-angiogenic poten- tial of substances and compounds. W.R. is the author of ImageJ. Lane JAE, Faulkner A, Finding EJT, Lynam EG, Wheeler-Jones CPD. xref B., Marquez-Curtis, L. A., Elliott, J. We also thank Dr Fabien Chevalier for providing some images of ETFA used for the first developments of AA. was at the origin of the ETFA experiments at the Gly-CRRET lab, she supervised the ETFA experiments and contributed to writing the paper. J Transl Med. The tube assay has been used to define angiogenic and anti-angiogenic factors and genes important in angiogenesis. Sakr, O. S. et al. 50, 311322, https://doi.org/10.1006/mvre.1995.1061 (1995). Methods Mol Biol. Primary or immortalized endothelial cells are mixed with conditioned media and plated on basement membrane matrix. government site. Use seeding densities between 5X10 3 cells/cm 2 and 2X10 4 cells/cm 2 as recommended in the respective product manual. S.F. Correspondence to FOIA ADS Article WebEndothelial Cell Tube Formation Assay. Image analysis for the FBA and ETFA were performed using a program developed for the ImageJ software19. Please enable it to take advantage of the complete set of features! The endothelial cell tube formation assay on basement membrane turns 20: state of the science and the art. We previously separately validated Angiogenesis Analyzer for both ETFA29 and FBA models14,15,30. 3F). We thank Profs. The Number of Anchorage Junction/Sphere (AJN/S), which are specific measurements of FBA and can be associated to the capacity to initiate sprouting, gave the smallest SEM for the maximum activation at the dose VEGF-A 10 compared to control conditions (AJN/S VEGF-A 10/Ctrl = 1.35 (p<0.001)). This assay has several advantages, namely: ease of set up, the ability to test numerous angiogenic/anti-angiogenic factors simultaneously, quick network formation, ability to view live or fixed tube networks, and quantifiability. Katahira Y, Murakami F, Inoue S, Miyakawa S, Sakamoto E, Furusaka Y, Watanabe A, Sekine A, Kuroda M, Hasegawa H, Mizoguchi I, Yoshimoto T. Front Immunol. In order to quantify the sprouting pseudo-microvessel network, samples were automatically scanned with a high-throughput imager (IXM, Molecular Device) at a 4x magnification with definition of 4 quadrants (images) per well to cover the entire surface of each well. MCPIP1 Downregulation in Clear Cell Renal Cell Carcinoma Promotes Vascularization and Metastatic Progression. Endothelial cell tube formation assay for the in vitro study of angiogenesis. 0000031111 00000 n Interestingly, the TMA and TSL reflected a slight decrease with 5nM sunitinib (TMA Ctrl/Sunitinib = 1.20 NS) (Fig. Lengths of the red arrows correspond to loss of size in the initial Segment of which only two green branches remain. This can be explained by considering the kinetic of the meshing network structuration during HUVEC culture in Matrigel. HUVECs were seeded at a density of 25,000 cells/cm 2 on Matrigel-coated plates, Tube formation assay of control and QD 2-labeled HUVECs. Soltani, A. et al. WebOne of the most widely used in vitro assays to model the reorganization stage of angiogenesis is the tube formation assay. WebCommon methods for studying angiogenesis in vitro include the tube formation assay, the migration assay, and the study of the endothelial genome. 84, 653661, https://doi.org/10.1055/a-0587-5991 (2018). HUVEC network analysis in ETFA. To obtain Consensus guidelines for the use and interpretation of angiogenesis assays. BMC Complement Altern Med. (E) The junction between the three binary segments is composed of the four nodes (red) materialized by circles of seven pixels in diameter. (A) (i) Higher expression of pERK in HUVECs in response to a cell-free (B) Binary image after segmentation (6-8). In vitro angiogenesis: endothelial cell tube formation on gelled basement membrane extract. Except for the Anchorage Number (AJN/S), other parameters exhibited a higher treatment/control ratio. The PubMed wordmark and PubMed logo are registered trademarks of the U.S. Department of Health and Human Services (HHS). IL-17A Enhances Retinal Neovascularization. 0000030679 00000 n Increased signaling by the autism-related Engrailed-2 protein enhances dendritic branching and spine density, alters synaptic structural matching, and exaggerates protein synthesis. The number of branch sites/nodes, loops/meshes, or number or length of tubes formed can be easily quantified as a measure of in vitro angiogenesis. PubMed WebBecton Dickinson endothelial cell ec tube formation Endothelial Cell Ec Tube Formation, supplied by Becton Dickinson, used in various techniques. Eight wells were analyzed per condition. WebVII. While this tendency was still visible with Total Segment Length (TSL) (VEGF-A 10/Ctrl = 1.13 NS) (Fig. The present comparison of the ETFA and FBA methods demonstrates their robustness. J Periodontol. Examples of this phenomenon are shown in Fig. Small intestinal submucosa-derived extracellular matrix as a heterotopic scaffold for cardiovascular applications. Endothelial cells on Matrigel form tubules [ 5, 22, 23 ]. Temperature affects the silicate morphology in a diatom. Beads and HUVEC were then co-incubated in a humidified incubator at 37C and 5% CO2 and gently manually shaken every 20min for 4h to allow cell adherence to the bead surface. PubMed The formation of capillary-like tubes in vitro on basement membrane matrix mimics many steps of the angiogenesis process in vivo and is used widely as a screening test for angiogenic or antiangiogenic factors. Crtl, medium only (black bars); increasing concentrations of VEGF-A (5, 10, 25 and 50ng/ml) and sunitinib (5 and 25nM) (white bars). Drug. Berndt, S., Issa, M. E., Carpentier, G. & Cuendet, M. A Bivalent Role of Genistein in Sprouting Angiogenesis. ISSN 2045-2322 (online). WebBackground: Here, we aimed to assess the inhibitory effect of a new compound from Panax ginseng on the migration of human ovarian cancer cells and tube formation of human umbilical vein endothelial cells (HUVECs).