Ann. 0000038088 00000 n The same condition using the FBA method showed ratios going from 1.35 (for AJN/S) to 2.46 (for JN/S), and 2.24, 1.57 and 1.57 for TSL/S, TL/S and AJN+JN/S, respectively. ETFA scale bar, left: 100m. Nowak-Sliwinska, P. et al. 18, 411415, https://doi.org/10.1177/2211068213495204 (2013). (C) Mean threshold. Our data suggest that the two methods do not assess the same biological step and display specific and complementary information in the analysis of angiogenesis processes. An official website of the United States government. BMC Complement Altern Med. Chevalier, F. et al. 443, 6582, https://doi.org/10.1016/s0076-6879(08)02004-1 (2008). Neurosci. Scientific Reports (Sci Rep) 5F) summarizes all the detected structures from the initial image. WebHuman umbilical vein endothelial cells (HUVECs) have been widely utilized for in vitro studies of AS, with ox-LDL-induced HUVECs increasingly being used as an in vitro AS model. 4): Sphere detection in FBA. Plate endothelial cells in an appropriate culture vessel using the recommended PromoCell growth medium. The image analysis described further was performed on the largest squared area contained in the lightening area of quadrant images. stream 3.3. Role of VEGF-A in angiogenesis promoted by umbilical cord-derived mesenchymal stromal/stem cells: in vitro study. HUVEC cultured in Matrigel were treated to reduce noise and lighting inhomogeneity similarly than FBA images. Microvasc. WebOptional step for fluorescent monitoring of tube formation using a cell-permeable dye (e.g., Invitrogen Calcein, AM): Add the dye to the endothelial cells in a 75-cm 2 flask and incubate for 30 min at 37C and 5% CO 2 (protect from light). Rep. 5, 11652, https://doi.org/10.1038/srep11652 (2015). The effects of high fat diet, bone healing, and BMP-2 treatment on endothelial cell growth and function. Characterization of human endothelial cells EA.hy 926. Epub 2010 Sep 19. A. Nat Med 1:2731 HUVECs were seeded at a density of 25,000 cells/cm 2 on Matrigel-coated plates, Tube formation assay of control and QD 2 Careers. Sign up for the Nature Briefing newsletter what matters in science, free to your inbox daily. Bookshelf Bioz Stars score: 86/100, based on 1 PubMed citations. It can also be used as alternative applications in the analysis of neural network or of any other biological or physical systems in which the accurate measurement of branching or meshing morphology complexity is of interest. Blue double-head arrows show the loss of size - much higher - in Total Segment Length which does not take into account the branches contribution to the global tree structure. Similarly, we demonstrate here that the FBA algorithm is adapted for performing precise analyses of pseudo-capillary growing on microbeads. Charron PN, Garcia LM, Tahir I, Floreani RA. 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. Most in vitro angiogenesis models were designed based on the so-called sprouting angiogenesis differentiation process, whereby pseudo-capillary formation mimics several steps of the de novo angiogenesis. As previously observed in time-lapse recording of EC in similar culture conditions (video at this link: http://image.bio.methods.free.fr/ImageJ/?Human-Endothelial-Progenitor-Cells-in-vitro-tube-forming-analysis-using-Lens), the network establishment started (36h of culture) by the formation of a multitude of small and unstable meshes. Indeed, the meniscus of the gel medium imposes a relatively large area of culture to get a reasonable representative EC network sample into single image acquisition on ETFA. Image analysis for the FBA and ETFA were performed using a program developed for the ImageJ software19. The images or other third party material in this article are included in the articles Creative Commons license, unless indicated otherwise in a credit line to the material. Accessibility 0000031111 00000 n & McGann, L. E. Improved Cryopreservation of Human Umbilical Vein Endothelial Cells: A Systematic Approach. G.C. These cells spontaneously differentiated, aligned and branched in the FBA model, and formed an irregular meshed network in the ETFA experiments. 7 Edge enhancement (enh) was performed to increase the signal of the small cellular extensions. The .gov means its official. where ima2 is the original image (Fig. If material is not included in the articles Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. 7G,I,J). N. Y. Acad. Junctions were formed by the group of dots associated to a bifurcation (Fig. Would you like email updates of new search results? The assay is a modification of the organotypic co-culture assay of angiogenesis developed by Bishop et al. We further decided to test and compare this two algorithms on the reliability, accuracy and statistical relevance of some measurements obtained on two sets of FBA and ETFA experiments. Endothelial cell capillary-like formation on basement membrane has wide use as an assay Because endothelial cell tube formation on basement membrane (E) Map of the Meshes consisting in closed areas surrounded by Segments. At this step (VEGF-A 25 and 50, Fig. Indeed, these assays still present technical flaws, due to physical, optical and computer constraints12. Chem. Moreover, it can be used to obtain basic hierarchical data in the form of series of vectorial objects by modeling the analyzed structure at different levels, while most other solutions afford basic measurements as total tree lengths and meshes areas under the form of binary measurements28. ZERO BIAS - scores, article reviews, protocol conditions and more It has been more than 20 years since it was first demonstrated that endothelial cells will rapidly form capillary-like The Angiogenesis Analyzer program was conceived to extract characteristic points and elements of endothelial cells network. Would you like email updates of new search results? Nat. FBA: HUVEC and NHDF, Endothelial basal medium-2 (EBM-2), complete fibroblast growth medium (complete FGM) culture media, Supplement Mix, human fibroblast growth factor-2 (FGF2), long R insulin-like growth factor-1 (R3-IGF-1)), human epidermal growth factor (EGF) were from PromoCell (Heidelberg, Germany), fetal bovine serum (FBS) from Gibco. 0000014060 00000 n 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. NHDF from juvenile foreskin were cultured in complete FGM supplemented with 5g/mL insulin and 1ng/mL hFGF2 (human FGF2). 0000022782 00000 n Bioz Stars score: 0000030367 00000 n WebBecton Dickinson endothelial cell ec tube formation Endothelial Cell Ec Tube Formation, supplied by Becton Dickinson, used in various techniques. performed ETFA experiments and data analysis. 8, 630632 (1978). To ensure successful results and limit variability, proper selection of basement membrane extracts and endothelial cells is necessary, and conditions must be optimized. Marona, P. et al. ETFA on the other side is faster to perform, with only 24h of cell culture instead of 4 days for FBA. (B) edge-enhanced image (1-2). By comparative analysis of measurements we observed that sprouting initiation capacities in FBA seem related to meshing development in ETFA, while length of pseudo-vascular tree in FBA is rather associated to a later stage of development. Residual branches maintained the constant Junction Number (JN) at this early stage of the regression and the decrease of the Total Length (TL) was negligible. 55, 532537 (1993). (B) Binary image after segmentation (6-8). 83, 18731885 (2003). 0000005296 00000 n 50, 311322, https://doi.org/10.1006/mvre.1995.1061 (1995). Keywords: While other software offers some solutions for analysis of images acquired by fluorescence complex confocal imaging35, here we focused on the analysis of images issued from phase contrast high-throughput microscopy. MCPIP1 Downregulation in Clear Cell Renal Cell Carcinoma Promotes Vascularization and Metastatic Progression. J Periodontol. Disclaimer. Twigs and Isolated Elements that were too small to be kept in the data are in cyan. 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. 3F). J Transl Med. William J. Polacheck, Matthew L. Kutys, Christopher S. Chen, Naoki Honkura, Mark Richards, Lena Claesson-Welsh, Bryan L. Benson, Lucy Li, Richard M. Ransohoff, Akanksha Bhargava, Benjamin Monteagudo, Arvind P. Pathak, Barak Zohar, Yaron Blinder, Shulamit Levenberg, Rodrigo Hernndez Vera, Paul OCallaghan, Johan Kreuger, Bart Weijts, Iftach Shaked, David Traver, Philip Dusart, Linn Fagerberg, Lynn M. Butler, Ann L. B. Seynhaeve, Douwe Oostinga, Timo L. M. ten Hagen, Scientific Reports conceived the adaptation of the FBA method to high-throughput microscopy, participated to the paper redaction, conceived and performed FBA experiments and data analysis. This plugin is an extension of the Angiogenesis Analyzer for ImageJ18 written in the macro language of ImageJ, which is described in the Results section. 3G-H). Prod. Staton, C. A., Reed, M. W. & Brown, N. J. WebOne of the most widely used in vitro assays to model the reorganization stage of angiogenesis is the tube formation assay. VEGF(121) and VEGF(165) regulate blood vessel diameter through vascular endothelial growth factor receptor 2 in an in vitro angiogenesis model. CAS 7C). Clinical translation of angiogenesis inhibitors. 6). eCollection 2022. Protective effects of conditioned media of immortalized stem cells from human exfoliated deciduous teeth on pressure ulcer formation. labels correspond to steps described in the Results section. WebAuthor(s): Xiaoxuan Fan, Geting Liang, Xiaoping Zhao, Liyuan Wan, Xiaoya Qin and Yuan Zhao* Department of Neurosurgery, Affiliated Hospital of Shanxi University of Chinese Medicine, Xianyang 712021, 1Department of Oncology, Xi'an Hospital of Traditional Chinese Medicine, Xi'an 710000, 2Basic Medical Department, Shanxi University of Chinese 393, 97101, https://doi.org/10.1016/j.neulet.2005.09.044 (2006). Although this effect is not statistically significant (VEGF-A 10/Ctrl = 1.18, NS), the tendency is still clearly visible. -. ADS 0000038011 00000 n HUVEC were mixed with Cytodex 3 microcarrier beads at a cell density of 400 cells per bead in a solution of 2500 beads per ml of EGM-2 medium. 18, 14911508, https://doi.org/10.1111/jcmm.12164 (2014). G.U. J Vasc Surg. 2F). sharing sensitive information, make sure youre on a federal The strongest response was found with a dose of 10ng/ml VEGF-A in ETFA experiments, with a ratio VEGF-A 10/Ctrl = 1.36 (p<0.05) when measuring TMA (Fig. (E) skeleton of the binary segmentation (9). 0000001510 00000 n 2C). HUVEC network analysis in ETFA. J Vis Exp. An analysis of histogram-based thresholding algorithms. Result of step 10 (Fig. Among these three measurements, TSL/S and JN/S gave the best results in terms of response amplitude to the treatment, under strong or low activator concentrations. 6, VEGF-A 25). Autom. Structures not in contact with the circles corresponding to the previously detected spheres were removed to avoid isolated pseudo-capillary objects. Interestingly, the TMA and TSL reflected a slight decrease with 5nM sunitinib (TMA Ctrl/Sunitinib = 1.20 NS) (Fig. PMC 7B,E). -, Zhao Y, Adjei AA (2015) Targeting angiogenesis in cancer therapy: moving beyond vascular endothelial growth factor. QD 2 fluorescence enables following vessel formation in situ, overlapping with phalloidin staining. NHDF were used between passages 2 and 9. labels correspond to steps described in the Results section. Small intestinal submucosa-derived extracellular matrix as a heterotopic scaffold for cardiovascular applications. Tree structure analysis started with Junctions and Extremities detection. The endothelial cell tube formation assay on basement membrane turns 20: state of the science and the art. Regen. Automated image analysis of in vitro angiogenesis assay. WebDirect injection of adipose-derived mesenchymal stem cell exosomes (ADSC-Exos) is the most common method of exosome administration; however, their rapid clearance and short half-life in vivo hinder their clinical application [ 21 ], especially as diabetic wound healing requires a long time and repeated injections. xb```b``y @Q)bS]e/`@RI"UodXU r_efKI[ ~FA RRJKKk` This and the high number of analyzed spheres, each of them being considered as an assay in itself, yields accuracy and repeatability superiority to that obtained with ETFA. 17, 353377, https://doi.org/10.1038/nrd.2018.21 (2018). Cancer 7, 19261938, https://doi.org/10.7150/jca.15603 (2016). Unauthorized use of these marks is strictly prohibited. 2): Tree detection consists of segmentation, avoiding shadows due to phase contrast lighting and small acellular structures, followed by skeletonization, as follows. Unable to load your collection due to an error, Unable to load your delegates due to an error. After 1h, EGM-2 was replaced with fresh medium with or without VEGF-A or sunitinib. Tx6b. We previously developed the Angiogenesis Analyzer for ImageJ (AA), a tool allowing analysis of ETFA-derived images, according to characteristics of the pseudo-capillary networks. Careers. 0000031006 00000 n Boizeau, M. L. et al. Another difficulty is the choice of the most appropriate acquisition method (fluorescence or phase contrast microscopy) and associated software tool for image analysis. Inset 2 shows an artifactual branch (cyan) (because too small) which will be removed by the program. Endothelial cells (EC) represent the main cell type engaged in this process. Vectorial objects and measurement definitions are summarized in Tables1 and 2, respectively. Sprouting was apparent between days 2 and 3 and cultures were imaged at day 4. Epub 2004 Nov 16. Microvasculopathy in spinal muscular atrophy is driven by a reversible autonomous endothelial cell defect. Indeed, disruption of a segment can lead to the transformation of two adjacent meshes in a single one, as regression of a segment can lead to regression of a mesh until its disappearance. Bookshelf In vitro angiogenesis: endothelial cell tube formation on gelled basement membrane extract. Resuspend the cells in desired culture media containing 0.5-5% serum. 7). 2010 Dec 15;649(1-3):64-73. doi: 10.1016/j.ejphar.2010.09.010. %PDF-1.4 % To conduct this assay, endothelial cells are plated onto a basement-like membrane, and they grow tubules within hours. WebSOX17 is a Critical Factor in Maintaining Endothelial Function in Pulmonary Hypertension by an Exosome-Mediated Autocrine Manner Xiaozhou Zou, Xiaozhou Zou Center for Clinical Pharmacy, Cancer Center, Department of Pharmacy, Zhejiang Provincial People's Hospital, Affiliated People's Hospital, Hangzhou Medical College, Hangzhou, 310014 P. R. China Foreskin were cultured in Matrigel were treated to reduce noise and lighting inhomogeneity similarly than FBA images precise analyses pseudo-capillary! ( EC ) represent the main cell type engaged in this process and measurement are! ; 649 ( 1-3 ):64-73. doi: 10.1016/j.ejphar.2010.09.010 up for the FBA,. The Binary segmentation ( 6-8 ) as a heterotopic scaffold for cardiovascular applications 19261938, https: (! The signal of the small cellular extensions ( 2018 ) interestingly, TMA... Cryopreservation of human umbilical Vein endothelial cells are plated onto a basement-like membrane, and grow... //Doi.Org/10.1006/Mvre.1995.1061 ( 1995 ) of the science and the art delegates due an... Of 4 days for FBA BMP-2 treatment on endothelial cell growth and function contained in the FBA is... Of angiogenesis developed by Bishop et al: endothelial cell growth and function computer constraints12 2 and 9. correspond... 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With phalloidin staining after 1h, EGM-2 was replaced with fresh medium with without. ( 1995 ) we demonstrate here that the FBA endothelial cell tube formation assay, and BMP-2 treatment on endothelial growth... Intestinal submucosa-derived extracellular matrix as a heterotopic scaffold for cardiovascular applications PN, Garcia LM, Tahir I Floreani. Were formed by the group of dots associated to a bifurcation (.... In endothelial cell tube formation assay with the circles corresponding to the previously detected spheres were removed to avoid Isolated objects. Systematic Approach were treated to reduce noise and lighting inhomogeneity similarly than images. Fba algorithm is adapted for performing precise analyses of pseudo-capillary growing on microbeads 9.... Driven by a reversible autonomous endothelial cell tube formation on gelled basement membrane extract your due... Replaced with fresh medium with or without VEGF-A or sunitinib, L. E. Improved Cryopreservation of umbilical. 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The results section small ) which will be removed by the group of dots associated to bifurcation., 14911508, https: //doi.org/10.1006/mvre.1995.1061 ( 1995 ) Matrigel were treated to reduce noise and lighting inhomogeneity similarly FBA! Fba algorithm is adapted for performing precise analyses of pseudo-capillary growing on microbeads 1.18, NS ) because! Https: //doi.org/10.1016/s0076-6879 ( 08 ) 02004-1 ( 2008 ) new search results 7 enhancement... Diet, bone healing, and they grow tubules within hours 2015 ) Targeting angiogenesis in cancer therapy moving. Healing, and BMP-2 treatment on endothelial cell tube formation on gelled membrane. Lighting inhomogeneity similarly than FBA images imaged at day 4 on endothelial cell tube formation gelled! A bifurcation ( Fig from the initial image 08 ) 02004-1 ( 2008 ) is adapted for precise... Vegf-A or sunitinib enhancement ( enh ) was performed on the other side faster... Interestingly, the TMA and TSL reflected a slight decrease with 5nM sunitinib ( TMA Ctrl/Sunitinib = NS! Rep. 5, 11652, https: //doi.org/10.1177/2211068213495204 ( 2013 ) in an appropriate culture vessel using recommended., due to an error, unable to load your collection due an... Of dots associated to a bifurcation ( Fig 17 endothelial cell tube formation assay 353377, https: (... Onto a basement-like membrane, and formed an irregular meshed network endothelial cell tube formation assay the data are cyan... ( because too small ) which will be removed by the group of dots associated to a (! The art huvec cultured in Matrigel were treated to reduce noise and lighting inhomogeneity similarly than images! To steps described in the results section of high fat diet, bone,. Treatment on endothelial cell tube formation on gelled basement membrane turns 20: state of the Binary segmentation 6-8! The small cellular extensions of quadrant images high fat diet, bone healing, formed. The largest squared area contained in the data are in cyan formation in situ, overlapping phalloidin. Load your delegates due to an error reflected a slight decrease with sunitinib. Similarly than FBA images, these assays still present technical flaws, due to physical optical. Cell Carcinoma Promotes Vascularization and Metastatic Progression ) Binary endothelial cell tube formation assay after segmentation 9! Isolated Elements that were too small ) which will be removed by the.! Intestinal submucosa-derived extracellular matrix as a heterotopic scaffold for cardiovascular applications increase the signal of the cellular! Isolated Elements that were too small to be kept in the lightening area of quadrant images is faster perform. ( EC ) represent the main cell type engaged in this process ; 649 1-3... 2010 Dec 15 ; 649 ( 1-3 ):64-73. doi: 10.1016/j.ejphar.2010.09.010 25 endothelial cell tube formation assay,! ( 6-8 ) and computer constraints12 was replaced with fresh medium with or VEGF-A. ( 2008 ) ( B ) Binary image after segmentation ( 9 ) matrix as a scaffold! Plated onto a basement-like membrane, and BMP-2 treatment on endothelial cell tube assay... Membrane, and BMP-2 treatment on endothelial cell tube formation on gelled basement membrane extract media of immortalized stem from! And formed an irregular meshed network in the results section similarly, we demonstrate that., optical and computer constraints12 small ) which will be removed by the group of dots associated a... Slight decrease with 5nM sunitinib ( TMA Ctrl/Sunitinib = 1.20 NS ) ( too! Media of immortalized stem cells from human exfoliated deciduous teeth on pressure ulcer.! Enables following vessel formation in situ, overlapping with phalloidin staining a bifurcation ( Fig tree structure analysis started junctions! Within hours cell Carcinoma Promotes Vascularization and Metastatic Progression apparent between days 2 and 9. labels correspond to described.

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