The adaptation of the FBA model into 96-well plates allows high-throughput drug screening using small amounts of the tested products 12, 14– 17. procollagen C endopeptidase enhancer 1, secreted protein acidic and cysteine-rich (SPARC), transforming growth factor-β-induced protein Ig-H3 (βIgH3) and insulin-like growth factor binding protein 7 (IGFBP7)) 12. These stromal cells provide a perfect mix of various angiogenic growth factors: hepatocyte growth factor (HGF), transforming growth factor alpha (TGF-α), angiopoietin-1 (Ang-1), as well as matrix molecules, matrix-modifying proteins and matricellular proteins (e.g. This test uses a culture of EC at the surface of ∼200 µm-sized Cytodex-3 microspheres embedded in a 3D fibrin matrix, in the presence of normal human dermal fibroblast (NHDF) as feeder cells. is now extensively used and adapted to various models for the screening and study of pro- or anti-angiogenic compounds 11, 12.Īn alternative in vitro model, named the Fibrin Bead Assay (FBA) is now widely used since its initial description 13. The “Endothelial Tube Formation Assays” (ETFA), based on the original design by Montesano and coll 9, 10. 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. In this context, in vitro as well as in vivo experimental models have been developed to evaluate angiogenesis features, to screen a variety of new angiostatic molecules and to study their properties 7, 8. Other fields of interest, such as stem cell research, also require the evaluation of angiogenic capacities of EC progenitors as well as quantitative tools to monitor their proliferation and differentiation capacities 3, 6. For example, the use of anti-angiogenic molecules still represents a promising therapeutic strategy to impede the development of solid tumors 4, 5.
#Angiogenesis analyze for imagej software full#
Accurate and objective evaluation of these phenomena is necessary for the full comprehension of this process, for both fundamental approaches and pharmacological applications. Under appropriate stimuli, these cells sprout from a root vessel, migrate, proliferate, then align and ultimately form tubes 2, 3. Endothelial cells (EC) represent the main cell type engaged in this process. Although the two methods do not assess the same biological step, our data suggest that they display specific and complementary information on the angiogenesis processes analysis.Īngiogenesis, 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 pathologies 1, 2. ETFA and FBA) in their efficiency, accuracy and statistical relevance to model angiogenesis patterns of Human Umbilical Vein EC (HUVEC).
![angiogenesis analyze for imagej software angiogenesis analyze for imagej software](https://d3i71xaburhd42.cloudfront.net/09f15edb832085ba3a616181f219f3ebef3a7d0c/10-Figure8-1.png)
We detailed these two algorithms and used the new AA version to compare both methods ( i.e. Such a method is presented for the first time in fully automated mode and using non-destructive image acquisition. 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.
![angiogenesis analyze for imagej software angiogenesis analyze for imagej software](https://d32ogoqmya1dw8.cloudfront.net/images/eyesinthesky2/week2/sample_images_imagej.png)
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. The analytical evaluation of these two widely used assays still remains challenging in terms of observation method and image analysis. An alternative approach to ETFA is the “Fibrin Bead Assay” (FBA), based on the use of Cytodex 3 microspheres, which promote the growth of 3D capillary-like patterns from coated EC, suitable for high throughput in vitro angiogenesis studies. In suitable culture conditions, EC form two-dimensional (2D) branched structures that can lead to a meshed pseudo-capillary network. Among the different methods applied to study angiogenesis, the most commonly used is the “Endothelial Tube Formation Assay” (ETFA). Angiogenesis assays based on in vitro capillary-like growth of endothelial cells (EC) are widely used, either to evaluate the effect of anti- and pro-angiogenesis drugs of interest, or to test and compare the functional capacities of various types of EC and progenitor cells.