Name: a simple bioassay for the evaluation of vascular endothelial growth factors
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Description: Watch this Scientific Journal Video about A Simple Bioassay for the Evaluation of Vascular Endothelial Growth Factors at JoVE.com

SAS and MGA are supported by Project Grants, a Program Grant and Research Fellowships from the National Health and Medical Research Council of Australia (NHMRC), and by funds from the Operational Infrastructure Support Program provided by the Victorian Government, Australia. MMH has support from a Peter MacCallum Foundation Grant.

Source of IL-3 and Preparation of WEHI-3D-conditioned Medium
Note: The mouse granulocytic leukemia cell line WEHI-3D is cultured to generate a conditioned medium containing IL-3.
<ol>
	<li>Culture WEHI-3D in Dulbecco&#39;s Modified Eagle&#39;s Medium (DMEM), 10% fetal bovine serum (FBS), 1% long-life glutamine supplement, 50 &#956;g/ml gentamicin. Inoculate 5 &#215; 106 cells in the log phase of growth into 50 ml of fresh culture medium in a T175 cm3 tissue culture flask and grow for about 7 days or until the cells have passed their log phase of growth by about 24 - 48 hr (avoid excessive cell death in the culture). Conditioned media is capable of being stored for multiple years at &#8722;20 &#176;C, so batches of 200 &#8722; 500 ml can be produced at one time.</li>
	<li>Decant fluid and cells from flasks and spin at 1,000 x g for 15 min to remove cells and cellular debris. Remove the top 90% of supernatant. Filter through a 0.22 &#956;m filter unit.</li>
	<li>Aliquot WEHI-3D conditioned medium (CM) into 1 ml, 50 ml and 200 ml volumes for storage at &#8722;20 &#176;C or &#8722;70 &#176;C. Smaller aliquots are useful for assay preparation, intermediate volumes for making culture medium for passage of factor-dependent cell lines, and larger volumes for long-term storage. IL-3 is relatively stable at 4 &#176;C so thawed medium can be stored for a few weeks if used under sterile conditions.</li>
	<li>Alternatively, use recombinant mouse IL-3 at levels of 50 ng/ml diluted into culture medium, after being filtered through a 0.22 &#181;m filter unit.</li>
</ol>
2. Culture and Evaluation of VEGFR-2-EpoR-Ba/F3 and Control Ba/F3 Cell Lines
<ol>
	<li>Culture control Ba/F3 cells in DMEM, 10% FBS, 50 &#181;g/ml gentamicin or penicillin/streptomycin supplement, 1% stabilized L-glutamine and 10% WEHI-3D-CM. Passage cells at 1:15 dilutions about every three days from cells growing in log phase.</li>
	<li>Culture VEGFR2-EpoR-Ba/F3 cells in DMEM, 10% FBS, 50 &#181;g/ml gentamycin, 1% stabilized L-glutamine and 10% WEHI-3D-CM and 1 mg/ml G418. Passage cells at 1:15 dilutions from cells growing in log phase. See 24 for more details about the construction and expression of the chimeric receptor.</li>
	<li>Harvest control Ba/F3 or VEGFR2-EpoR-Ba/F3 cells from mid log-phase cultures. Gently pipette to remove these non-adherent cells from the bottom of the flask. Wash three times in mouse tonicity phosphate-buffered saline, pH 7.3 (PBS), (10 ml, centrifuge at 750 x g for 5 min to recover cell pellet) to remove medium containing IL-3.</li>
	<li>Wash cells once with DMEM and additives, without the WEHI-3D-CM or recombinant IL-3, and then resuspend in this medium at a concentration of 7.4 &#215; 104 cells/ml (i.e., 1,000 cells per 13.5 &#181;l; 10,000 cells per 135 &#181;l as determined by counting using a hemocytometer) for use in the semi-quantitative or quantitative versions of the assay respectively.</li>
	<li>Assess cells for viability by Trypan Blue exclusion (CAUTION). Mix Trypan Blue in PBS (0.4%) 1:1 with the cell population and count a minimum of 100 cells on a hemocytometer. Cells that take up the dye are considered dead or dying. Viability of greater than 98% is required to perform the assay.</li>
</ol>
3. Semi-quantitative Assay
<ol>
	<li>Add washed cells (1,000 cells) contained in 13.5 &#956;l of IL-3-deficient medium to the wells of a 72-well microwell plate at RT. Take care to mix the cell suspension during aliquotting to ensure cell settling by gravity does not bias cell concentration. Use a well-calibrated P20 automated pipette, and autoclaved tips.</li>
	<li>Add test samples and controls to the wells at 10% volume (1.5 &#956;l, making a final volume of 15 &#181;l containing 1,000 cells per well) using a calibrated P20 pipette or, preferably, P2 pipette. Take care to ensure that samples are compatible with the culture conditions for Ba/F3 cells in terms of pH, salt and other potentially cytotoxic/cytostatic substances. Where possible, use a compatible medium or buffer (e.g.,&#160;DMEM or PBS, respectively) or dilute in such medium or buffer. Trituration with the same tip will ensure mixing.</li>
	<li>For control samples, include (i) Medium alone-containing no IL-3; (ii) WEHI-3BD-CM added to medium alone at 10% final volume; and/or (iii) VEGF-A diluted to 100 ng/ml in medium alone, containing no IL-3.</li>
	<li>Fill any unused wells of the microwell plate with sterile water, PBS or medium and place the plate within a humidified container (with water soaked tissue paper) allowing gas exchange. Incubate cells within the containers in a humidified atmosphere of 10% CO2. This assay can be comfortably set up in 3 hr by one person if test samples and media components are available.</li>
	<li>Assess plates typically after 16 hr of incubation at which time discrimination between positive and negative samples is evident. See Figure 3 for examples of how cells appear in culture. Analyze plates using a standard inverted phase-contrast microscope at 40 &#8722; 100&#215; magnification. 
	Note: Wells containing supportive growth factors such as IL-3 or VEGF-A will contain cells which appear round and translucent. Wells without supportive growth factors or with medium alone will have reduced numbers of round cells, as well as dead or dying cells and cellular debris (e.g.,&#160;Figure 3C). Evaluating the assay at 24&#8722;48 hr post incubation is the optimum for sensitivity.</li>
</ol>
4. Quantitative Bioassay
<ol>
	<li>Add washed cells (10,000 cells in 135 &#956;l) in IL-3-deficient medium to the wells of a standard 96-well microtiter plate at RT. Take care to mix the cell suspension during aliquotting to ensure cell settling by gravity does not affect cell concentration.</li>
	<li>Add test samples and controls to the wells at 10% volume (15 &#956;l) using a calibrated pipette (P20). Take care to ensure that samples are compatible with the culture conditions for Ba/F3 cells in terms of pH, salt or other potentially cytotoxic/cytostatic substances. Where possible, use a compatible medium or buffer (e.g., DMEM or PBS) or dilute in such medium or buffer. Filter sterilize small volumes using a 0.22 &#181;m pore cellulose acetate centrifuge tube filter unit.</li>
	<li>Incubate the mixture of cells, growth factors and/or inhibitor agents in a humidified atmosphere of 10% CO2 for 48 hr. Perform the assay within a humidified container that has water-soaked tissue paper and allows gas-exchange. At the completion of the incubation period, evaluate the assay using one of the alternative methods listed below which are surrogate markers of the viable cell number in the well.</li>
	<li>Alternative#1: 3H-thymidine Incorporation 
	Note: This quantification is designed for the 96-well plate format.
	<ol>
		<li>After incubation of assay plates for 48 hr at 37 &#176;C (150 &#181;l volume per well), add 3H-thymidine in a volume of 50 &#181;l of assay medium (Ba/F3 culture medium without IL-3/WEHI-3D CM) per well at a concentration of 20 &#181;Ci/ml, giving a final dose of 1 &#181;Ci per well.</li>
		<li>Incubate plates for a further 4 hr at 37 &#176;C before harvesting cells using a cell harvester. Extract individual samples by placing the filters in scintillant and quantify with a Liquid Scintillation Counter.</li>
	</ol>
	</li>
	<li>Alternative#2: Bioluminescence Detection of Cellular ATP 
	Note: This approach uses an ATP monitoring reagent which when combined with cell lysate can generate a bioluminescence signal which reflects the viable cells in the population.
	<ol>
		<li>Lyse cells to extract ATP and use an ATP Monitoring Reagent to generate a luminescent signal according to the manufacturer&#39;s instructions. Read luminescence using a luminometer compatible with a 96-well plate format.</li>
	</ol>
	</li>
	<li>Alternative#3: Enzymatic Reduction of an Indicator Dye by Viable Cells 
	Note: Resazurin based assays show good correlation to cell viability.
	<ol>
		<li>Combine cultured cells with the resazurin based dye and quantify assays using a fluorescence plate reader according to the manufacturer&#39;s instructions.</li>
	</ol>
	</li>
</ol>

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Steven A. Stacker and Marc M. Achen are shareholders in Circadian Technologies Ltd., a company developing therapeutics by targeting the VEGF family of growth factors.

The assay described here relies on using cells of high viability, which are dependent on growth factors. Cells therefore need to be carefully cultured to ensure they are factor-dependent, and retain expression of the chimeric receptor. Ensuring that the medium is freshly made and not stored for an excessively long period and that WEHI-3D CM is highly active is important. Cells need to be thoroughly washed from IL-3 containing medium into the assay medium to ensure that no residual IL-3 contaminates the assay when exposin...

The vascular endothelial growth factor (VEGF) family of secreted protein growth factors and their cognate cell surface receptors is an important and diverse group of soluble ligands and membrane-embedded receptors, respectively that function in transducing signals across cellular membranes. They function mainly in endothelial cells but also in cells of epithelial origin and those of the immune system 1,2. Signaling pathways engaged by ligand-activated VEGF receptors (VEGFRs) are critical in major pathologies, such as age-related macular degeneration and cancer, and therapeutics targeting them are in frequent clinical use (e.g., the monoclonal antibody bevacizumab that targets VEGF-A) 3,4.
One of the complexities of the VEGF family is the diversity of soluble ligands present in nature (VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF proteins encoded by the parapox virus family orf and snake venom VEGF, plus other inhibitory isoforms of VEGF-A)2.
These ligands interact with three members of the receptor tyrosine kinase family, namely VEGFR-1, VEGFR-2 and VEGFR-3. These receptors are variably expressed on different cell types but are often co-expressed on the surface of endothelial cells that line blood and lymphatic vessels of all sizes 5. VEGFR-2 can bind the mammalian ligands VEGF-A 6, VEGF-C 7 and VEGF-D 8,9 as well as orf virus VEGF10 and snake venom VEGF11. VEGFR-2 plays a major role in driving angiogenesis (the growth of new blood vessels from pre-existing vessels) in embryonic development, wound healing, cancer and eye diseases. In these contexts, ligands such as VEGF-A, -C and -D bind and activate the receptor on blood vascular endothelial cells12-15. On lymphatic endothelial cells, VEGFR-2 plays a role in lymphangiogenesis, the formation of new lymphatic vessels 16. VEGFR-2 can also promote dilation and expansion of major arteries and lymphatics in healthy tissues and disease 17. A complete understanding of VEGFR-2:ligand interactions is therefore important for the development of inhibitors for use in treating angiogenesis-dependent diseases18. While most isoforms of VEGF-A bind to VEGFR-2, proteolytic cleavage of VEGF-C and VEGF-D is required to release a fragment consisting of the VEGF-homology domain that exhibits high affinity binding to VEGFR-2 19,20.
We have developed a bioassay to monitor ligands of VEGFR-2 that is designed to circumvent the need for primary endothelial cells, which are technically difficult to passage, expensive to purchase and culture (requiring specialized medium) 21 and express multiple VEGFRs and associated co-receptors 22. Heterodimerization of VEGFR-2 with other VEGF receptors or co-receptors can cause unwanted complexity when aiming to study binary receptor-ligand interactions, evaluating activity attributable to a specific receptor, or assessing the effect of inhibitory reagents.23. The bioassay retains mobility of the relevant receptor in the cell membrane and allows evaluation of a ligand&#39;s ability to bind and cross-link the VEGFR-2 extracellular region.
The bioassay relies on the creation of a chimeric receptor in which the extracellular region of a VEGF receptor (in this case VEGFR-2) is fused to the transmembrane and intracellular regions of the erythropoietin receptor (EpoR), a member of the cytokine receptor family 8,24. This fusion protein is then expressed in the factor-dependent pro-B cell line Ba/F3, upon which stimulation with a ligand capable of binding and cross-linking the extracellular domain of the receptor causes activation of the cytoplasmic effector region, which is capable of transducing a survival signal via Janus kinases (JAKs) to promote cell survival and/or proliferation. In contrast, expression of full-length VEGFR-2 in the same cell type, and stimulation with ligand, does not promote cell survival and proliferation, indicating that the proximal signaling effectors of the VEGFR-2 pathway are not available in this cell type.
We have used the assay in a variety of contexts to explore binding of novel VEGFR-2 ligands 10,19,20,24-29. In combination with a VEGFR-3-EpoR-Ba/F3 assay, we have compared the relative activities of the VEGF-C and VEGF-D growth factors for binding and cross-linking VEGFR-2 and VEGFR-3 30. The assay has been used to characterize the inhibitory activity of neutralizing monoclonal antibodies to VEGFR-2 or VEGF-D, soluble VEGFR-2 trap and peptidomimetics targeting the VEGF family31. The assay was also used to show the ability of VEGFs from different orf virus strains to bind and cross-link VEGFR-2 prior to testing in primary endothelial cells 10,26. The assay is particularly useful for the rapid screening of mutants of VEGFs which can be quickly assessed for activity before they are introduced to the more laborious endothelial cell assays 25 or when assessing protocols for purifying growth factors 27.
The assay we describe is easy to perform, and the semi-quantitative version allows for quick determinations that are sometimes required when monitoring the production or purification of growth factors, antibodies or soluble receptor domains for other experiments. The ease of use of the assay makes it an ideal complement to further and more complete studies performed with primary endothelial cells derived from blood or lymphatic vessels from specific tissues or organ systems.

<table><tbody><tr><td>Trypan Blue</td><td>Sigma-Aldrich</td><td>T8154</td><td>0.4% solution in PBS is used 1:1 with cell suspensions to measure viable cells. Hazard-may cause cancer</td></tr><tr><td>G418 Sulphate (Geneticin)</td><td>Invivogen</td><td>ant-gn-5</td><td>Agent for selecting transfected eukaryotic cells. Hazard-may cause allergy or asthma symptoms or breathing difficluties.</td></tr><tr><td>&lt;sup&gt;3&lt;/sup&gt;H-Thymidine</td><td>PerkinElmer</td><td>NET-027</td><td>This radioactive nucleoside is incorporated into chromosomal DNA during mitosis. Hazard-radiation</td></tr><tr><td>Vialight Plus Kit</td><td>Lonza</td><td>LT07-221</td><td>Bioluminescent detection of cellular ATP to quantify viability, using ATP Monitoring Reagent</td></tr><tr><td>Prestoblue Cell Viability Reagent</td><td>Invitrogen</td><td>A13261</td><td>Resazurin-based indicator of cell viability. Turns red in color in the reducing environment of the cell</td></tr><tr><td>Nunc Minitray with Nunclon Delta Surface (72 well)</td><td>Thermo Scientific</td><td>136528</td><td>Small microtitre plate</td></tr><tr><td>96 well Tissue Culture Plate</td><td>Falcon, Corning Inc.</td><td>353072</td><td></td></tr><tr><td>DMEM (1X)</td><td>Gibco</td><td>11965-92</td><td></td></tr><tr><td>GlutaMAX (100X)</td><td>Gibco</td><td>35050-061</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Gibco</td><td>&amp;nbsp;10099-141</td><td></td></tr><tr><td>Cell Harvester</td><td>Tomtec Life Sciences</td><td></td><td>Tomtec Harvester, 96 Mach 3M Cell Harvester</td></tr><tr><td>Liquid Scintillation Counter</td><td>LKB Wallac</td><td>1205</td><td>LKB Wallac 1205 Betaplate Scintillation Counter</td></tr><tr><td>UniFilter-96 GF/B</td><td>Perkin Elmer</td><td>6005177</td><td>White 96-well Barex Microplate with GF/B filterof 1 &amp;micro;m poresize</td></tr><tr><td>Gentamicin</td><td>Gibco, Life Technologies</td><td>15750-060</td><td></td></tr><tr><td>Penicillin/Streptomycin</td><td>Gibco, Life Technologies</td><td>15140-122</td><td></td></tr><tr><td>0.22 um pore cellulose acetate centrifuge tube filter unit</td><td>Costar, Corning Inc.</td><td>8160</td><td>Centrifuge tube filters have a 0.22 &amp;micro;m pore CA membrane-containing filter unit within a 500 &amp;micro;l capacity polypropylene microcentrifuge tube.</td></tr><tr><td>Fluorescence Reader</td><td>BioTek</td><td></td><td>BioTek Synergy 4 Hybrid Microplate Reader&amp;nbsp;</td></tr></tbody></table>

a simple bioassay for the evaluation of vascular endothelial growth factors

The analysis of receptor tyrosine kinases and their interacting ligands involved in vascular biology is often challenging due to the constitutive expression of families of related receptors, a broad range of related ligands and the difficulty of dealing with primary cultures of specialized endothelial cells. Here we describe a bioassay for the detection of ligands to the vascular endothelial growth factor receptor-2 (VEGFR-2), a key transducer of signals that promote angiogenesis and lymphangiogenesis. A cDNA encoding a fusion of the extracellular (ligand-binding) region of VEGFR-2 with the transmembrane and cytoplasmic regions of the erythropoietin receptor (EpoR) is expressed in the factor-dependent cell line Ba/F3. This cell line grows in the presence of interleukin-3 (IL-3) and withdrawal of this factor results in death of the cells within 24 hr. Expression of the VEGFR-2/EpoR receptor fusion provides an alternative mechanism to promote survival and potentially proliferation of stably transfected Ba/F3 cells in the presence of a ligand capable of binding and cross-linking the extracellular portion of the fusion protein (i.e., one that can cross-link the VEGFR-2 extracellular region). The assay can be performed in two ways: a semi-quantitative approach in which small volumes of ligand and cells permit a rapid result in 24 hr, and a quantitative approach involving surrogate markers of a viable cell number. The assay is relatively easy to perform, is highly responsive to known VEGFR-2 ligands and can accommodate extracellular inhibitors of VEGFR-2 signaling such as monoclonal antibodies to the receptor or ligands, and soluble ligand traps.

In this section, we show the results of an experiment demonstrating the essential features of a VEGFR-2-EpoR-Ba/F3 bioassay (see Figure 1 for principles of the assay). Other published studies demonstrate broader applications of the assay for alternative VEGFR-2 ligands, mutant VEGF molecules and inhibitory monoclonal antibodies 8,10,19,24-30.
The data presented here represent an assay in which the VEG...

Watch this Scientific Journal Video about A Simple Bioassay for the Evaluation of Vascular Endothelial Growth Factors at JoVE.com

A Simple Bioassay for the Evaluation of Vascular Endothelial Growth Factors

We describe a simple cell-based bioassay for detecting, quantifying and monitoring the activity of members of the vascular endothelial growth factor family of ligands. The assay uses chimeric receptors expressed in a factor-dependent cell line to provide a semi-quantitative or quantitative assessment of receptor binding and cross-linking by the ligand.

The analysis of receptor tyrosine kinases and their interacting ligands involved in vascular biology is often challenging due to the constitutive ...

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JoVE Journal

Biology

9.6K Views.  Peter MacCallum Cancer Centre. We describe a simple cell-based bioassay for detecting, quantifying and monitoring the activity of members of the vascular endothelial growth factor family of ligands. The assay uses chimeric receptors expressed in a factor-dependent cell line to provide a semi-quantitative or quantitative assessment of receptor binding and cross-linking by the ligand.

Video: A Simple Bioassay for the Evaluation of Vascular Endothelial Growth Factors

Optimized Fibrin Gel Bead Assay for the Study of Angiogenesis

Angiogenesis is a complex multi-step process, where, in response to angiogenic stimuli, new vessels are created from the existing vasculature.  These steps include: degradation of the basement membrane, proliferation and migration (sprouting) of endothelial cells (EC) into the extracellular matrix, alignment of EC into cords, branching, lumen formation, anastomosis, and formation of a new basement membrane.  Many in vitro assays have been developed to study this process, but most only mimic certain stages of angiogenesis, and morphologically the vessels within the assays often do not resemble vessels in vivo.  Based on earlier work by Nehls and Drenckhahn, we have optimized an in vitro angiogenesis assay that utilizes human umbilical vein EC and fibroblasts.  This model recapitulates all of the key early stages of angiogenesis and, importantly, the vessels display patent intercellular lumens surrounded by polarized EC. EC are coated onto cytodex microcarriers and embedded into a fibrin gel.  Fibroblasts are layered on top of the gel where they provide necessary soluble factors that promote EC sprouting from the surface of the beads.  After several days, numerous vessels are present that can easily be observed under phase-contrast and time-lapse microscopy. This video demonstrates the key steps in setting up these cultures.

This video demonstrates the protocol of an in vitro angiogenesis assay that recapitulates several stages of angiogenesis. Time-lapse images of sprouting, lumen formation, branching and anastomosis - key features of angiogenesis - are shown.

Angiogenesis is a complex multi-step process, where, in response to angiogenic stimuli, new vessels are created from the existing vasculature.  These ...

The Aortic Ring Co-culture Assay: A Convenient Tool to Assess the Angiogenic Potential of Mesenchymal Stromal Cells In Vitro

Angiogenesis is a complex, highly regulated process responsible for providing and maintaining adequate tissue perfusion. Insufficient vasculature maintenance and pathological malformations can result in severe ischemic diseases, while overly abundant vascular development is associated with cancer and inflammatory disorders. A promising form of pro-angiogenic therapy is the use of angiogenic cell sources, which can provide regulatory factors as well as physical support for newly developing vasculature.
Mesenchymal Stromal Cells (MSCs) are extensively investigated candidates for vascular regeneration due to their paracrine effects and their ability to detect and home to ischemic or inflamed tissues. In particular, first trimester human umbilical cord perivascular cells (FTM HUCPVCs) are a highly promising candidate due to their pericyte-like properties, high proliferative and multilineage potential, immune-privileged properties, and robust paracrine profile. To effectively evaluate potentially angiogenic regenerative cells, it is a requisite to test them in reliable and &#34;translatable&#34; pre-clinical assays. The aortic ring assay is an ex vivo angiogenesis model that allows for easy quantification of tubular endothelial structures, provides accessory supportive cells and extracellular matrix (ECM) from the host, excludes inflammatory components, and is fast and inexpensive to set up. This is advantageous when compared to in vivo models (e.g., corneal assay, Matrigel plug assay); the aortic ring assay can track the administered cells and observe intercellular interactions while avoiding xeno-immune rejection.
We present a protocol for a novel application of the aortic ring assay, which includes human MSCs in co-cultures with developing rat aortic endothelial networks. This assay allows for the analysis of the MSC contribution to tube formation and development through physical pericyte-like interactions and of their potency for actively migrating to sites of angiogenesis, and for evaluating their ability to perform and mediate ECM processing. This protocol provides further information on changes in MSC phenotype and gene expression following co-culture.

The Aortic Ring Co-culture Assay: A Convenient Tool to Assess the Angiogenic Potential of Mesenchymal Stromal Cells In Vitro

Here, we present a novel application of the aortic ring assay where prelabelled mesenchymal cells are co-cultured with rat aorta-derived endothelial networks. This novel method allows visualization of Mesenchymal Stromal Cells (MSCs) homing and integration with endothelial networks, quantification of network properties, and evaluation of MSC immunophenotypes and gene expression.

Angiogenesis is a complex, highly regulated process responsible for providing and maintaining adequate tissue perfusion. Insufficient vasculature ...

Developmental Biology

Screening Assays to Characterize Novel Endothelial Regulators Involved in the Inflammatory Response

The endothelial layer is essential for maintaining homeostasis in the body by controlling many different functions. Regulation of the inflammatory response by the endothelial layer is crucial to efficiently fight against harmful inputs and aid in the recovery of damaged areas. When the endothelial cells are exposed to an inflammatory environment, such as the outer component of gram-negative bacteria membrane, lipopolysaccharide (LPS), they express soluble pro-inflammatory cytokines, such as Ccl5, Cxcl1 and Cxcl10, and trigger the activation of circulating leukocytes. In addition, the expression of adhesion molecules E-selectin, VCAM-1 and ICAM-1 on the endothelial surface enables the interaction and adhesion of the activated leukocytes to the endothelial layer, and eventually the extravasation towards the inflamed tissue. In this scenario, the endothelial function must be tightly regulated because excessive or defective activation in the leukocyte recruitment could lead to inflammatory-related disorders. Since many of these disorders do not have an effective treatment, novel strategies with a focus on the vascular layer must be investigated. We propose comprehensive assays that are useful to the search of novel endothelial regulators that modify leukocyte function. We analyze endothelial activation by using specific expression targets involved in leukocyte recruitment (such as, cytokines, chemokines, and adhesion molecules) with several techniques, including: real-time quantitative polymerase chain reaction (RT-qPCR), western-blot, flow cytometry and adhesion assays. These approaches determine endothelial function in the inflammatory context and are very useful to perform screening assays to characterize novel endothelial inflammatory regulators that are potentially valuable for designing new therapeutic strategies.

Vascular endothelium tightly controls leukocyte recruitment. Inadequate leukocyte extravasation contributes to human inflammatory diseases. Therefore, searching for novel regulatory elements of endothelial activation is necessary to design improved therapies for inflammatory disorders. Here, we describe a comprehensive methodology to characterize novel endothelial regulators that can modify leukocyte trafficking during inflammation.

The endothelial layer is essential for maintaining homeostasis in the body by controlling many different functions. Regulation of the inflammatory ...

Immunology and Infection

Strategic Endothelial Cell Tube Formation Assay: Comparing Extracellular Matrix and Growth Factor Reduced Extracellular Matrix

Malignant tumors require a blood supply in order to survive and spread. These tumors obtain their needed blood from the patient's blood stream by hijacking the process of angiogenesis, in which new blood vessels are formed from existing blood vessels. The CXCR2 (chemokine (C-X-C motif) receptor 2) receptor is a transmembrane G-protein-linked molecule found in many cells that is closely associated with angiogenesis1. Specific blockade of the CXCR2 receptor inhibits angiogenesis, as measured by several assays such as the endothelial tube formation assay. The tube formation assay is useful for studying angiogenesis because it is an excellent method of studying the effects that any given compound or environmental condition may have on angiogenesis. It is a simple and quick in vitro assay that generates quantifiable data and requires relatively few components. Unlike in vivo assays, it does not require animals and can be carried out in less than two days. This protocol describes a variation of the extracellular matrix supporting endothelial tube formation assay, which tests the CXCR2 receptor.

This manuscript describes a tube formation assay to quantify the effects of a given compound or condition on angiogenesis by using endothelial cell tube formation in a controlled environment.

Malignant tumors require a blood supply in order to survive and spread. These tumors obtain their needed blood from the patient's blood stream by ...

Medicine

Rat Mesentery Angiogenesis Assay

The adult rat mesentery window angiogenesis assay is biologically appropriate and is exceptionally well suited to the 
study of sprouting angiogenesis in vivo [see review papers], which is the dominating form of angiogenesis in human tumors and non-tumor 
tissues, as discussed in invited review papers1,2. Angiogenesis induced in the membranous mesenteric parts by intraperitoneal (i.p.) injection of a pro-angiogenic factor can be modulated
by subcutaneous (s.c.), intravenous (i.v.) or oral (p.o.) treatment with modifying agents of choice. Each membranous part of the mesentery is 
translucent and framed by fatty tissue, giving it a window-like appearance.
The assay has the following advantageous features: (i) the test tissue is natively vascularized, albeit 
sparsely, and since it is extremely thin, the microvessel network is virtually two-dimensional, which allows the entire network 
to be assessed microscopically in situ; (ii) in adult rats the test tissue lacks significant physiologic angiogenesis, which characterizes
 most normal adult mammalian tissues; the degree of native vascularization is, however, correlated with age, as discussed in1; 
(iii) the negligible level of trauma-induced angiogenesis ensures high sensitivity; (iv) the 
assay replicates the clinical situation, as the angiogenesis-modulating test drugs are administered systemically and the responses 
observed reflect the net effect of all the metabolic, cellular, and molecular alterations induced by the treatment; (v) the assay 
allows assessments of objective, quantitative, unbiased variables of microvascular spatial extension, density, and network pattern 
formation, as well as of capillary sprouting, thereby enabling robust statistical analyses of the dose-effect and molecular 
structure-activity relationships; and (vi) the assay reveals with high sensitivity the toxic or harmful effects of treatments in 
terms of decreased rate of physiologic body-weight gain, as adult rats grow robustly.
Mast-cell-mediated angiogenesis was first demonstrated using this assay3,4. The model demonstrates a high level of 
discrimination regarding dosage-effect relationships and the measured effects of systemically administered chemically or functionally closely related drugs and proteins, 
including: (i) low-dosage, metronomically administered standard chemotherapeutics that yield diverse, drug-specific effects (i.e., 
angiogenesis-suppressive, neutral or angiogenesis-stimulating activities5); (ii) natural iron-unsaturated human lactoferrin, which stimulates 
VEGF-A-mediated angiogenesis6, and natural iron-unsaturated bovine lactoferrin, which inhibits VEGF-A-mediated angiogenesis7; and (iii) 
low-molecular-weight heparin fractions produced by various means8,9. Moreover, the assay is highly suited to studies of the combined 
effects on angiogenesis of agents that are administered systemically in a concurrent or sequential fashion.
The idea of making this video originated from the late Dr. Judah Folkman when he visited our laboratory and witnessed the methodology being demonstrated.
Review papers (invited) discussing and appraising the assay
Norrby, K. In vivo models of angiogenesis. J. Cell. Mol. Med. 10, 588-612 (2006).
Norrby, K. Drug testing with angiogenesis models. Expert Opin. Drug. Discov. 3, 533-549 (2008).

Normal adult vascularized mammalian tissue that lacks physiologic angiogenesis and that has not been exposed to surgical intervention is used to study: (i) the initiation and development of angiogenesis following intraperitoneal administration of test agents; and (ii) modification of angiogenesis following systemic administration of selected test agents.

The adult rat mesentery window angiogenesis assay is biologically appropriate and is exceptionally well suited to the 
study of sprouting angiogenesis in ...

Endothelial Cell Tube Formation Assay for the In Vitro Study of Angiogenesis

Angiogenesis is a vital process for normal tissue development and wound healing, but is also associated with a variety of pathological conditions. Using this protocol, angiogenesis may be measured in vitro in a fast, quantifiable manner. Primary or immortalized endothelial cells are mixed with conditioned media and plated on basement membrane matrix. The endothelial cells form capillary like structures in response to angiogenic signals found in conditioned media. The tube formation occurs quickly with endothelial cells beginning to align themselves within 1&#160;hr and lumen-containing tubules beginning to appear within 2 hr. Tubes can be visualized using a phase contrast inverted microscope, or the cells can be treated with calcein AM prior to the assay and tubes visualized through fluorescence or confocal microscopy. 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. In summary, this assay can be used to identify genes and pathways that are involved in the promotion or inhibition of angiogenesis in a rapid, reproducible, and quantitative manner.

Endothelial Cell Tube Formation Assay for the In Vitro Study of Angiogenesis

The tube formation assay is a fast, quantifiable method for measuring in vitro angiogenesis. Endothelial cells are combined with conditioned media and plated on basement membrane extract. Tube formation occurs within hours and newly formed tubules easily quantified.

Angiogenesis is a vital process for normal tissue development and wound healing, but is also associated with a variety of pathological conditions. Using ...

Monitoring Functionality and Morphology of Vasculature Recruited by Factors Secreted by Fast-growing Tumor-generating Cells

The subcutaneous matrigel plug assay in mice is a method of choice for the in vivo evaluation of pro- and anti-angiogenic factors. In this method, desired factors are introduced into cold-liquid ECM-mimic gel which, after subcutaneous injection, solidifies to form an environment mimicking the cancer milieu. This matrix permits the penetration of host cells, such as endothelial cells, and therefore, the formation of vasculature.
Herein we propose a new modified matrigel plug assay, which can be exploited to illustrate the angiogenic potential of a pool of factors secreted by cancer cells, as opposed to a specific factor (e.g., bFGF and VEGF) or agent. The plug containing ECM-mimic gel is utilized to introduce the host (i.e., mouse) with a pool of factors secreted to the C.M. of fast-growing tumor-generating glioblastoma cells. We have previously described an extensive comparison of the angiogenic potential of U-87 MG human glioblastoma and its dormant-derived clone, in this system model, showing induced angiogenesis in the U-87 MG parental cells. The C.M. is prepared by filtering collected media from confluent tissue culture plates of either cell line following 48 hr incubation. Hence, it contains only factors secreted by the cells, without the cells themselves. Described here is the combination of two imaging modalities, microbubbles contrast-enhanced ultrasound imaging and intravital fibered-confocal endomicroscopy, for an accurate, real-time characterization of the extent, morphology and functionality of newly-formed blood vessels within the plugs.

We describe a matrigel plug assay to illustrate angiogenic potential of a pool of factors secreted by cancer cells using two complementary imaging modalities, ultrasound and endomicroscopy. The matrigel, an extracellular matrix (ECM)-mimic gel, is utilized to introduce the host (mouse) with angiogenic factors secreted to the conditioned media (C.M.).&#160;

The subcutaneous matrigel plug assay in mice is a method of choice for the in vivo evaluation of pro- and anti-angiogenic factors. In this method, desired ...

Evaluating Vascular Hyperpermeability-inducing Agents in the Skin with the Miles Assay

The primary function of the vascular endothelium in vertebrate organisms is to serve as a barrier between the blood and each tissue of the body, whereby the permeability of the endothelium to blood cells, plasma macromolecules, and water can be adapted according to the physiological need. In certain diseases, cytokines and growth factors are released that target the endothelial barrier to transiently increase vascular permeability; however, their prolonged presence may cause chronic vascular hyperpermeability and thereby tissue-damaging edema. The Miles assay is an in vivo technique that allows researchers to study vascular hyperpermeability through the proxy measurement of vascular leakage. Here, we provide a detailed protocol on how to perform this procedure in the mouse, which is the most widely used model organism to study mammalian physiology and pathology. The procedure involves the intravenous injection of Evans blue dye to label the circulating albumin followed by multiple intradermal injections of permeability-inducing agents and vehicle control solutions into opposing flanks of the mouse. Consequently, Evans blue dye gradually leaks into the dermis, where it accumulates and can be extracted for quantification as leakage induced by the permeability-inducing agent relative to the vehicle. The Miles assay can be performed in wild type or genetically modified mouse models and may be combined with drug administration to study molecular mechanisms that regulate vascular permeability and identify agents/targets capable of inducing or blocking hyperpermeability.

Here, we present a protocol to measure the vascular leakage induced by intradermal administration of permeability promoting agents into the murine skin. This technique can be used to determine the ability of molecules to promote or inhibit vascular leakage or to study the molecular mechanisms that regulate vascular permeability.

The primary function of the vascular endothelium in vertebrate organisms is to serve as a barrier between the blood and each tissue of the body, whereby ...

Study of Angiogenesis Using Scratch Wound Migration and Spheroid Sprouting Assays

Investigating Angiogenesis on a Functional and Molecular Level by Leveraging the Scratch Wound Migration Assay and the Spheroid Sprouting Assay

Angiogenesis plays a crucial role in both physiological and pathological processes within the body including tumor growth or neovascular eye disease. A detailed understanding of the underlying molecular mechanisms and reliable screening models are essential for targeting diseases effectively and developing new therapeutic options. Several in vitro assays have been developed to model angiogenesis, capitalizing on the opportunities a controlled environment provides to elucidate angiogenic drivers at a molecular level and screen for therapeutic targets.
This study presents workflows for investigating angiogenesis in vitro using human umbilical vein endothelial cells (HUVECs). We detail a scratch wound migration assay utilizing a live cell imaging system measuring endothelial cell migration in a 2D setting and the spheroid sprouting assay assessing endothelial cell sprouting in a 3D setting provided by a collagen matrix. Additionally, we outline strategies for sample preparation to enable further molecular analyses such as transcriptomics, particularly in the 3D setting, including RNA extraction as well as immunocytochemistry. Altogether, this framework offers scientists a reliable and versatile toolset to pursue their scientific inquiries in in vitro angiogenesis assays.

Author Spotlight: Investigating Angiogenesis Through Challenges and Innovations in Assay Development

This study presents the two-dimensional (2D) scratch wound migration assay and the three-dimensional (3D) spheroid sprouting assay, along with their respective downstream analysis methods, including RNA extraction and immunocytochemistry, as suitable assays to study angiogenesis in vitro.

Angiogenesis plays a crucial role in both physiological and pathological processes within the body including tumor growth or neovascular eye disease. A ...

In Vitro Three-Dimensional Sprouting Assay of Angiogenesis Using Mouse Embryonic Stem Cells for Vascular Disease Modeling and Drug Testing

Recent advances in induced pluripotent stem cells (iPSC) and gene editing technologies enable the development of novel human cell-based disease models for phenotypic drug discovery (PDD) programs. Although these novel devices could predict the safety and efficacy of investigational drugs in humans more accurately, their development to the clinic still strongly rely on mammalian data, notably the use of mouse disease models. In parallel to human organoid or organ-on-chip disease models, the development of relevant in vitro mouse models is therefore an unmet need for evaluating direct drug efficacy and safety comparisons between species and in vivo and in vitro conditions. Here, a vascular sprouting assay that utilizes mouse embryonic stem cells differentiated into embryoid bodies (EBs) is described. Vascularized EBs cultured onto 3D-collagen gel develop new blood vessels that expand, a process called sprouting angiogenesis. This model recapitulates key features of in vivo sprouting angiogenesis-formation of blood vessels from a pre-existing vascular network-including endothelial tip cell selection, endothelial cell migration and proliferation, cell guidance, tube formation, and mural cell recruitment. It is amenable to screening for drugs and genes modulating angiogenesis and shows similarities with recently described three-dimensional (3D) vascular assays based on human iPSC technologies.

In Vitro Three-Dimensional Sprouting Assay of Angiogenesis Using Mouse Embryonic Stem Cells for Vascular Disease Modeling and Drug Testing

This assay utilizes mouse embryonic stem cells differentiated into embryoid bodies cultured in 3D-collagen gel to analyze the biological processes that control sprouting angiogenesis in vitro. The technique can be applied for testing drugs, modeling diseases, and for studying specific genes in the context of deletions that are embryonically lethal.

Recent advances in induced pluripotent stem cells (iPSC) and gene editing technologies enable the development of novel human cell-based disease models for ...

A Simple Bioassay for the Evaluation of Vascular Endothelial Growth Factors

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Optimized Fibrin Gel Bead Assay for the Study of Angiogenesis

The Aortic Ring Co-culture Assay: A Convenient Tool to Assess the Angiogenic Potential of Mesenchymal Stromal Cells In Vitro

Screening Assays to Characterize Novel Endothelial Regulators Involved in the Inflammatory Response

Strategic Endothelial Cell Tube Formation Assay: Comparing Extracellular Matrix and Growth Factor Reduced Extracellular Matrix

Rat Mesentery Angiogenesis Assay

Endothelial Cell Tube Formation Assay for the In Vitro Study of Angiogenesis

Monitoring Functionality and Morphology of Vasculature Recruited by Factors Secreted by Fast-growing Tumor-generating Cells

Evaluating Vascular Hyperpermeability-inducing Agents in the Skin with the Miles Assay

Investigating Angiogenesis on a Functional and Molecular Level by Leveraging the Scratch Wound Migration Assay and the Spheroid Sprouting Assay

In Vitro Three-Dimensional Sprouting Assay of Angiogenesis Using Mouse Embryonic Stem Cells for Vascular Disease Modeling and Drug Testing