We thank Drs. Victoria Bautch and Joshua Boucher for helpful discussions and advice on optimizing standard bead sprouting assay conditions and a sprouting assay staining protocol.&#160;S.H.A. was supported in part by a grant from the National Institute of General Medical Sciences under award 5T32 GM007092.

Day 1:
1. Transient Transfection of Endothelial Cells
<ol>
	<li>If desired, perform a transient transfection of Human Umbilical Vein Endothelial Cells (HUVEC) using gene-regulatory oligonucleotides (e.g. micro-RNAs or small interfering RNA (siRNA)) and the appropriate lipofectamine reagent according to manufacturer&#39;s instructions. 
	NOTE: This protocol has had great success performing reverse transfections with custom siRNA sequences and a commercial transf...

<ol>
	<li>Hanahan, D., Folkman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patterns+and+emerging+mechanisms+of+the+angiogenic+switch+during+tumorigenesis.">Patterns and emerging mechanisms of the angiogenic switch during tumorigenesis.</a> Cell. 86 (3), 353-364 (1996).</li><li>Semenza, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Angiogenesis+in+ischemic+and+neoplastic+disorders.">Angiogenesis in ischemic and neoplastic disorders.</a> Annu Rev Med. 54, 17-28 (2003).</li><li>Zhang, Z. G., Chopp, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurorestorative+therapies+for+stroke:+underlying+mechanisms+and+translation+to+the+clinic.">Neurorestorative therapies for stroke: underlying mechanisms and translation to the clinic.</a> Lancet Neurol. 8 (5), 491-500 (2009).</li><li>Nakatsu, M. N., Hughes, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+optimized+three-dimensional+in+vitro+model+for+the+analysis+of+angiogenesis.">An optimized three-dimensional in vitro model for the analysis of angiogenesis.</a> Methods Enzymol. 443, 65-82 (2008).</li><li>Nehls, V., Drenckhahn, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel,+microcarrier-based+in+vitro+assay+for+rapid+and+reliable+quantification+of+three-dimensional+cell+migration+and+angiogenesis.">A novel, microcarrier-based in vitro assay for rapid and reliable quantification of three-dimensional cell migration and angiogenesis.</a> Microvasc Res. 50 (3), 311-322 (1995).</li><li>Sims, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pericyte--a+review.">The pericyte--a review.</a> Tissue Cell. 18 (2), 153-174 (1986).</li><li>Armulik, A., Genove, G., Betsholtz, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pericytes:+developmental,+physiological,+and+pathological+perspectives,+problems,+and+promises.">Pericytes: developmental, physiological, and pathological perspectives, problems, and promises.</a> Dev Cell. 21 (2), 193-215 (2011).</li><li>Tattersall, I. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+modeling+of+endothelial+interaction+with+macrophages+and+pericytes+demonstrates+Notch+signaling+function+in+the+vascular+microenvironment.">In vitro modeling of endothelial interaction with macrophages and pericytes demonstrates Notch signaling function in the vascular microenvironment.</a> Angiogenesis. 19 (2), 201-215 (2016).</li><li>Jain, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Normalization+of+tumor+vasculature:+an+emerging+concept+in+antiangiogenic+therapy.">Normalization of tumor vasculature: an emerging concept in antiangiogenic therapy.</a> Science. 307 (5706), 58-62 (2005).</li><li>Tahergorabi, Z., Khazaei, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+review+on+angiogenesis+and+its+assays.">A review on angiogenesis and its assays.</a> Iran J Basic Med Sci. 15 (6), 1110-1126 (2012).</li><li>Popson, S. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interferon-induced+transmembrane+protein+1+regulates+endothelial+lumen+formation+during+angiogenesis.">Interferon-induced transmembrane protein 1 regulates endothelial lumen formation during angiogenesis.</a> Arterioscler Thromb Vasc Biol. 34 (5), 1011-1019 (2014).</li><li>Coultas, L., Chawengsaksophak, K., Rossant, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+cells+and+VEGF+in+vascular+development.">Endothelial cells and VEGF in vascular development.</a> Nature. 438 (7070), 937-945 (2005).</li><li>Newman, A. C., Nakatsu, M. N., Chou, W., Gershon, P. D., Hughes, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+requirement+for+fibroblasts+in+angiogenesis:+fibroblast-derived+matrix+proteins+are+essential+for+endothelial+cell+lumen+formation.">The requirement for fibroblasts in angiogenesis: fibroblast-derived matrix proteins are essential for endothelial cell lumen formation.</a> Mol Biol Cell. 22 (20), 3791-3800 (2011).</li></ol>

The authors have nothing to disclose.

This protocol presents a method for characterizing the complex stages and heterotypic cellular interactions of sprouting angiogenesis by enabling the researcher to employ genetic and imaging approaches to conduct thorough mechanistic investigations. When performing the assay, it is essential that efficient endothelial coating of the beads takes place during the bead agitation steps. Poor endothelial coating will be made evident, if the beads do not appear to have a golf ball-like rough surface the next morning prior to g...

Angiogenesis is vital to appropriate embryogenesis and wound healing, and it also plays key roles in numerous diseases including cancer progression1 and coronary artery disease.2,3 Having a better understanding of how angiogenesis occurs during normal development, and how it is reactivated in pathologic contexts, is critical for the development of novel, effective therapeutics. Faithful in vitro models that recapitulate the important stages and cell types involved in angiogenesis in vivo are needed to allow researchers to better characterize the molecular mechanisms driving angiogenesis and make novel discoveries in endothelial regulation.
Nakatsu and Hughes have optimized a sprouting bead assay that they have demonstrated undergoes the many known stages of sprouting angiogenesis.4,5 The purpose of the method presented here is to build upon the assay optimized by Nakatsu and Hughes by incorporating perictyes into the assay, so that the paracrine and juxtracrine roles of mural cells in endothelial cell sprouting can be incorporated in novel angiogenesis studies. Pericytes are mural cells that are defined by their role as cells that maintain close physical contact with endothelial cells due to their being embedded in the vascular basement membrane.6 Pericytes and endothelial cells engage in complex cross-talk via signaling pathways including Notch signaling, Ang-Tie2, PDGFR&#946;, TGFR&#946;, and many others.7,8 Mouse models deficient in these signaling pathways demonstrate poor pericyte coverage of developing vasculature in embryogenesis, leading to poor vascular remodeling and dysfunctional vasculature.7 In addition, the role of pericytes in pathologic angiogenesis is important but oftentimes under-appreciated. For example, a unique feature of tumor vasculature is that the vessels are more immature, leaky, and dysfunctional due to poor pericyte coverage.9 It has been proposed that the presence or absence of pericytes dramatically impacts the phenotype of tumor blood vessels and is an important mediator of responses to antiangiogenic and antitumor therapies.9 Thus, including the role of pericytes in in vitro assays is key to more completely capture the important mechanisms of endothelial regulation.
Although there are many in vitro and ex vivo assays currently employed to study angiogenesis, there are shortcomings to consider in each. Some, such as endothelial proliferation and endothelial migration assays, are overly simplified and focus on one endothelial function in an isolated setting on tissue culture plastic.10 Other assays occur in a more 3 dimensional (3D) setting, such as the Matrigel tube formation assay,10 but these assays are still oversimplified and focus more on the ability of endothelial cells to migrate and form de novo vascular structures, as opposed to sprouting from pre-existing vasculature. Furthermore, none of these assays incorporate mural cell types. There are ex vivo models such as the ring aorta assay that do incorporate pericytes present in the host organ, but genetic manipulation of these models is much more challenging due to the necessity of generating knockout or transgenic mouse models of the pathways of interest. The bead sprouting assay is ideal because it models endothelial sprouting, proliferation, migration, and even anastomosis and lumen formation in a 3D matrix.4 The assay faithfully allows for mechanistic assessment of the many different stages of sprouting, while still allowing for direct genetic modification of either the endothelial cells or pericytes in a more controlled setting. The fibrin clots containing the sprouting beads can be easily fixed, stained, and imaged at different stages of sprouting; these sprouts can also be placed in a live imaging chamber to perform real-time imaging of sprouting. The methodology presented here is ideal for studying basic mechanisms of angiogenesis through in depth phenotyping and thorough analysis of the pathways activated during angiogenesis.

<table>
	<tbody>
		<tr>
			<td>Sterile Pipette tips</td>
			<td>VWR</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pipettors</td>
			<td>Eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Complete EGM2 Media Bullet Kit</td>
			<td>Lonza</td>
			<td>CC-3162</td>
			<td>HUVEC Media</td>
		</tr>
		<tr>
			<td>MEM</td>
			<td>Gibco</td>
			<td>11095114</td>
			<td>10T1/2 Media</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Gibco</td>
			<td>11965118</td>
			<td>NHLF Media</td>
		</tr>
		<tr>
			<td>Tissue culture-grade PBS</td>
			<td>Gibco</td>
			<td>14190-144</td>
			<td>Magnesium and calcium free</td>
		</tr>
		<tr>
			<td>Accutase</td>
			<td>Life Technologies</td>
			<td>A1110501</td>
			<td>For lifting HUVEC</td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Life Technologies</td>
			<td>15050065</td>
			<td>For lifting 10T 1/2 and NHLF</td>
		</tr>
		<tr>
			<td>Customs siRNAs</td>
			<td>Sigma</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Lipofectamine RNAiMax</td>
			<td>Life Technologies</td>
			<td>13778150</td>
			<td></td>
		</tr>
		<tr>
			<td>HUVEC</td>
			<td>Lonza</td>
			<td>C2517A</td>
			<td></td>
		</tr>
		<tr>
			<td>10T 1/2</td>
			<td>ATCC</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NHLF</td>
			<td>ATCC</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cytodex 3 microcarrier beads</td>
			<td>Sigma</td>
			<td>C3275</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue culture-coated 6 and 10 cm plates</td>
			<td>Corning</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fibrinogen from bovine plasma</td>
			<td>Sigma</td>
			<td>F8630</td>
			<td></td>
		</tr>
		<tr>
			<td>Thrombin</td>
			<td>Sigma</td>
			<td>t9549</td>
			<td></td>
		</tr>
		<tr>
			<td>Aprotinin</td>
			<td>Sigma</td>
			<td>a3428</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon Round-Bottom Tubes</td>
			<td>Corning</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue culture incubator and hood</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>24-well glass bottom plates</td>
			<td>MatTek</td>
			<td>P24G1.513F</td>
			<td>Glass-bottom plates are needed only if the sprouts are going to be imaged. If not, tissue culture plastic is also acceptable.</td>
		</tr>
		<tr>
			<td>Sterile Filtration Device</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

incorporating pericytes into an endothelial cell bead sprouting assay

Angiogenesis is the growth of new vessels from pre-existing vasculature and is an important component of many biological processes, including embryogenesis and development, wound healing, tumor growth and metastasis, and ocular and cardiovascular diseases. Effective in vitro models that recapitulate the biology of angiogenesis are needed to appropriately study this process and identify mechanisms of regulation that can be ultimately targeted for novel therapeutic strategies. The bead angiogenesis assay has been previously demonstrated to recapitulate the multiple stages of endothelial sprouting in vitro. However, a limitation of this assay is a lack of endothelial &#8211; mural cell interactions, which are key to the molecular and phenotypic regulation of endothelial cell function in vivo. The protocol given here presents a methodology for the incorporation of mural cells into the bead angiogenesis assay and demonstrates a tight association of endothelial cells and pericytes during sprouting in vitro. The protocol also details a methodology for effective silencing of target genes using siRNA in endothelial cells for mechanistic studies. Altogether, this protocol provides an in vitro assay that more appropriately models the diverse cell types involved in sprouting angiogenesis, and provides a more physiologically-relevant platform for therapeutic assessment and novel discovery of mechanisms of angiogenesis regulation.

This protocol allows for a tight association of the two cell types in vitro, and the presence of the pericytes complements the occurrence of sprouting (Figure 1A, B). The protocol also enables effective silencing (e.g. via RNA interference) of a gene of interest in a cell type of interest (such as VEGFA specifically in endothelial cells or PDGFR&#946; in pericytes)7,12</su...

Watch this Scientific Journal Video about Incorporating Pericytes into an Endothelial Cell Bead Sprouting Assay at JoVE.com

Incorporating Pericytes into an Endothelial Cell Bead Sprouting Assay

This protocol presents a novel in vitro bead assay that more appropriately models the process of in vivo sprouting angiogenesis by incorporating pericytes. This modification enables the bead assay to more faithfully recapitulate the heterotypic cellular interactions between endothelial cells and mural cells that are critical for angiogenesis.

Angiogenesis is the growth of new vessels from pre-existing vasculature and is an important component of many biological processes, including ...

The overall goal of this assay is to incorporate heterotypic cellular interactions in vitro that encapsulate the complexity of angiogenesis in vivo. This method can help answer key questions in the vascular biology field about the importance of pericyte endothelial cell interactions during angiogenesis. The main advantage of this technique is that it integrates the different stages of sprouting angiogenesis, including proliferation, migration and anastomosis while still permitting the study of endothelial cell pericyte interactions.Begin by reconstituting the microcarrier beads at a six times 10 to the fourth beads per milliliter concentration in PBS, followed by autoclave sterilization of the microcarrier bead suspension. Next, aliquot one milliliter of complete medium into the appropriate number of round-bottom Fluorescence Activated Cell Sorting, or FACS, tubes, and add 20 microliters of room temperature-cooled, autoclave sterilized microcarrier bead suspension to each tube. Add one milliliter of cells to each tube.Agitate tubes and incubate the tubes at 37 degrees Celsius for 2 1/2 to three hours with gentle agitation every 20 minutes. At the end of the incubation, rest the cells in beads at room temperature for five minutes to allow the cells and beads to settle to the bottom of the tubes. Then, use a P1000 pipette to replace 0.5 milliliters of medium from each tube with two times 10 to the fifth pericytes and 0.5 milliliters of complete medium.Agitate the cell-bead suspension, and return the cells to the 37 degrees Celsius incubator with gentle agitation every 20 minutes until the beads have been agitated for a total of four hours. At the end of the incubation, gently agitate the tubes one last time, and immediately transfer the entire two milliliter volume of solution from each tube into individual six centimeter plates. Then, add an additional three milliliters of complete medium to each plate, and incubate the plates at 37 degrees Celsius overnight.The next morning, examine the dishes of microcarrier-coated beads under the microscope at a 20 times magnification to confirm that all of the beads have been sufficiently coated by the endothelial cells. Transfer the plates to a laminar flow hood, and use a P1000 pipette to vigorously aspirate and dispense the coated-bead solutions to detach the cells from the plate bottoms and transfer the supernatants into individual tubes. Allow the beads to settle to the bottoms of the tubes for three minutes.Then use the P1000 pipette to remove as much of the supernatants as possible without disturbing the bead clusters. Re-suspend the beads in one milliliter of complete medium per tube, and allow the beads to settle for 30 to 60 seconds. Use the P1000 pipette to aspirate the supernatant again, and wash the free-floating endothelial cells from the bead suspension with fresh complete medium two more times as just demonstrated.After the third wash, re-suspend the beads in 2.5 milliliters of fibrinogen solution. Add 13 microliters of thrombin solution per well in a 24-well, glass-bottom plate. Then add 0.5 milliliters of the bead fibrinogen solution to each well, taking care to carefully pipette the beads directly into the thrombin, and slowly pipette up and down two to three times to thoroughly mix the solutions.When all of the wells have been seeded, allow the thrombin-fibrinogen solution to preclot in the laminar flow hood for 30 minutes at room temperature. At the end of the incubation, carefully transfer the plate to a 37 degree Celsius incubator for an additional 1.5 to two hours. When the gel has fully solidified, re-suspend normal, human lung fibroblasts at a two times 10 to the fourth cells per milliliter concentration in complete medium, and add one milliliter of fibroblasts to each well.Then return the plate to the cell culture incubator. Strict adherence to the coating protocol will result in cell-coated beads with a rough, golf ball-like appearance, as illustrated in this representative image of sufficiently coated beads that are ready for fibrin gel implantation. Further, this protocol facilitates a tight association of the two cell types in vitro with the presence of the pericytes complementing the occurrence of sprouting.While attempting this procedure, it is important to remember to completely coat the beads with cells during the agitation steps, and to carefully embed the beads in the fibrin gel without introducing bubbles during the gel implantation. Following this procedure, other analyses like mouse studies of angiogenesis can be performed to answer additional questions, such as what is the in vivo relevance of the mechanisms and phenotypes explored in the pericyte beads sprouting assay in vitro? This technique will be useful for researchers in the field of cancer biology and exploring endothelial cell pericyte interactions and functions in a more physiologically relevant in vitro model of angiogenesis.

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7.9K Views.  University of North Carolina at Chapel Hill. The overall goal of this assay is to incorporate heterotypic cellular interactions in vitro that encapsulate the complexity of angiogenesis in vivo. This method can help answer key questions in the vascular biology field about the importance of pericyte endothelial cell interactions during angiogenesis. The main advantage of this technique is that it integrates the different stages of sprouting angiogenesis, including proliferation, migration and anastomosis while still permitting the study o...