Incorporating Pericytes into an Endothelial Cell Bead Sprouting Assay

Summary

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.

Abstract

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 – 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.

Introduction

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 dr....

Protocol

Day 1:

1. Transient Transfection of Endothelial Cells

1. 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's instructions.
   NOTE: This protocol has had great success performing reverse transfections with custom siRNA sequences and a commercial transfection reagent (See Materials Table). The authors have not tested other transfection protocols and reagents with this assay. The steps for the reverse....

Results

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 PDGFRβ in pericytes)^7,12 du.......

Discussion

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.......

Materials

List of materials used in this article

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