Thin layer angiogenesis: a modified basement matrix assay for assessment of endothelial cell differentiation

Ashton Faulkner1, Robert Purcell1, Andrew Hibbert1, Sally Latham1, Scott Thomson1, Wendy Hall2, Caroline Wheeler-Jones1, David Bishop-Bailey1. 1Royal Veterinary College, London, UK, 2Kings College London, London, UK

The in vitro assay of endothelial cell morphogenesis on a basement membrane is widely used for first-pass screening of potential pro- and anti-angiogenic compounds (1,2). Traditional volumes of basement membrane used in this assay (in the order of 150μl/cm2) can make it costly, limits working distances for microscopy, and requires further cell manipulation (enzymatic cell detachment) for subsequent molecular analysis. Here, we describe a validated thin-layer modification of the endothelial cell morphogenesis assay that overcomes these limitations.

A thin layer of GeltrexTM basement membrane was spread in either 24-well plates (10μl/well), 96-well plates (2μl/well), or for imaging experiments 35mm glass-bottom dishes (10μl/ well). Human umbilical vein endothelial cells (HUVEC) were then seeded (25,000 cells/cm2) and stimulated with or without vascular endothelial cell growth factor (VEGF; 25ng/ml; medium-only control contained 1% FCS). After 16 hours incubation, tube-like structures were imaged using Leica DMIRB inverted microscope (x10 objective) and quantified by manual counting with ImageJ software. RNA was extracted from tube-forming cells by direct extraction using the Qiagen RNA isolation kit according to the manufacturer’s recommended protocol. GAPDH and COX-2 mRNA were then assessed by qRT-PCR (2). For single cell confocal imaging, HUVEC differentiated on 35mm glass-bottom dishes for 16h were incubated with the live mitochondrial dye MitoTraker green (200nM) for 40min. Confocal fluorescence images were obtained using Leica SP5 confocal microscope (x63 objective).

VEGF significantly increased the number of tube-like structures formed at 16 hours in 24-well plates (33±3 tubes control vs. 54±7 tubes VEGF; p=0.02 by students t-test); data represents mean ± SEM from n=4-6 independent experiments. Similar results were also observed in the 96-well format assay (p<0.05). HUVEC expressed detectable GAPDH and COX-2 mRNA, however there was no significant regulation of COX-2 relative to GAPDH in cells stimulated by VEGF. Z-stack 3D images of the mitochondrial network in differentiated HUVEC mitochondria were then successfully obtained (Figure 1).

Here we present a modified thin-layer version of the traditional tube-formation assay which provides qualitatively comparable results with the traditional method in terms of identifying the pro-angiogenic effect of VEGF but has the added benefit of permitting direct RNA extraction allowing for molecular analysis of tube-forming cells. When coupled with high-contrast microscopy, the significantly reduced working distance allows for the investigation of organelles (such as mitochondria) within cells undergoing morphogenesis, an ability that may be useful in the first-past screening of new pro- or anti-angiogenic compounds.

(1) Piqueras L, Reynolds AR, Hodivala-Dilke KM et al., (2007) Arterioscler. Thromb. Vasc. Biol. 27:63-69

(2) Garonna E, Botham KM, Birdsey GM et al., (2011) PloS One. 6(4):e18823