- To obtain a representative evaluation for each plug, monitor
two sections from at least four levels, respectively, and calculate
the final average from all values. - Representative images from Matrigel plugs in wild-type and
AMPKα1 knockout mice as published by Stahmann et al. [2]
are shown in Fig.5.
4 Notes
- HUVEC are prepared from umbilical cords by filling the vein
with 0.01% collagenase and incubating it for 3 min at 37C.
Dissociated cells are collected and seeded in endothelial growth
medium on culture flasks coated with 0.2% gelatine. After
reaching confluency, cells are detached with Trypsin/EDTA
and split at a 1:3 ratio. Cells of the first passage are used to
generate spheroids. - We have good experiences with endothelial mitogen from BTI
Inc. or endothelial growth supplement from Sigma-Aldrich.
Fig. 5VEGF-induced tube formation is inhibited in AMPKα1 knockout mice}. 500μL of Matrigel including
200 ng of VEGF and 200μg of heparin or 500μL Matrigel without VEGF was injected subcutaneously into the
right or left flank, respectively, of wild-type or AMPKα1 knockout mice. Plugs were removed after 7 days, fixed
in zinc fixative, and embedded in paraffin. Tissue sections were stained with an anti-CD31 antibody and
evaluated by fluorescence microscopy. Representative images (400) and an analysis of the vascularized
area are shown. MeanSEM;n¼4 for control plugs,n¼13 or 14 for VEGF-containing plugs in wild-type or
AMPKα1 knockout mice, respectively.+p<0.05 versus non-VEGF-treated controls; *p<0.05 versus the
respective values in wild-type mice.}This research was originally published in The Journal of Biological
Chemistry: Stahmann N, Woods A, Spengler K, Heslegrave A, Bauer R, Krause S, Viollet B, Carling D, Heller
R. Activation of AMP-activated protein kinase by vascular endothelial growth factor mediates endothelial
angiogenesis independently of nitric-oxide synthase.J Biol Chem2010; 285: 10638-52.©the American
Society for Biochemistry and Molecular Biology
532 Katrin Spengler et al.