AMPK Methods and Protocols

Chapter 33

Studying the Role of AMPK in Angiogenesis

Katrin Spengler, Silke Große, Nderim Kryeziu, Anne Knierim,

and Regine Heller

Abstract

The role of AMPK in angiogenesis can be studied using in vitro and in vivo assays. The endothelial spheroid
assay is a robust three-dimensional in vitro test, which allows investigation of tubular morphogenesis by
integrating cell-cell as well as cell-matrix interactions. The Matrigel plug assay validates the process of
angiogenesis in vivo and allows studies in genetically modified mice. Here, we give a detailed description of
both assays and their application in AMPK research.

Key wordsAngiogenesis, Spheroid assay, Sprouting, Tube formation, Matrigel plug assay

1 Introduction

AMPK has been demonstrated to play a role in angiogenesis in vitro

and in vivo [1]. Our own study has shown that vascular endothelial

growth factor (VEGF) activates AMPK via Ca2+/calmodulin-

dependent protein kinase kinase 2 and that AMPKα1 depletion

reduces VEGF-induced angiogenesis in cultured endothelial cells

and in mice [2]. The mechanisms underlying angiogenic effects of

AMPK are still obscure and may involve endothelial NO synthase

(eNOS) activation [3] and eNOS-independent pathways

[2]. Recent data from our group suggested that the suppression

of the hexosamine biosynthetic pathway by AMPK promotes

VEGF-induced angiogenesis [4]. However, additional processes

may contribute to angiogenic effects of AMPK, and more detailed

knowledge about the role of AMPK in angiogenesis is required.

In general, angiogenesis is monitored in in vitro and in vivo

settings with each having advantages and disadvantages. While

in vitro assays allow the evaluation of distinct processes and com-

ponents of angiogenesis, in vivo assays reflect the complexity of

angiogenesis and verify the biological relevance of specific observa-

tions [5]. In vitro angiogenesis assays include the evaluation of

endothelial functions required for angiogenesis, i.e., proliferation,

Dietbert Neumann and Benoit Viollet (eds.),AMPK: Methods and Protocols, Methods in Molecular Biology, vol. 1732,
https://doi.org/10.1007/978-1-4939-7598-3_33,©Springer Science+Business Media, LLC 2018

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