migration, and differentiation into tubular structures. Differentia-
tion is best measured in three-dimensional assays, which integrate
cell-cell and cell-matrix interactions and allow lumen formation in
the developing sprouts. In these assays, endothelial cells are placed
within an extracellular matrix such as collagen, fibrin, or Matrigel,
which may contain pro- or antiangiogenic compounds [5]. Cells
are added as single-cell suspension, spheroids (cell aggregates sta-
bilized by addition of a scaffold molecule such as methylcellulose),
or endothelial cell-coated beads, and tube formation is monitored
over time by counting the length and/or number of the formed
sprouts. However, given the heterogeneity of endothelial cells, the
alteration of their phenotype in culture, and the lack of interaction
with mural cells, it is essential that results from in vitro assays are
validated in vivo. In vivo angiogenesis assays utilize an intact organ-
ism to model vessel formation. The most common in vivo assays
include chorioallantoic membrane assay, fluorescent zebrafish assay,
and tests, which assess mammalian angiogenesis, i.e., matrix inva-
sion assays, dorsal air sac or chamber assays, and tumor mouse
models [5]. All of these assays can be employed for studying the
effect of compounds on angiogenesis, but molecular mechanisms
of sprouting are preferentially investigated in the mammalian mod-
els. Among them, in vivo matrix invasion assays facilitate angiogen-
esis in natural extracellular biomaterials, e.g., Matrigel. They allow
not only the analysis of a given molecule that is incorporated into
the matrix but can also reveal the impact of host molecules on
angiogenesis when performed in genetically modified mice.1.1 Endothelial Cell
Spheroid Assay
The use of endothelial spheroids to study angiogenesis was first
described by Korff et al., who discovered that in contrast to an
endothelial monolayer, the surface layer of endothelial spheroids is
able to differentiate in response to VEGF [6] and that VEGF-
treated spheroids form lumenized capillary-like tubes when embed-
ded in collagen or fibrin [7]. Tubulogenesis is controlled byα 2 β 1
integrins in collagen and by αvβ2 andα 5 β1 in fibrin matrices
[8, 9]. Spheroid assays can be performed with endothelial cells
from different sources. Among them, human umbilical vein endo-
thelial cells (HUVEC) are widely used due to their availability, the
relatively easy preparation, and the high purity of cultures. The
HUVEC-based spheroid assay has been characterized as a versatile
tool to study the impact of pro- and antiangiogenic determinants
on angiogenesis under standardized conditions (i.e., low passage
numbers or immortalized cells) [10]. HUVEC gene expression can
be modified efficiently using siRNA techniques [2, 4], which is
another requirement for a qualified in vitro angiogenesis assay
[11]. In line with this, we performed assays with HUVEC spher-
oids, in which both AMPKαisoforms were individually or jointly
downregulated to investigate the role of AMPK in angiogenesis
[2, 4].520 Katrin Spengler et al.