Biology Reference
In-Depth Information
on factors produced by the EC themselves in coordination with exogenous ang-
iogenic agonists or antagonists in the culture medium. We utilized the matrigel
assay to test the angiogenic activity of the HCMV secretome. Low-passage primary
human umbilical vein endothelial cells (HUVECs) were nutrient-starved in serum
and growth factor-free endothelial medium (SFM) prior to harvest and resuspension
in this same medium. Cells were introduced into 24-well trays containing polymer-
ized plugs of growth-factor-reduced matrigel in the presence of 300 µl of control
and test supernatants. Control supernatants included SF-SFM and complete SFM
containing 10% human serum and endothelial cell growth supplement. Each super-
natant was tested in quadruplicate. Cell phenotype was digitally recorded at 24 h to
evaluate the degree of EC migration and differentiation into tubule structures, and
again after 2 weeks to evaluate vessel survival and stability. Figure 1a graphically
depicts two quantitative measures of angiogenesis: the number of enclosed polygonal
spaces delimited by complete tubules (lumens) and the number of nodes where
branching tubules meet (branch points). Figure 1b shows a representative example
of each culture condition as a low-power image. Figure 1c shows high-power
images to emphasize the differences in vessel integrity between conditions. Results
with control supernatants confirmed that exogenous angiogenic factors are required
to support the formation of a robust capillary network when ECs are plated on
GFR-matrigel. Specifically, ECs cultured in complete medium for 24 h aligned to
form a meshwork of anastomosing tubules with multinodal branch points and
enclosed lumens. In contrast, ECs cultured in SFM for 24 h were unable to form a
consistent network of interconnecting tubules, with many cells generating incom-
plete tubes or aggregating in clumps. In contrast, ECs cultured in the presence of
the HCMV secretome supported the formation of an extensive polygonal capillary
network. Vessels formed in the presence of the HCMV secretome appeared to be
more defined than those formed in the presence of complete medium, presumably
reflecting increased stability of intercellular junctions. To test the degree of stabili-
zation afforded by the HCMV supernatants, trays were kept in culture for 2 weeks
(Fig. 1d). By this time, networks induced by complete medium had degenerated,
but those induced by the HCMV secretome remained intact. Collectively, we show
that the HCMV secretome contains factors that promote angiogenesis and allow
stabilization of neovessels and that generation of this active secretome requires
HCMV replication (data not shown).
HCMV Secretome Induces Wound Healing in Endothelial Cells
For the WH assays we used an electric cell-substrate impedance sensing (ECIS)
system available from Applied Biophysics Inc., to monitor cell behavior. In
ECIS, cells are grown on eight-well chamber slides with 250-mm-diameter gold
electrodes microfabricated onto each well bottom. A larger counter-electrode
completes the circuit, using standard tissue culture medium as an electrolyte.
When a weak AC signal is applied to the system, the presence of a confluent
