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

Fig. 1 HCMV secretome mediates EC tubule formation. a Quantitation of two parameters of angiogenesis, lumen formation, and number of branch points at 24 h after plating on matrigel in the presence of control supernatants (SFM alone or SFM + HS/ECGS) and test supernatants conditioned by factors secreted by Mock- and HCMV-infected cells. b Low-power images of EC differentiation on matrigel. c High-power images, conditions as for (b), illustrating the integrity of individual tubules. d Tubule survival after 2 weeks on matrigel in the presence of SFM plus HS/ ECGS or supernatant conditioned by HCMV-infected cells

Fig. 1 HCMV secretome mediates EC tubule formation. a Quantitation of two parameters of angiogenesis, lumen formation, and number of branch points at 24 h after plating on matrigel in the presence of control supernatants (SFM alone or SFM + HS/ECGS) and test supernatants conditioned by factors secreted by Mock- and HCMV-infected cells. b Low-power images of EC differentiation on matrigel. c High-power images, conditions as for (b), illustrating the integrity of individual tubules. d Tubule survival after 2 weeks on matrigel in the presence of SFM plus HS/ ECGS or supernatant conditioned by HCMV-infected cells monolayer is reflected by a marked increase in impedance, since cells restrict the effective area available for current flow. Fluctuations in measured impedance occur in response to micromotions of cells and can be used as indications of cell viability or morphology change (Keese et al. 2004). ECIS technology has been adapted for wound healing assays (Charrier et al. 2005). Cells are grown to confluence on the arrays to achieve high impedance values and a transient voltage spike is applied to kill only the cells on the electrode. Normal ECIS measurements are then used to monitor of the rate of repopulation of the wounded area from cells surrounding the electrode, which is superior to traditional WH assays since it is entirely automated and allows continuous monitoring of the cellular response to wounding. We have used the ECIS system to measure the influence of the HCMV secretome on WH. For these assays, primary HUVECs were plated in two arrays in complete medium and incubated overnight to allow establishment of a confluent monolayer. The next day, cells were serum-starved prior to the addition of secretome samples from mock- and AD169- (plus or minus UV or foscarnet treatments) infected or HSV-1-infected HFs to duplicate wells. SFM and complete medium were used as negative and positive controls, respectively. Immediately after application of supernatants, arrays were placed in the electrode holders and impedance was measured for a few minutes. This confirmed the existence of confluent cell monolayers over each electrode and verified that the cells were healthy. All but one of the chambers of cells were wounded for 30 s. As expected, the electrical wounding resulted in an immediate drop in impedance to the level of an open electrode. The subsequent rise in impedance due to migration and repopulation of the wound was monitored. As shown in Fig. 2 under the presence of complete medium, the wound was repopulated in about 6 h. Similarly,

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