Figure 3.

HRGP reverses inhibition of endothelial cell migration and tube formation by Vstat120. A: Scratch assays. A scratch was made within a confluent monolayer of HDMVECs and the cells were incubated overnight with normal media (media only – lane 1) showing moderate migratory growth into the acellular area of about 40% total closure. When CM from Vstat120-expressing cells (6A8 – lane 7 and LN229Vstat120 – lane 3) was added, fewer cells were seen migrating from the original cell boundary, especially when compared with the parental LN229 line (lane 2). An average of 30.3% and 31.7% closure, respectively, was calculated for the Vstat120-rich CMs used versus 57% for LN229. When CM from lines expressing both Vstat120 and HRGP (line 5 – lane 4, line 22 – lane 5, and line 23 – lane 6) was added cell migration equal to or greater than cells treated with normal growth media only was seen and approximately doubled that of the 6A8 control. The percent wound was 65.1%, 64.6%, and 56.0% for the three HRGP lines compared with 30.3% for the 6A8 control (*P < 0.003 for all cases). B–F: Matrigel tube formation assays. Equal numbers of HDMVECs were seeded onto a matrigel support and incubated overnight with Vstat120-only CM (LN229Vstat120 or the HRGP control line 6A8), normal growth media (media only), or CM from three lines expressing both Vstat120 and HRGP (lines 5, 22, and 23.) With no CM added, after 14 hours some cells have begun to spread and form tube-like structures with an average of 12.7 branch points per 10× field (F – lane 1 – media only; 2 fields in each of 3 separate experiments were counted). This is reduced when Vstat120 is present in the original Vstat120 cell line or in the HRGP control line (6A8 – E) with 6.8 and 7.3 branch points counted for each (F – lanes 3 and 7). On addition of HRGP (B–D), increased numbers of web-like structures are observed consistent with tube formation. The average number of branch points was 18.2, 17.3, and 17.1 for the three HRGP lines 5, 22, and 23, respectively (*P < 0.002 when compared with the control 6A8 line). HRGP attenuates Vstat120-induced apoptosis. G: Western blot of caspase-3 activation of HDMVECs after treatment with CMs containing either Vstat120 or Vstat120 and HRGP and quantification of caspase-3 activation (H). CMs with Vstat120 show a 2.5-fold (HRGP control 6A8) and fourfold (LN229Vstat120) increase in caspase-3 activation over serum-free media. When HRGP is present with Vstat120, caspase-3 activity returns to basal levels (*P < 0.04 when comparing HRGP lines 5, 22, and 23 to the control line 6A8).