FIG. 4.

PI3 kinase and Akt kinase activities are required for eNOS- and cGMP-dependent protein kinase-promoted endothelial cell migration. (A) HAEC were infected with either a control adenovirus expressing β-galactosidase (Ad-LacZ) at 30 MOI or an adenovirus expressing a constitutively active form of the cGMP-dependent protein kinase (Ad-PKGca) at 30 or 100 MOI. Cell migration through the membrane filter in the presence or absence of VEGF (10 ng/ml) after 24 h is shown. Cell migration was quantified as described in Materials and Methods. The data are shown as mean ± standard error of the mean and expressed as a percent of that of the Ad-LacZ-infected cells without VEGF. Asterisks indicate a statistically significant difference (P < 0.05) from Ad-LacZ-infected cells without VEGF, and # indicates a statistically significant difference (P < 0.05) from Ad-LacZ-infected cells with VEGF. (B) PKG catalytic domain (≈42 kDa) transgene products were detected in lysates from HAEC infected with Ad-PKGca at 100 MOI. (C) BAEC were infected with Ad-LacZ or an adenovirus expressing a wild-type cGMP-dependent protein kinase 1α (Ad-PKG) at 50 MOI for 16 h before measurement of migration as described in the text. Cell migration through the membrane filter in the presence and absence of 8-bromo-cGMP was measured. The data are shown as mean ± standard error of the mean and expressed as a percentage of the control. Asterisks indicate a statistically significant difference (P < 0.05) from Ad-LacZ-infected cells without 8-bromo-cGMP, and # indicates a statistically significant difference (P < 0.05) from Ad-LacZ-infected cells with 8-bromo-cGMP. (D) PKG1α (≈76 kDa) transgene products were detected in lysates from BAEC infected with Ad-PKG. (E) BAEC were infected with Ad-LacZ or wild-type Ad-PKG at 50 MOI for 16 h. Akt phosphorylation at the Ser⁴⁷³ position was measured after BAEC were incubated in the presence or absence of 8-bromo-cGMP (300 μm) for 1 h. (F) BAEC were infected with Ad-LacZ (100 MOI), Ad-PKG (50 MOI) plus Ad-LacZ (50 MOI), Ad-PKG (50 MOI) plus Ad-dnPI3K (50 MOI), or Ad-dnPI3K (50 MOI) plus Ad-LacZ (50 MOI) for 16 h before measurement of migration as described in the text. Asterisks indicate a statistically significant difference (P < 0.05) from Ad-PKG-infected cells without 8-bromo-cGMP. (G) BAEC were incubated with Ad-LacZ (100 MOI), Ad-eNOS (50 MOI) plus Ad-LacZ (50 MOI), Ad-eNOS (50 MOI) plus Ad-dnPI3K (50 MOI), or Ad-dnPI3K (50 MOI) plus Ad-LacZ (50 MOI) for 16 h before measurement of migration as described in the text. Migration was quantified by counting cells which migrated through the membrane filter. The data are shown as mean ± standard error of the mean and expressed as a percentage of the control. Asterisks indicate a statistically significant difference (P < 0.05) from Ad-LacZ-infected cells.