Angiopoietin-1-mediated phosphorylation and functional inhibition of FKHR in primary endothelial cells. (A) MVECs were serum-starved for 1 h and then either untreated or stimulated for the indicated times with VEGF, bFGF, or Ang-1. Cell lysates were subjected to Western blot with antibodies against phospho-Akt (Ser 473), Akt, phospho-FKHR (Thr 24 or Ser 256), FKHR, phospho ERK 1/2(Thr 202/Tyr 204) and ERK 1/2. (B) MVECs were serum-starved overnight and then either untreated or stimulated with Ang-1 for the indicated times. Cytoplasmic and nuclear fractions were prepared and subjected to Western blot with antibodies against phospho-FKHR (Thr 24 or Ser 256) or FKHR. Antibodies against GAPDH and CREB were used to validate the fractionation procedure. (C) MVECs were serum-starved overnight and then either untreated or stimulated with Ang-1 for 30 min. Following treatment, cells were fixed and stained with antibody against phospho-FKHR Ser 256. (D) MVECs were transfected with FHRE-Luc reporter plasmid along with plasmids encoding: eGFP, FKHR + eGFP, or FKHR + myrAkt. At 24 h after transfection, cell lysates were prepared and luciferase activity was measured. Bars show luciferase activity relative to eGFP control (assigned a value of 1.0). Bars represent the mean and S.E.M., n = 3.(E) MVECs were transfected with FHRE-Luc reporter plasmid along with plasmids encoding either eGFP or FKHR. At 8 h after transfection, cultures expressing FKHR were either untreated or stimulated with Ang-1. At 24 h, cell lysates were prepared and luciferase activity was measured. Bars show luciferase activity relative to eGFP control (assigned a value of 1.0). Bars represent the mean and S.D., n = 9. The difference in luciferase activity between the FKHR and FKHR + Ang-1 samples was statistically significant by t-test, p < 0.001.

Angiopoietin-1 inhibits FKHR-induced apoptosis in endothelial cells. (A, top panel) HUVECs were infected with adenoviruses encoding either GFP or FKHR-TM. At 24, 32, or 48 h after infection, cell lysates were prepared and subjected to Western blot with antibodies against myc tag (detects virally encoded FKHRTM), GFP, active caspase-3, or ERK (as a loading control). (Bottom panel) HUVECs were infected with adenoviruses encoding either FKHR-DB or FKHR-TM for 24 or 48 h. Cell lysates were prepared and subjected to Western blot with antibodies against myc tag (detects both FKHR-TM and FKHR-DB), active caspase-3 or ERK. (B) HUVECs were infected with adenoviruses encoding either GFP or FKHR-TM for 32 h. Cells were fixed and nuclear morphology was assessed by staining with Hoechst 33342. Arrows indicate apoptotic nuclei. (C) HUVECs were infected with adenoviruses encoding either GFP, FKHR, or FKHR-TM for 24, 32, or 45 h. At each time point the relative number of living cells was determined by MTS assay. The data points represent the number of living cells relative to the GFP control cultures (assigned a value of 1.0). Each data point is the mean and S.D. of three experiments. (D) MVECs were infected with adenoviruses encoding either GFP or FKHR. At 9, 22, and 30 h after infection, FKHR expressing cultures were either untreated or stimulated with Ang-1. At 48 h after infection, the relative number of viable cells was determined by MTS assay. The bars represent the mean and S.D., n = 6. The difference in the number of viable cells between the FKHR and the FKHR + Ang-1 cultures was statistically significant by t-test, p < 0.005.

Validation of microarray data. (A) HUVECs were infected with adenoviruses encoding GFP, FKHR-DB, or FKHRTM for 16 or 24 h. Total RNA was isolated and subjected to Northern blot using probes specific for Ang-2, MMP7, semaphorin 3C, or survivin. Ethidium bromide-stained rRNA is shown to confirm equal loading. (B, top panel) HUVECs were infected with adenoviruses encoding either GFP or FKHR-TM for 8, 12, or 24 h. Cell lysates were prepared and subjected to Western blot with antibodies against Ang-2, Id2, myc tag (detects FKHR-TM), GFP, or ERK. (Bottom panel) HUVECs were infected with adenoviruses encoding either GFP or FKHR-TM for 16, 20, or 24 h. Cell lysates were prepared and subjected to Western blot with antibodies against survivin, grancalcin, or myc tag (FKHR-TM). (C) HUVECs were infected with viruses encoding either GFP, FKHR-TM, or FKHR-TM H212R for 24 h. Cell lysates were prepared and subjected to Western blot with antibodies against myc tag (FKHR), Ang-2, grancalcin (gca), and ERK. Each infection was done in triplicate, with similar results (two samples from each set are shown).

Angiopoietin-1 inhibits FKHR-mediated changes in gene expression. MVECs were infected with adenoviruses encoding either GFP or FKHR. At 9 h after infection, FKHR-expressing cultures were either untreated or stimulated with Ang-1. At 14 h after infection, total RNA was isolated and the relative levels of Ang-2, decorin, and Id2 RNAs (normalized to GAPDH) were determined by real-time PCR. The data are expressed as relative RNA level versus GFP control (assigned a value of 1.0). Bars represent the mean and S.D., n = 3. The effect of Ang-1 on the levels of Ang-2, decorin, and Id2 RNAs was statistically significant by t-test, p < 0.005.

Regulation of gene expression by the endogenous FKHR protein. (A) HUVECs were infected with adenoviruses encoding either GFP or myrAkt (the samples in lanes 1,2 and 3,4 are duplicates) for 24 h. Cell lysates were prepared and subjected to Western blot with antibodies against phospho-Akt (Ser 473), Akt, phospho-FKHR (Thr 24), or FKHR. The myrAkt protein is larger than the endogenous Akt because it is tagged with eGFP. (B) HUVECs were infected with adenoviruses encoding either GFP or myrAkt for 16 or 24 h. Total RNA was isolated and the relative levels (normalized to actin) of Ang-2, CTGF, and ESM-1 RNAs were determined by real-time PCR. For each RNA analyzed, the level in the GFP-expressing cells was assigned a value of 1.0. (C) HUVECs were infected with adenoviruses encoding either a negative control siRNA or siRNA against FKHR. At 48 and 72 h after infection, cell lysates were prepared and subjected to Western blot with antibodies against FKHR, Ang-2, Akt, and ERK. (Right) The relative levels of FKHR and Ang-2 proteins (assigned a value of 1.0 in the control samples) were quantitated and plotted. The bars represent the mean and S.D., n = 3. The effect of the FKHR siRNA on FKHR and Ang-2 protein levels was statistically significant at both time points by t-test, p < 0.001. (D) HUVECs were infected with adenoviruses encoding either a negative control siRNA or siRNA against FKHR. At 48 h after infection, total RNA was isolated and the relative levels (normalized to cyclophilin) of FKHR, Ang-2, ESM-1, and CTGF RNAs were determined by real-time PCR. Bars represent the mean and S.D., n = 4. The effect of FKHR siRNA on the levels of each RNA was statistically significant by t-test, p < 0.0001.

Angiopoietin-1 regulates endothelial cell gene expression via FKHR. Ang-1, via activation of the Akt pathway, inhibits FKHR-dependent changes in the expression of genes that regulate vascular remodeling and endothelial cell apoptosis. The induction of Ang-2 expression by FKHR (or by other signals) might initiate a positive feedback loop in which Ang-2, by blocking Ang-1/Akt signaling, induces further increases in its own expression and in the expression of genes like semaphorin 3C and slit2, which may directly promote vessel destabilization and remodeling.