Figure 4
Figure 4. PGE2 induces angiogenesis through PKA. (A) PGE1-OH induces the VASP phosphorylation. HMVECs were stimulated with PGE1-OH (in nM) for 5 minutes. Cell monolayers were lysed and subjected to Western blot analysis with the use of anti-VASP Abs. Note that the phosphorylation of VASP retards its migration on SDS-PAGE. (B) Knockdown of EP4 attenuates the PGE2-induced VASP phosphorylation. Cells transiently transfected with siCon or siEP4 were treated with PGE2 (100nM) for 5 minutes and analyzed for VASP phosphorylation content as in panel A. Inhibition of PKA activity with H89 (C) or PKI (D) attenuates the prostaglandin-mediated VASP phosphorylation and tube formation. HMVECs were pretreated, or not, with PKA inhibitor H89 for 30 minutes (C) or PKI for 1 hour (D) followed by stimulation with PGE2 (100nM for H89 and 10nM for PKI) or PGE1-OH (100nM) for 5 minutes for the VASP phosphorylation (top) or 6 hours for the tube formation (bottom). Each point represents the mean ± SEM of values obtained from 3 experiments performed in triplicate. *P < .05, **P < .01, compared with corresponding vehicle sample; #P < .05, ##P < .01, compared with corresponding treatment sample. (E) Knockdown of PKA catalytic subunits. HMVECs were transiently transfected with siCon or siRNA targeting genes encoding the individual PKA catalytic subunits. Quantitative PCR analysis was performed 60 hours after transfection. *P < .05, **P < .01, compared with corresponding siCon sample. (F) Knockdown of PKA Cγ attenuates the PGE2-induced tube formation. Cells transfected with siCon or siRNA targeting gene encoding the individual PKA catalytic subunit for 72 hours were used for the tube formation assay. Each point represents the mean ± SEM of values obtained from 3 experiments performed in triplicate. **P < .01, compared with vehicle-treated samples; ##P < .01, compared with corresponding treatment samples. (G) Detection of PKA catalytic subunit proteins. Recombinant PKA Cα, Cβ, and Cγ GST-fusion proteins were subjected to Western blot analysis with the use of anti-Cα, anti-Cβ, or anti-Cγ Abs. Numbers on the left indicate loaded amount of each protein. (H) Expression of PKA catalytic subunit proteins in HMVECs. Recombinant PKA Cα, Cβ, and Cγ GST-fusion proteins were used as standards. Intensity of bands was determined with ImageJ (National Institutes of Health). Data are presented as fold change in expression of PKA catalytic subunit, where the value of Cα was arbitrarily assigned 1.0. Numbers on the right indicate molecular mass in kDa. The * denotes endogenous catalytic subunit protein. (I) Effect of knocking down expression of PKA catalytic subunits on PGE2-induced VASP phosphorylation as determined by Western blotting with the use of anti-VASP Abs.

PGE2 induces angiogenesis through PKA. (A) PGE1-OH induces the VASP phosphorylation. HMVECs were stimulated with PGE1-OH (in nM) for 5 minutes. Cell monolayers were lysed and subjected to Western blot analysis with the use of anti-VASP Abs. Note that the phosphorylation of VASP retards its migration on SDS-PAGE. (B) Knockdown of EP4 attenuates the PGE2-induced VASP phosphorylation. Cells transiently transfected with siCon or siEP4 were treated with PGE2 (100nM) for 5 minutes and analyzed for VASP phosphorylation content as in panel A. Inhibition of PKA activity with H89 (C) or PKI (D) attenuates the prostaglandin-mediated VASP phosphorylation and tube formation. HMVECs were pretreated, or not, with PKA inhibitor H89 for 30 minutes (C) or PKI for 1 hour (D) followed by stimulation with PGE2 (100nM for H89 and 10nM for PKI) or PGE1-OH (100nM) for 5 minutes for the VASP phosphorylation (top) or 6 hours for the tube formation (bottom). Each point represents the mean ± SEM of values obtained from 3 experiments performed in triplicate. *P < .05, **P < .01, compared with corresponding vehicle sample; #P < .05, ##P < .01, compared with corresponding treatment sample. (E) Knockdown of PKA catalytic subunits. HMVECs were transiently transfected with siCon or siRNA targeting genes encoding the individual PKA catalytic subunits. Quantitative PCR analysis was performed 60 hours after transfection. *P < .05, **P < .01, compared with corresponding siCon sample. (F) Knockdown of PKA Cγ attenuates the PGE2-induced tube formation. Cells transfected with siCon or siRNA targeting gene encoding the individual PKA catalytic subunit for 72 hours were used for the tube formation assay. Each point represents the mean ± SEM of values obtained from 3 experiments performed in triplicate. **P < .01, compared with vehicle-treated samples; ##P < .01, compared with corresponding treatment samples. (G) Detection of PKA catalytic subunit proteins. Recombinant PKA Cα, Cβ, and Cγ GST-fusion proteins were subjected to Western blot analysis with the use of anti-Cα, anti-Cβ, or anti-Cγ Abs. Numbers on the left indicate loaded amount of each protein. (H) Expression of PKA catalytic subunit proteins in HMVECs. Recombinant PKA Cα, Cβ, and Cγ GST-fusion proteins were used as standards. Intensity of bands was determined with ImageJ (National Institutes of Health). Data are presented as fold change in expression of PKA catalytic subunit, where the value of Cα was arbitrarily assigned 1.0. Numbers on the right indicate molecular mass in kDa. The * denotes endogenous catalytic subunit protein. (I) Effect of knocking down expression of PKA catalytic subunits on PGE2-induced VASP phosphorylation as determined by Western blotting with the use of anti-VASP Abs.

Close Modal
This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal