PGE2 promotes angiogenesis through EP4 and PKA Cγ pathway by Yushan Zhang, and Yehia Daaka Blood Volume 118(19):5355-5364 November 10, 2011 ©2011 by American Society of Hematology

PGE2 promotes angiogenesis. PGE2 promotes angiogenesis. (A-B) PGE2 promotes the in vitro tube formation. HMVECs were seeded on growth factor–reduced Matrigel-coated wells in the absence or presence of PGE2 or VEGF. The tube formation images at 50× magnification in the central field of the well were taken 6 hours later (A), and the branching points were counted with ImageJ software (National Institutes of Health). (B) Each point represents the mean ± SEM of 3 experiments performed in triplicate. (C-D) PGE2 promotes angiogenesis. Fertilized chicken eggs were incubated for 10 days, and CAMs were treated with PGE2 (1μM) or VEGF (100 ng/mL) twice per day for a total of 3 days. Blood vessel images at a 50× magnification were photographed (C), and branching points quantified with the ImageJ software (National Institutes of Health; D). The experiments were initiated with 12 eggs for each group and were culminated with 4-6 evaluable CAMs. Each point represents the mean ± SEM of 3 independent experiments. (E-F) PGE2 promotes aortic ring vessel outgrowth. Mouse thoracic aorta were sectioned into 1-mm rings and placed into growth factor–reduced Matrigel-coated wells. The rings were treated daily with PGE2 (100nM) or VEGF (50 ng/mL) for a total of 6 days, and 50× magnification images were recorded. The area of the cell outgrowth was quantified with the ImageJ (National Institutes of Health). (F) Two rings per aorta from a total of 4 aortas were used for each group, and the assay was repeated 3 times. Each point represents the mean ± SEM of 5 evaluable outgrowths. (B,D,F) *P < .05 and **P < .01 versus vehicle groups. Yushan Zhang, and Yehia Daaka Blood 2011;118:5355-5364 ©2011 by American Society of Hematology

PGE2 promotes the tube formation by activating EP4. PGE2 promotes the tube formation by activating EP4. (A) Effect of EP agonists on the tube formation. HMVECs were stimulated with PGE2, sulprostone (Sulp), butaprost (Buta), or PGE1-OH (PGE1). Tubular networks were quantified with ImageJ software (National Institutes of Health). Each point represents the mean ± SEM of values obtained from 3 independent experiments performed in triplicate. (B) Effect of EP antagonists on the tube formation. HMVECs were treated with AH23848 or AH6809 in the presence or absence of PGE2 or PGE1-OH. Tubular networks were quantified with ImageJ software (National Institutes of Health). Each point represents the mean ± SEM of values obtained from 3 independent experiments performed in triplicate. (C) Knockdown of endogenous EP4 expression. HMVECs were transfected with control scrambled siRNA (siCon) or siRNA targeting EP4 (siEP4) and allowed to grow for 60 hours. Total RNA was extracted and subjected to quantitative real-time PCR to determine expression of the EP4 gene. Expression of EP4 gene in the siEP4 sample is presented as a fraction of that in the siCon sample. Each point represents the mean ± SEM of 3 independent experiments. (D) Knockdown of EP4 expression inhibits the PGE2-induced tube formation. Representative images were obtained after 78 hours of transfection with siRNA. (E) Quantification of the tube formation after EP4 knockdown. Each point represents the mean ± SEM of triplicate samples, and experiments were repeated 3 times. ##P < .01 versus corresponding treatment in the siCon group. (A-C,E) *P < .05 and **P < .01, in comparison to corresponding control groups. Yushan Zhang, and Yehia Daaka Blood 2011;118:5355-5364 ©2011 by American Society of Hematology

PGE2 induces angiogenesis via cAMP. PGE2 induces angiogenesis via cAMP. (A) PGE2 and PGE1-OH treatment increases cAMP accumulation. HMVECs were treated with PGE2 (in nM), PGE1-OH (in nM), or isoproterenol (ISO; 1μM), and cAMP levels were determined with a cAMP enzyme immunoassay. Each point represents the mean ± SEM of triplicate samples, and experiments were repeated 3 times. (B) cAMP mimetic db-cAMP induces the tube formation. HMVECs were treated with db-cAMP for 6 hours followed by quantitation of branching points, as described. (C) cAMP competitive analog Rp-cAMPS inhibits the PGE2-induced tube formation. HMVECs were treated with Rp-cAMPS in the presence or absence of PGE2 (100nM) for 6 hours followed by quantitation of branching points. Each point represents the mean ± SEM of triplicate samples, and experiments were repeated 3 times. #P < .05 and ##P < .01 versus corresponding treatment in the control group. For all panels, *P < .05, **P < .01, compared with appropriate control group. Yushan Zhang, and Yehia Daaka Blood 2011;118:5355-5364 ©2011 by American Society of Hematology

PGE2 induces angiogenesis through PKA PGE2 induces angiogenesis through PKA. (A) PGE1-OH induces the VASP phosphorylation. 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. Yushan Zhang, and Yehia Daaka Blood 2011;118:5355-5364 ©2011 by American Society of Hematology

PKA substrates mediate the PGE2 proangiogenic signal. PKA substrates mediate the PGE2 proangiogenic signal. (A) Knockdown efficiency of representative targets. HMVECs were transiently transfected with scrambled siRNA (CN) or siRNA targeting indicated genes (KD) and allowed to recover for 72 hours. Cell lysates were collected and analyzed by immunoblotting with the use of the corresponding Abs. (B) Quantitation of HMVEC tube formation. HMVECs transfected with siRNA corresponding to the indicated gene or scramble siRNA for 72 hours were seeded into growth factor–reduced Matrigel in the presence of vehicle DMSO or PGE2 (100nM) for 6 hours, followed by counting the branching points, as described. The branching points of the tubular network of cells transfected with scramble siRNA and treated with DMSO were set to 100. All other branching point counts are presented as the fraction of this control value. Each point represents the mean ± SEM of values obtained from 3 experiments performed in triplicate. *P < .05, compared with vehicle in the control group; ##P < .01, compared with corresponding treatment in the control group. (C) Representative images depicting tube formation of HMVECs transfected with siRNA targeting GSK3β (siGSK3β) or scrambled siRNA (siCon) after treatment, or not, with PGE2 (100nM) for 6 hours. (D) PGE2 promotes the dose-dependent GSK3β phosphorylation. HMVECs were treated with PGE2 (in nM) for 5 minutes (left) or LiCl (in mM) for 5 minutes (right). Cell lysates were analyzed by Western blotting with the use of phospho-Ser9 or total GSK3β Abs. (E) LiCl promotes the HMVEC tube formation. HMVECs were subjected to tube formation assay in the presence of LiCl or NaCl (both in mM) for 6 hours, followed by measurement of branching points of the tubular network, as described. Each point represents the mean ± SEM of values obtained from 3 experiments performed in triplicate. *P < .05, **P < .01, compared with NaCl group. (F) Knockdown of PKA Cγ attenuates PGE2-induced GSK3β phosphorylation. HMVECs transfected with siCon or siPKA that targeted individual catalytic subunits were stimulated, or not, with PGE2 (100nM) for 5 minutes, and cell lysates were subjected to Western blot analysis with the use of anti–phospho-Ser9 (top) or total (bottom) GSK3β Abs. Yushan Zhang, and Yehia Daaka Blood 2011;118:5355-5364 ©2011 by American Society of Hematology

EP4 and PKA mediate the in vivo angiogenesis. EP4 and PKA mediate the in vivo angiogenesis. (A) Images of polyvinyl acetal sponges harvested from mice. The sponges were implanted in the flank of C57BL/6 mice and injected with PGE2 (8.8 ng/g body weight), AH23848 (25 or 125 ng/g body weight), or H89 (11 or 55 ng/g body weight) every 2 days for 2 weeks. (B) Hemoglobin content in the sponges as determined with the Drabkin reagent. Each point represents the mean ± SEM of values obtained from 4 to 5 sponges. *P < .05, **P < .01, compared with corresponding vehicle group. Low, AH23848 (25 ng/g body weight) and H89 (11 ng/g body weight) and high, AH23848 (125 ng/g body weight) and H89 (55 ng/g body weight), groups. (C) Expression of CD31+ cells by immunohistochemistry. Harvested sponges were sectioned and stained with the endothelial cell marker CD31 Ab. Sections were counterstained with hematoxylin to visualize nuclei. (D) Blood vessel density in harvested sponges as quantitated by counting red-staining–positive cells or cell clusters. Three 200× magnification fields containing the most prominent hotspots were counted for each section. Each point represents the mean ± SEM of values obtained from 4 to 5 sponges. *P < .05, **P < .01, compared with corresponding vehicle group. Low, AH23848 (25 ng/g body weight) and H89 (11 ng/g body weight) and high, AH23848 (125 ng/g body weight) and H89 (55 ng/g body weight), groups. (E) Schematic presentation of PGE2-induced angiogenic signal in HMVECs (see text for details). Yushan Zhang, and Yehia Daaka Blood 2011;118:5355-5364 ©2011 by American Society of Hematology