Profiles of prostanoid producing enzymes in EPCs and CAECs. Cells were cultured in EBM-2 for 24 h, protein samples were collected for Western blotting. Data are presented as ratio to CAECs. A-C, n=4–5; D, n=3; * P< 0.05, compared with CAECs. E. Cells were treated with the indicated concentrations of TNF-α for 24 h. n=6, data are presented as ratio to EPCs baseline (EBM-2 alone). * P< 0.05.

Effects of CM of EPCs and CAECs on the resting membrane potentials of CSMCs A: A representative tracing of membrane potentials was recorded continuously from CSMCs showing the effects of CMs derived from CAECs, EPCs, or EPCs + indomethacin (Indo, 10 μmol/L). B: Change in membrane potential (ΔmV) after treatment with CMs derived from CAECs, EPCs or EPCs+Indo is shown in the bar graph. Treatment with CM of EPCs significantly hyperpolarized the membrane potential by 9.29±1.95 mV, which was reversed in the presence of 10 μmol/L Indo. n=5–6. * P<0.05, compared with EBM-2, CM-CAECs, or CM-EPCs+Indo.

Role of PPARδ in PGI₂-mediated EPC proliferation. EPCs were transfected with 30 nmol/L Ct-siRNA or PGIS-siRNA (A) or COX-1-siRNA (B) for 48 h. Protein samples were collected for Western blotting. A, n=5, *P<0.05, compared to Ct-siRNA. B, n=3, *P<0.05, compared to Ct-siRNA. C: After EPCs were treated with PGIS- siRNA or Ct-siRNA for 24 h, cells were seeded on 24-well plates (20000/well, in triplicate) and cultured in EGM-2 in the absence or presence of 3 μmol/L iloprost for 48 h. Number of attached cells was counted (n=4–6, **P<0.05, compared with 2 other groups, * P<0.05, compared with Ct-siRNA). D: After EPCs were transfected with COX-1-siRNA or Ct-siRNA for 24 h, cells were seeded in 24-well plates and treated with 3 μmol/L of iloprost or GW501516, or 1 μmol/L cicaprost for 48 h (n=5–12, *P<0.05, compared with COX-1-siRNA, cox-1-siRNA+cicapr, or Ct-siRNA, **P<0.05, compared with Ct-siRNA). E: EPCs were transfected with PGIS-siRNA, COX-1-siRNA, or Ct-siRNA for 48h, then seeded on 96-well plates and assayed for BrdU incorporation [n=3, *P<0.05, compared with control (EGM-2 alone) or Ct-siRNA]. F: After EPCs were transfected with COX-1-siRNA for 30 h, cells were seeded on 96-well plates and incubated in the absence (EGM-2 alone) or presence of indicated treatments for 14 h. Cells were then labeled with BrdU in the same treatment as before, for 24 h (n=5, *P<0.05, compared with COX-1-siRNA in EGM-2 alone or COX-1-siRNA+cicaprost). G: EPCs were transfected with 100 nmol/L of PPARδ-siRNA or Ct-siRNA for 48, protein samples were assayed for Western blotting. Quantification of 4 independent experiments is presented under the representative blot (*P<0.05, compared to Ct-siRNA). H: EPCs were transfected with PPARδ-siRNA or Ct-siRNA for 30 h, cells were then seeded in 96-well plates and cultured in the absence (EGM-2 alone) or presence of 3 μmol/L iloprost for 14 h. Cells were then labeled with BrdU in the same incubations as before, for 24 h (n=5, *P<0.05, compared with Ct-siRNA).

In vivo capillary formation. EPCs were treated with 30 nmol/L Ct-siRNA (A, B), PGIS-siRNA (C), or COX-1-siRNA (D) for 48 h, and then transplanted with Matrigel into nude mice. The gel plugs were excised 2 weeks after transplantation. A, control staining for mouse IgG. B-D, immunostaining for human VEGFR-2 (40X magnification). The arrow heads indicate capillaries, whereas the capillaries containing red blood cells are indicated by arrows. E, quantification of capillary formation (data presented as % of Ct-siRNA), n=4–7, *P<0.05, compared with Ct-siRNA. F: EPCs were transfected with Ct-siRNA or PPARδ-siRNA (n=4, 2 pairs at a concentration of 100 nmol/L, 2 pairs at 30 nmol/L) for 48 h, and transplanted into the nude mice (n=4, *P<0.05, compared to Ct-siRNA).

Phenotyping of human EPCs. A: A single outgrowth colony formed 2–3 weeks after seeding of mononuclear cells on fibronectin-coated plates (4X magnification). B: Around week 4, confluent cells grew into a monolayer with cobblestone appearance (10X magnification). C: FCAS analysis of cell surface markers on EPCs. Shown are representative data from at least 3 independent experiments for each marker. The open black-lined histograms represent test antibodies, and filled histograms represent the control IgG antibodies. Percentage positive cells are shown in each marker panel. D: Cells were cultured in EBM-2 for 24 h, and assayed for total NOS activity. n=4–7, *P<0.05, compared with CAECs. E: Western blotting for eNOS in cells in the presence or absence of TNF-α for 24 h. n=4, * P<0.05.

Production of prostanoids in EPCs and CAECs. Cells were cultured in EBM-2 (A-D) in the presence or absence of 0.5 ng/ml TNF-α for 24 h. CMs were collected and assayed for 6-keto PGF_1α (A), TXB₂ (B), and PGE₂ (C). n=3, * P< 0.05. D: Ratio of 6-keto PGF_1α/TXB₂ under basal EBM-2 and TNF-α conditions. * P< 0.05. n=3. E: EPCs were incubated with EBM-2 (control), or EBM-2 + 0.1 μmol/L SC-560 for 24 h. CMs were collected for measuring of 6-keto PGF_1α. Data are present as percentage of control. * P< 0.05. n=5. F: EPCs were transfected with COX-1-siRNA or Ct-siRNA for 48 h. Cells were then incubated with EGM-2 (2 ml/60 mm dish) for 2 h. The supernatants were collected for measurement of 6-keto PGF_1α, n=5 *P<0.05. In all panels, open columns present EPCs and hatched columns, CAECs.

Role of COX-1 in EPC proliferation. A. EPCs (20000/well of 24-well plate, seeded in triplicate) were cultured in EGM-2 in the absence (control) or presence of indomethacin (Indo), or Indo + iloprost (ilopr) for 3 days. The number of attached cells in each well was counted in a hemocytometer. A: n=7. *P<0.05, compared to the other 2 groups. **P<0.05, compared to control. B: EPCs were treated with the indicated concentrations of SC560 or NS398 for 3 days. n=3, *P<0.05, compared with EGM-2 alone (control). **P<0.05, compared to other 4 groups.

In vitro tube formation. A-B: EPCs were incubated in EBM-2 in the absence (control) or presence of indomethacin (Indo), or Indo+iloprost (Ilopr) for 18 h. Cells were then seeded on Matrigel^TM coated well and continued the treatment for another 4 h. A, representative images of tube formation. B: quantification of tube formation (n=4–6, *P<0.05, compared with all other groups, **P<0.05, compared to control). C-E: After transfected with Ct-siRNA, PGIS-siRNA, or COX-1-siRNA (C, D), or PPARδ (E) for 48 h, EPCs were treated with 3 μmol/L of iloprost or GW501516, or 1 μmol/L cicaprost for 18 h. Tube formation assay was then performed in the same incubations as before. D, Data are presented as % of control (n=3–6, *P<0.05, compared to COX-1-siRNA or COX-1-siRNA+cicapr, **P<0.05, compared with PGIS-siRNA, COX-1-siRNA, or COX-1-siRNA+cicapr). E, n=3, *P<0.05, compare to Ct-siRNA.