Angiogenic function of prostacyclin biosynthesis in human endothelial progenitor cells

Abstract

The role of prostaglandin production in the control of regenerative function of endothelial progenitor cells (EPCs) has not been studied. We hypothesized that activation of cyclooxygenase (COX) enzymatic activity and the subsequent production of prostacyclin (PGI(2)) is an important mechanism responsible for the regenerative function of EPCs. In the present study, we detected high levels of COX-1 protein expression and PGI(2) biosynthesis in human EPCs outgrown from blood mononuclear cells. Expression of COX-2 protein was almost undetectable under basal conditions but significantly elevated after treatment with tumor necrosis factor-alpha. Condition medium derived from EPCs hyperpolarized human coronary artery smooth muscle cells, similar to the effect of the PGI(2) analog iloprost. The proliferation and in vitro tube formation by EPCs were inhibited by the COX inhibitor indomethacin or by genetic inactivation of COX-1 or PGI(2) synthase with small interfering (si)RNA. Impaired tube formation and cell proliferation induced by inactivation of COX-1 were rescued by the treatment with iloprost or the selective peroxisome proliferator-activated receptor (PPAR)delta agonist GW501516 but not by the selective PGI(2) receptor agonist cicaprost. Downregulation of PPARdelta by siRNA also reduced angiogenic capacity of EPCs. Iloprost failed to reverse PPARdelta siRNA-induced impairment of angiogenesis. Furthermore, transfection of PGI(2) synthase siRNA, COX-1 siRNA, or PPARdelta siRNA into EPCs decreased the capillary formation in vivo after transplantation of human EPCs into the nude mice. These results suggest that activation of COX-1/PGI(2)/PPARdelta pathway is an important mechanism underlying proangiogenic function of EPCs.

Figure 1

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.

Figure 1

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.

Figure 2

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.

Figure 3

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.

Figure 4

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.

Figure 5

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.

Figure 6

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).

Figure 6

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).

Figure 6

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).

Figure 7

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.

Figure 7

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.

Figure 8

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).