Fig 1.
Effects of the periostin domain 1 and its fragments on migration of ECFCs.
(A) Schematic diagram of human periostin D1 domain (amino acid 97~234) and its six truncated protein constructs (amino acids 97~216, 97~196, 97~176, 97~156, 97~136, 97~116). The red square box indicates 20 amino acid of the periostin amino acid sequence. (B) Purification and SDS-PAGE analysis of the recombinant fragments of periostin D1 domain. (C) Effects of the Periostin D1 domain and its fragments on migration of ECFCs. Chemotactic migration of ECFCs was determined after treatment with VEGF, full length recombinant periostin, the periostin D1 domain or its fragments (each 10 μg/ml). Data represent mean ± S.D. (n = 9), * indicates P < 0.05 vs. control.
Fig 2.
Effects of the periostin peptide 136~156 and its fragments on migration and tube formation of ECFCs.
(A) Effects of the periostin peptide 136~151 and its fragments on chemotactic migration of ECFCs. (B) Dose-dependent effects of the periostin peptide 136 ~ 151 on chemotactic migrations of ECFCs. Migration of ECFCs in response to increasing concentrations of the periostin peptide 136~151 was determined. (C) Effects of the periostin peptide 136~151 on the tube formation of ECFCs (scale bar = 100 μm). (D) Tube formation was quantified by measuring the length of tubes in four random fields from each well and normalizing the values relatives to those of the corresponding control. Data represent mean ± S.D. (n = 12), #, P <0.05; * indicates P < 0.05 vs. control.
Fig 3.
Effects of the periostin peptide 136~151 and its fragments on angiogenic activities of ECFCs in vitro.
(A) Amino acid sequence of the periostin peptide 136~151. (B, C) Effects of periostin peptide 136~151 and its fragments on migration (B) and adhesion (C) of ECFCs. Migration and adhesion of ECFCs were measured in response to VEGF, recombinant periostin FL (10 μg/ml), the periostin D1 domain (10 μg/ml), or various fragments of periostin 136~151 (each 0.5 μM) after treatment for 12 h. (D, E) Effects of various periostin peptides on tube formation of ECFCs. The length of tubes was quantified in four random fields from each well and the values was normalized relative to those of the corresponding control (scale bar = 100 μm). Data are expressed as mean ± SD. #, P <0.05; * indicates P < 0.05 vs. control.
Fig 4.
Effect of periostin peptide 142~151 on ECFC proliferation.
(A, B) ECFCs were incubated with the periostin peptide 142~151 for 24 h and labeled with anti-Ki67 antibody (green) and nuclei were counterstained with DAPI (blue). The number of Ki67-positive cells was quantified per DAPI. (scale bar = 50 μm). (C) Effects of the periostin peptide 142~151 on proliferation of ECFCs. Human ECFC were treated with the periostin peptide 142~151 and VEGF for 24 h, followed by MTT analysis. Data are expressed as mean ± SD. * indicates P < 0.05.
Fig 5.
Effects of neutralizing antibodies against integrins β1, β3, and β5 on ECFC migration.
ECFCs were pre-incubated with control antibody or neutralizing antibodies against integrins β1, β3, and β5, followed treatment with periostin FL (10 μg/ml), Periostin D1 (10 μg/ml), or 0.5 μM the periostin peptide 142~151 for determination of cell migration. Data represent mean ± S.D. (n = 6). * indicate P <0.05 vs. anti-control.
Fig 6.
Role of PI3K and MEK1/2 pathway in the periostin peptide 142~151-induced migration of ECFCs.
(A) Effects of the periostin peptide 142~151 on phosphorylation (AktSer473 and ErkThr202/Tyr204) and expression levels of Akt and Erk were determined by Western blot analysis. The cells were treated with VEGF (10 ng/ml), the periostin FL (10 μg/ml), periostin D1 domain 1 (10 μg/ml), or the periostin 142~151 amino acid peptide (0.5 μM) for 1 minutes. Representative data from three independent experiments. (B) ECFCs were pre-incubated with the PI3K inhibitors Wortmannin and LY294002 or the ERK inhibitor U0126, followed by treatment with VEGF (10 ng/ml), periostin FL (10 μg/ml), the periostin D1 domain (10 μg/ml), or the periostin peptide 142~151 (0.5 μM). Data represent mean ± S.D. (n = 6). * indicate P <0.05 vs. control.