PLoS One. 2012;7(2):e30503. doi: 10.1371/journal.pone.0030503. Epub 2012 Feb 15.

Over-expression of PDGFR-β promotes PDGF-induced proliferation, migration, and angiogenesis of EPCs through PI3K/Akt signaling pathway.

Wang H, Yin Y, Li W, Zhao X, Yu Y, Zhu J, Qin Z, Wang Q, Wang K, Lu W, Liu J, Huang L.

Source

Institute of Cardiovascular Science Xinqiao Hospital, Third Military Medical University, Shapingba District, Chongqing, People's Republic of China.

Abstract

The proliferation, migration, and angiogenesis of endothelial progenitor cells (EPCs) play critical roles in postnatal neovascularization and re-endothelialization following vascular injury. Here we evaluated whether the over-expression of platelet-derived growth factor receptor-β (PDGFR-β) can enhance the PDGF-BB-stimulated biological functions of EPCs through the PDGFR-β/phosphoinositide 3-kinase (PI3K)/Akt signaling pathway. We first confirmed the expression of endogenous PDGFR-β and its plasma membrane localization in spleen-derived EPCs. We then demonstrated that the PDGFR-β over-expression in EPCs enhanced the PDGF-BB-induced proliferation, migration, and angiogenesis of EPCs. Using AG1295 (a PDGFR kinase inhibitor), LY294002 (a PI3K inhibitor), and sc-221226 (an Akt inhibitor), we further showed that the PI3K/Akt signaling pathway participates in the PDGF-BB-induced proliferation, migration, and angiogenesis of EPCs. In addition, the PI3K/Akt signaling pathway is required for PDGFR-β over-expression to enhance these PDGF-BB-induced phenotypes.

PMID:: 22355314; [PubMed - indexed for MEDLINE]
PMCID:: PMC3280261

Free PMC Article

Images from this publication.See all images (11)Free text

Figure 2

PDGFR-β expression and localization in EPCs.

(A)RT- PCR analysis of PDGFR-β (296 bp) in EPCs at Day 4, 7, 14, and 21. (B) Western blot analysis of PDGFR-β protein (124 kDa) in EPCs at Day 4, 7, 14, and 21. (C, D, E) Subcellular localization of PDGFR-β in EPCs. EPCs were stained for PDGFR-β (C) and nuclei were stained with DAPI (D). PDGFR-β was localized predominantly to the plasma membrane of EPCs. PDGFR-β positive cells in the total cells were 88.6±4.3% (n = 5; three random fields per well).Scale bar = 25 µm.

Over-Expression of PDGFR-? Promotes PDGF-Induced Proliferation, Migration, and Angiogenesis of EPCs through PI3K/Akt Signaling Pathway

PLoS One. 2012;7(2):e30503.

Figure 4

ELISA of PDGF-BB secreted by EPCs in response to PDGFR-β transfection.

The concentration of PDGF-BB in the supernatant of culture medium was measured using ELISA. The concentration of PDGF-BB was significantly lower in the pEGFP-N2-PDGFR-β group than in the control or pEGFP-N2 group at 48 h and 72 h post-transfection. * P<0.01 vs. control or pEGFP-N2 (n = 6).

Over-Expression of PDGFR-? Promotes PDGF-Induced Proliferation, Migration, and Angiogenesis of EPCs through PI3K/Akt Signaling Pathway

PLoS One. 2012;7(2):e30503.

Figure 6

Effects of PDGFR-β overexpression on PDGF-BB-induced EPC migration.

EPCs (untransfected,or transfected with either pEGFP-N2 or pEGFP-N2-PDGFR-β) were incubated with PDGF-BB of different concentrations. EPC migration was examined by the Transwell system. * P<0.01 vs. pEGFP-N2-PDGFR-β cells under 0 ng/mL PDGF-BB stimulation.

Over-Expression of PDGFR-? Promotes PDGF-Induced Proliferation, Migration, and Angiogenesis of EPCs through PI3K/Akt Signaling Pathway

PLoS One. 2012;7(2):e30503.

Figure 8

The role of PI3K/Akt pathway in PDGF-BB-induced proliferation, migration, and tube formation of EPCs.

(A, B, C) Cells in the control group (bright bars, left half) and the pEGFP-N2-PDGFR-β group (dark bars, right half) were without pretreatment or pretreated with AG1295, LY294002, or sc-221226 for 1 h and then treated with 20 ng/mL PDGF-BB (for the control cells) or 80 ng/mL PDGF-BB (for the pEGFP-N2-PDGFR-β cells). PDGF-BB-induced proliferation (A), migration (B), and angiogenesis (C) of EPCs were significantly inhibited by pretreatment with AG1295, LY294002, or sc-221226. ** P<0.01 vs. control cells under 0 ng/mL PDGF-BB stimulation; * P<0.01 vs. control cells under 20 ng/mL PDGF-BB stimulation. ## P<0.01 vs. pEGFP-N2-PDGFR-β cells under 0 ng/mL PDGF-BB stimulation; # P<0.01 vs. pEGFP-N2-PDGFR-β cells under 80 ng/mL PDGF-BB stimulation.

Over-Expression of PDGFR-? Promotes PDGF-Induced Proliferation, Migration, and Angiogenesis of EPCs through PI3K/Akt Signaling Pathway

PLoS One. 2012;7(2):e30503.

Figure 10

Representative photos of PDGF-BB-induced EPC angiogenesis in vitro by PI3K/Akt pathway.

Cells in the control group (A) and the pEGFP-N2-PDGFR-β group (B) were without pretreatment or pretreated with AG1295, LY294002, or sc-221226 for 1 h and then treated with 20 ng/mL PDGF-BB (for the control cells) or 80 ng/mL PDGF-BB (for the pEGFP-N2-PDGFR-β cells). Scale bar = 100 µm.

Over-Expression of PDGFR-? Promotes PDGF-Induced Proliferation, Migration, and Angiogenesis of EPCs through PI3K/Akt Signaling Pathway

PLoS One. 2012;7(2):e30503.

Figure 1

Spleen-derived MNCs differentiate into cells with characteristics of endothelial progenitor cells after cultured in vitro.

(A) After 4–7days in culture, spleen-derived MNCs exhibited a spindle-shaped, endothelial cell-like morphology. Scale bar = 100 µm. (B) After 10 days, cells formed cord-like structures. Scale bar = 100 µm. (C) After 20 days, cells became confluent, and looked like cobblestones. Scale bar = 200 µm. (D,E,F) Spleen-derived MNCs showed uptake of acetylated LDL (D) and UEA-1 binding (E) after 4 days in culture. Most adherent cells were LDL and UEA-1 double positive (91.2±1.8%; n = 5; three random fields per well). Scale bar = 75 µm. (G) FACS analysis of cultured spleen-derived MNCs for FITC-Sca-1 (a stem/progenitor cell marker), generating a positive rate of 71.7±0.93% (n = 3). (H) FACS analysis of cultured spleen-derived MNCs for PE-VEGFR-2 (an endothelial cell marker), generating a positive rate of 52.49±9.27% (n = 3). The left peak in each box denotes corresponding negative isotype control labeling, and the positive gate M1 is shown.

Over-Expression of PDGFR-? Promotes PDGF-Induced Proliferation, Migration, and Angiogenesis of EPCs through PI3K/Akt Signaling Pathway

PLoS One. 2012;7(2):e30503.

Figure 3

Overexpression of PDGFR-β in transfected EPCs.

(A,B,C) Transfected cells can be identified using an in-vector marker, EGFP 21 h after transfection, the expression of EGFP was detected by laser scanning confocal microscopy in the pEGFP-N2-PDGFR-β group (A), and nuclei were stained with DAPI (B). The transfection efficiency (number of green cells/total number of cells) was about 50-60%. Scale bar = 75 µm. (D,E) 72 h post-transfection, PDGFR-β mRNA (D) and protein levels (E) in the pEGFP-N2-PDGFR-β group were significantly higher than in the control group or pEGFP-N2 group.* P<0.01 vs. control or pEGFP-N2; NS, no significant vs. control (n = 3).

Over-Expression of PDGFR-? Promotes PDGF-Induced Proliferation, Migration, and Angiogenesis of EPCs through PI3K/Akt Signaling Pathway

PLoS One. 2012;7(2):e30503.

Figure 5

Effects of PDGFR-β overexpression on PDGF-BB-induced EPC proliferation.

EPCs (untransfected,or transfected with either pEGFP-N2 or pEGFP-N2-PDGFR-β) were incubated with PDGF-BB of different concentrations.EPC proliferation was examined by the MTS assay. * P<0.01 vs. pEGFP-N2-PDGFR-β cells under 0 ng/mL PDGF-BB stimulation.

Over-Expression of PDGFR-? Promotes PDGF-Induced Proliferation, Migration, and Angiogenesis of EPCs through PI3K/Akt Signaling Pathway

PLoS One. 2012;7(2):e30503.

Figure 7

Effects of PDGFR-β overexpression on PDGF-BB-induced EPC tube formation.

EPCs (untransfected,or transfected with either pEGFP-N2 or pEGFP-N2-PDGFR-β) were incubated with PDGF-BB of different concentrations. EPC tube formation was examined by the In Vitro Angiogenesis Assay Kit. * P<0.01 vs. pEGFP-N2-PDGFR-β cells under 0 ng/mL PDGF-BB stimulation.

Over-Expression of PDGFR-? Promotes PDGF-Induced Proliferation, Migration, and Angiogenesis of EPCs through PI3K/Akt Signaling Pathway

PLoS One. 2012;7(2):e30503.

Figure 9

Representative photos of PDGF-BB-induced EPC migration by PI3K/Akt pathway.

Over-Expression of PDGFR-? Promotes PDGF-Induced Proliferation, Migration, and Angiogenesis of EPCs through PI3K/Akt Signaling Pathway

PLoS One. 2012;7(2):e30503.

Figure 11

Pretreatment with various inhibitors attenuates PDGF-stimulated phosphorylation of PDGFR-β, PI3K, and Akt.

(A, B, C) Cells in the control group (bright bars) and the pEGFP-N2-PDGFR-β group (dark bars) were pretreated with AG1295, LY294002, or sc-221226 for 1 h and then treated with 20 ng/mL PDGF-BB (for the control cells) or 80 ng/mL PDGF-BB (for the pEGFP-N2-PDGFR-β cells). PDGF-BB-induced phospho-PDGFR-β (A), phospho-PI3K (B), and phospho-Akt (C) were normalized to total PDGFR-β, PI3K, or Akt, respectively. * P<0.05 and ** P<0.01 vs. control cells under 20 ng/mL PDGF-BB stimulation. # P<0.05 and ## P<0.01 vs. pEGFP-N2-PDGFR-β cells under 80 ng/mL PDGF-BB stimulation.

Over-Expression of PDGFR-? Promotes PDGF-Induced Proliferation, Migration, and Angiogenesis of EPCs through PI3K/Akt Signaling Pathway

PLoS One. 2012;7(2):e30503.

Publication Types, MeSH Terms, Substances

Publication Types

Research Support, Non-U.S. Gov't

MeSH Terms

1-Phosphatidylinositol 4-Kinase/genetics
1-Phosphatidylinositol 4-Kinase/metabolism*
Blotting, Western
Cell Movement/drug effects
Cell Movement/physiology*
Cell Proliferation*/drug effects
Cells, Cultured
Endothelium, Vascular/cytology
Endothelium, Vascular/metabolism*
Enzyme Inhibitors
Enzyme-Linked Immunosorbent Assay
Fluorescent Antibody Technique
Humans
Neovascularization, Physiologic*
Phosphorylation/drug effects
Proto-Oncogene Proteins c-akt/genetics
Proto-Oncogene Proteins c-akt/metabolism*
Proto-Oncogene Proteins c-sis/genetics
Proto-Oncogene Proteins c-sis/metabolism*
RNA, Messenger/genetics
Real-Time Polymerase Chain Reaction
Receptor, Platelet-Derived Growth Factor beta/genetics
Receptor, Platelet-Derived Growth Factor beta/metabolism*
Signal Transduction/drug effects
Stem Cells/cytology
Stem Cells/metabolism*

Substances

Enzyme Inhibitors
Proto-Oncogene Proteins c-sis
RNA, Messenger
platelet-derived growth factor BB
1-Phosphatidylinositol 4-Kinase
Receptor, Platelet-Derived Growth Factor beta
Proto-Oncogene Proteins c-akt

PubMed

Result Filters

Display Settings:

Send to:

Over-expression of PDGFR-β promotes PDGF-induced proliferation, migration, and angiogenesis of EPCs through PI3K/Akt signaling pathway.

Source

Abstract

Publication Types, MeSH Terms, Substances

Publication Types

MeSH Terms

Substances

LinkOut - more resources

Full Text Sources

Medical

Supplemental Content

Save items

Related citations in PubMed

Cited by 1 PubMed Central article

Related information

Recent activity

Simple NCBI Directory

Getting Started

Resources

Popular

Featured

NCBI Information