Platelet‑derived growth factor D promotes the angiogenic capacity of endothelial progenitor cells

Abstract

Neovascularization and re-endothelialization rely on endothelial progenitor cells (EPCs). However, the recruitment and angiogenic roles of EPCs are subject to regulation through the vascular microenvironment, which remains largely unknown. Platelet‑derived growth factor D (PDGF‑D) is a new member of the PDGF family that binds the PDGFR‑β homodimer. However, it remains unknown whether and how it affects the angiogenic capacity of EPCs and participates in tube‑like formation. EPCs were derived from rat bone marrow cells, and the gain‑of‑function approach was used to study the effects of PDGF‑D on the biological activities of EPCs. EPCs that stably express PDGF‑D were generated by lentiviral‑mediated transduction, and the expression levels were evaluated by western blotting and reverse transcription, followed by real‑time quantitative polymerase chain reaction (RT‑qPCR). The biological activities of EPCs evaluated in the present study included proliferation, adhesion, migration, tube formation and senescence. Furthermore, the downstream signaling of PDGF‑D was explored by western blot analysis. The results revealed that the lentiviral‑mediated expression of PDGF‑D in the microenvironment promoted the migration, proliferation, adhesion and tube formation of EPCs. In addition, PDGF‑D suppressed the senescence of EPCs. Mechanistically, PDGF‑D induced the phosphorylation of several signaling molecules, including STAT3, AKT, ERK1/2, mTOR and GSK‑3β, suggesting that PDGF‑D enhanced the angiogenic function of EPCs through PDGF receptor‑dependent and ‑independent signaling pathways. In conclusion, PDGF‑D promotes the angiogenic capacity of EPCs, including proliferation, migration, adhesion and tube formation, and thereby contributes to angiogenesis.

Keywords: therapeutic angiogenesis; platelet-derived growth factor D; endothelial progenitor cells.

Figure 1.

Characterization of EPCs. (A) Adherent cells with a spindle-shaped endothelial cell-like morphology incorporated Dil-AcLDL and FITC-UEA-lectin at day 7. (B) The representative images of EPCs for the positive immunofluorescence staining of CD34, VEGFR2 and CD133.

Figure 2.

Verification of EPCs stably expressing PDGF-D. (A) Determination of the infection efficiency of EPCs by lentiviruses using a fluorescence microscopy. GFP-positive cells indicated the infection and quantification of the GFP-positive cell number in three repeated experiments. (B) The expression of PDGF-D in PDGF-D-EPCs was measured by RT-qPCR. (C) The expression of PDGF-D in PDGF-D-EPCs was measured by western blot analysis. (D) The levels of PDGF-D in the culture medium of PDGF-D-EPCs at different time points were determined by ELISA. The expression levels were normalized to control expression. *P<0.05 vs. the respective control group.

Figure 3.

PDGF-D in the microenvironment promotes EPCs proliferation, migration, adhesion and tube formation, and inhibits senescence. (A) MTT assays were performed to evaluate the viability of EPCs co-cultured with PDGF-D. Cell growth was determined at the indicated time points. **P<0.01 vs. the control group. (B) EPCs co-cultured with PDGF-D were seeded in the top chamber for the migration assay. *P<0.05 and **P<0.01 vs. the control group. Scale bars, 100 µm. (C) EPCs co-cultured with PDGF-D were seeded on collagen type I and fibronectin-coated 96-well plates for the adhesion assays. **P<0.01 vs. the control group. Scale bars, 100 µm. (D) EPCs co-cultured with PDGF-D were seeded on a Matrigel layer. *P<0.05 and **P<0.01 vs. the control group. Scale bars, 100 µm. (E) B-gal staining was performed for the senescence analysis of EPCs co-cultured with PDGF-D. **P<0.01 vs. the control group. Scale bars, 100 µm.

Figure 4.

PDGF-D functions via PDGFR-dependent and -independent pathways. (A-E) Western blot analysis was performed on the total protein extracted from confluent PDGF-D-EPCs and GFP-EPCs. (1–1, 1–2, 1–3 stand for GFP-EPCs and 2–1, 2–2, 3–3 represent PDGF-D-EPCs). The expression and phosphorylation of STAT3, AKT, ERK1/2, mTOR and GSK-3β were measured by immunoblotting, and normalized to the levels of GAPDH. **P<0.01 vs. the control group. (F) The expression of VEGF, HGF and PDGF-B was examined by RT-qPCR. The expression levels were normalized to GAPDH; **P<0.01 vs. the control group.