Synergistic effect of VEGF and SDF-1α in endothelial progenitor cells and vascular smooth muscle cells

Abstract

Vascular endothelial growth factor (VEGF) is a potent agonist of angiogenesis that induces proliferation and differentiation of endothelial progenitor cells (EPCs) after vascular injury. Previous studies have suggested that stromal cell-derived factor 1-alpha (SDF-1α) and VEGF have a synergistic effect on vascular stenosis. The aim of the present study was to investigate whether VEGF and SDF-1α act synergistically in EPCs and vascular smooth muscle cells (VSMCs). In this study, EPCs were isolated from rat bone marrow and their morphology and function were studied. Subsequently, VEGF was delivered into EPCs using an adenoviral vector. Tube formation, migration, proliferation, and apoptosis of VEGF-overexpressing EPCs was analyzed. Then, EPCs were co-cultured with VSMCs in the presence or absence of SDF-1α, the migration, proliferation, apoptosis, and differentiation capacity of EPCs and VSMCs were analyzed respectively. The isolated EPCs showed typical morphological features, phagocytic capacity, and expressed surface proteins. While stable expression of VEGF remarkably enhanced tube formation, migration, and proliferation capacity of EPCs, apoptosis was decreased. Moreover, the proliferation, migration, and differentiation capacity of EPCs in the co-cultured model was enhanced in the presence of SDF-1α, and apoptosis was decreased. However, these effects were reversed in VSMCs. Therefore, our results showed that VEGF and SDF-1α synergistically increased the migration, differentiation, and proliferation capabilities of EPCs, but not VSMCs. This study suggests a promising strategy to prevent vascular stenosis.

Keywords: endothelial progenitor cell; stromal cell-derived factor-1α; synergistic effect; vascular endothelial growth factor; vascular smooth muscle cells.

FIGURE 1

Characterization of cultured EPCs. (A) Typical morphological appearance (cluster-like morphology) of EPCs after 7 days of culture. (B) Tube formation in matrigel after 14 days culture. The cultured cells were stained with UEA-1-FITC (C, green) and DiI-acLDL (D, red); merged (E, two-color overlay). (F) Representative immunofluorescence staining of endothelial cell markers CD31, CD34, VEGF-R2, and CD133 in red imaged using a laser confocal microscope (cell nuclei were counterstained with DAPI, shown in blue. “Merge” represents two-color overlay).

FIGURE 2

Ad5/VEGF transduction upregulates VEGF expression and improves tube-formation capacity of EPCs. (A) Identification of EPCs transducted by Non-Adv, Ad5/EGFP, and Ad5/VEGF by enumerating GFP-positive cells; the graph shows the transduction rate of GFP. (B) VEGF mRNA expression was determined using qPCR; actin was used as control. (C) VEGF protein expression was assessed by western blotting; GAPDH was used as control. The graph shows a remarkable increase in VEGF expression. (D) Tube formation in matrigel on day 14; the number of tubes formed are indicated in the graph.

FIGURE 3

VEGF expression increases proliferation and migration, and decreases apoptosis of EPCs. (A) Representative images showing cell migration (a); Bar graph indicates high migration rate of Ad5/VEGF-EPCs (b). (B) Proliferation capacity of Ad5/VEGF-EPCs was higher than that of non-Adv-EPCs or Ad5/EGFP-EPCs on days 1, 3, 5, and 7 after transduction. (C) Apoptotic rate of EPCs was analyzed by FCM using Annexin V/propidium iodide (PI) staining. Apoptotic cells are defined as Annexin V+/PI- (Quadrant three and 4) (a); (b) Apoptosis rate of Ad5/VEGF-EPCs was significantly lower than that of non-Adv-EPCs or Ad5/EGFP-EPCs.

FIGURE 4

SDF-1α improves EPC function in the co-culture model. (A) Proliferation capacity of EPCs in different groups. (B) Migration ability of EPCs. (C) Apoptosis rate of EPCs (Numbers 1–7 represent VSMCs and Non-Adv-EPCs; VSMCs and Ad5/EGFP-EPCs; VSMCs and Ad5/VEGF-EPCs; SDF-1α+ VSMCs and Non-Adv-EPCs; SDF-1α+ VSMCs and Ad5/EGFP-EPCs; SDF-1α+ VSMCs and Ad5/VEGF-EPCs; SDF-1α+ VSMCs and Ad5/VEGF-EPCs + AMD3100 respectively. *p < 0.05, **p < 0.01, n = 3 per group.).

FIGURE 5

SDF-1α decrease VSMC function in the co-culture model. (A) Proliferation capacity of VSMCs in different groups. (B) Migration ability of VSMCs. (C) Apoptosis rate of VSMCs (Numbers 1–7 represent VSMCs and Non-Adv-EPCs; VSMCs and Ad5/EGFP-EPCs; VSMCs and Ad5/VEGF-EPCs; SDF-1α+VSMCs and Non-Adv-EPCs; SDF-1α+ VSMCs and Ad5/EGFP-EPCs; SDF-1α+VSMCs and Ad5/VEGF-EPCs; SDF-1α+VSMCs and Ad5/VEGF-EPCs + AMD3100 respectively. *p < 0.05, **p < 0.01, n = 3 per group.).