Overexpression or silencing of FOXO3a affects proliferation of endothelial progenitor cells and expression of cell cycle regulatory proteins

Abstract

Endothelial dysfunction is involved in the pathogenesis of many cardiovascular diseases such as atherosclerosis. Endothelial progenitor cells (EPCs) have been considered to be of great significance in therapeutic angiogenesis. Furthermore, the Forkhead box O (FOXO) transcription factors are known to be important regulators of cell cycle. Therefore, we investigated the effects of changes in FOXO3a activity on cell proliferation and cell cycle regulatory proteins in EPCs. The constructed recombinant adenovirus vectors Ad-TM (triple mutant)-FOXO3a, Ad-shRNA-FOXO3a and the control Ad-GFP were transfected into EPCs derived from human umbilical cord blood. Assessment of transfection efficiency using an inverted fluorescence microscope and flow cytometry indicated a successful transfection. Additionally, the expression of FOXO3a was markedly increased in the Ad-TM-FOXO3a group but was inhibited in the Ad-shRNA-FOXO3a group as seen by western blotting. Overexpression of FOXO3a suppressed EPC proliferation and modulated expression of the cell cycle regulatory proteins including upregulation of the cell cycle inhibitor p27(kip1) and downregulation of cyclin-dependent kinase 2 (CDK2), cyclin D1 and proliferating cell nuclear antigen (PCNA). In the Ad-shRNA-FOXO3a group, the results were counter-productive. Furthermore, flow cytometry for cell cycle analysis suggested that the active mutant of FOXO3a caused a noticeable increase in G1- and S-phase frequencies, while a decrease was observed after FOXO3a silencing. In conclusion, these data demonstrated that FOXO3a could possibly inhibit EPC proliferation via cell cycle arrest involving upregulation of p27(kip1) and downregulation of CDK2, cyclin D1 and PCNA.

Figure 1. EPCs were isolated and double-labeled for Dil-ac-LDL and UEA1.

(A) EPCs(CD133⁺CD34⁺CD31⁺ cells) were isolated using fluorescence-activated cell sorting. (B)Fluorescence microscopy illustrates that endothelial progenitor cells are positive for Dil-ac-LDL(Red) and UEA1(Green) by HMC confocal microscopy.

Figure 2. Protein expression of FOXO3a in EPCs transfected with Ad-GFP, Ad-TM-FOXO3a or Ad-shRNA-FOXO3a; meanwhile, untransfected EPCs were operated as a negative control.

Blots were scanned, and expression of FoxO3a was quantified by densitometric analysis. The ratios for FoxO3a/Tubulin are shown. Data are mean±SE, n = 3, ^*p<0.05 vs. Ad-GFP or untransfected group.

Figure 3. Representative microscopy of the morphology of EPCs at 24-GFP, Ad-TM-FOXO3a or Ad-shRNA-FOXO3a.

Figure 4. Effects of changes in FOXO3a activity on the proliferation of EPCs via CCK-8 assay.

EPCs were transfected with the indicated vectors and cultured in compete EGM-2. Cell viability was determined by CCK-8 assay at the three time points. Data shown are the mean±SE of the ratio for light absorbance at 450 nm. n = 3, *p<0.01 vs. Ad-GFP Group.

Figure 5. Growth curve of EPCs within 72 hours after transfection.

1.5x10⁵ EPCs were seeded in a 6-well plate and transfected with different recombinant adenovirus vectors. Cell number was counted by using a haemocytometer for a consecutive three days. Growth curve of EPCs showed cell growth pattern within 72 hours after transfection.

Figure 6. Cell cycle analysis using flow cytometry.

(A) The representative FACS plots displayed differences in cell cycle phases of EPCs transfected with Ad-GFP, Ad-TM-FOXO3a or Ad-shRNA-FOXO3a. (B) Cell cycle analysis demonstrated that the proliferation index (the percentage of cells in S- and G2- phases) was significantly elevated after FOXO3a silencing when compared to the proliferation index in Ad-GFP group. Data were presented as the mean±SD of three independent experiments. *p<0.05 vs. Ad-GFP.

Figure 7. FOXO3a modulated the expression of cell cycle regulatory proteins in EPCs.

(A) EPCs were transfected with the indicated vectors. Cells were lysed and protein expression was determined by Western blotting. (B)Blots were scanned, and expression of protein was quantified by densitometric analysis. The ratios of CDK2, cyclinD1, P27 or PCNA to Tubulin are shown. Band intensity analysis was performed with Quantity One Software. Knockdown FOXO3a led to dramatic protein expression increases in CDK2, cyclinD1 and PCNA. Data are mean±SE, n = 3, *p<0.05 vs. Ad-GFP.