Androgen Modulates Functions of Endothelial Progenitor Cells through Activated Egr1 Signaling

Abstract

Researches show that androgens have important effects on migration of endothelial cells and endothelial protection in coronary heart disease. Endothelial progenitor cells (EPCs) as a progenitor cell type that can differentiate into endothelial cells, have a critical role in angiogenesis and endothelial protection. The relationship between androgen and the functions of EPCs has animated much interest and controversy. In this study, we investigated the angiogenic and migratory functions of EPCs after treatment by dihydrotestosterone (DHT) and the molecular mechanisms as well. We found that DHT treatment enhanced the incorporation of EPCs into tubular structures formed by HUVECs and the migratory activity of EPCs in the transwell assay dose dependently. Moreover, microarray analysis was performed to explore how DHT changes the gene expression profiles of EPCs. We found 346 differentially expressed genes in androgen-treated EPCs. Angiogenesis-related genes like Egr-1, Vcan, Efnb2, and Cdk2ap1 were identified to be regulated upon DHT treatment. Furthermore, the enhanced angiogenic and migratory abilities of EPCs after DHT treatment were inhibited by Egr1-siRNA transfection. In conclusion, our findings suggest that DHT markedly enhances the vessel forming ability and migration capacity of EPCs. Egr1 signaling may be a possible pathway in this process.

Figure 1

Characterization of EPCs. (a) Mononuclear cells were adhered and showed a spindle morphology in 72 hours. After 7 days, cells with oval and spindle shape formed cell colonies. (b) The spindle-shaped adherent cells that were positive for Dil-Ac-LDL uptake were identified as EPCs.

Figure 2

The expression of cell surface markers in EPCs. Adherent cells were positive for CD133 (93.3 ± 1.4%), CD105 (91.1 ± 1.3), CD31 (14.5 ± 1.4), CD34 (5.6 ± 1.2), CD45 (98.1 ± 0.2%), and CD11b (62.1 ± 6.5). All assays were triplicated and demonstrated similar results.

Figure 3

In vitro incorporation assay by HUVECs and incorporated EPCs. DHT-treated or nontreated EPCs were tracked with DiI. DiI-labeled cells and HUVECs were seeded onto Matrigel-coated 96-well plates in 10% FBS/EBM2-MV. After 24 hours in culture, incorporation of each cell population into tube-like structures formed with HUVECs was evaluated under fluorescence microscopy. (a) Incorporated DiI positive cells were indicated by arrows. (b) Number of incorporated cells into tube-like structures was counted and averaged. All assays were triplicated and demonstrated similar results. Data are presented in mean ± SD format. ^∗ P < 0.05 versus control group, ^∗∗ P < 0.01 versus control group, and ^∗∗∗ P < 0.001 versus control group.

Figure 4

Transwell assay of EPCs. DHT-treated or nontreated EPCs were incubated in the upper chamber with the serum-free medium. And the lower chamber was filled with 15% FBS/DMEM. Six hours later, the number of cells that migrated to the bottom of the membrane was quantified after staining with DAPI. (a) Migrating EPCs from serum-free upper chambers to the lower chambers filled with 15% FBS/DMEM. (b) The migrating EPCs were counted and averaged at high power field. All assays were triplicated and demonstrated similar results. Data are presented in mean ± SD format. ^∗∗∗ P < 0.001 versus control group.

Figure 5

Microarray data analysis workflow and cluster analysis of gene expression profiles in DHT-treated and nontreated EPCs. (a) EPCs were isolated from mouse bone marrow. RNA samples were extracted from DHT-treated and nontreated EPCs and hybridized to Affymetrix GeneChip microarrays. (b) The cluster analysis of gene expression profiles. High expression is indicated in red, whereas low expression is coded in green. Each row corresponds to the expression profile of a mouse sample, and each column corresponds to a gene.

Figure 6

The significant enriched GO terms and pathways.

Figure 7

Validation of gene expressions by qPCR. Genes were selected based on the fold change of expression differences or the association with the biological functions of EPCs and angiogenesis. Sixteen genes from the microarray result were validated by qPCR, and 11 of them were consistent with the results of microarray data. All assays were triplicated and demonstrated similar results. Data are presented in mean ± SD format. ^∗ P < 0.05 versus control group, ^∗∗ P < 0.01 versus control group, and ^∗∗∗ P < 0.001 versus control group.

Figure 8

The transfection of EPCs by Egr1-siRNA. (a) The sequences for mouse Egr1-siRNAs. (b) After 6 h of incubation at 37°C with siRNA, transfection efficiencies can be achieved up to 90%. (c) After more 48 h of incubation at 37°C, EPCs transfected with Egr1-siRNA showed a decreased expression level of Egr-1, and the inhibition efficiencies can be up to 60%. All assays were triplicated and demonstrated similar results. Data are presented in mean ± SD format. ^∗∗∗ P < 0.001 versus control group.

Figure 9

The in vitro incorporation assay of Egr1-silenced EPCs. Our data showed that Egr1-siRNA attenuated the boosting effects of DHT on the incorporation function of EPCs. (a) Incorporated DiI positive cells were indicated by arrows. (b) Number of incorporated cells into tube-like structures was counted and averaged. All assays were triplicated and demonstrated similar results. Data are presented in mean ± SD format. ^∗∗ P < 0.01 versus control group, ^∗∗∗ P < 0.001 versus control group.

Figure 10

The transwell assay of Egr1-silenced EPCs. The increased migratory capacity of EPCs by DHT treatment was also blocked by Egr1-siRNA transfection. (a) Transwell migration assay was employed to examine the migration ability of DHT-treated EPCs after transfection of Egr1-siRNA or control. (b) The migrating EPCs were counted and averaged at high power field. All assays were triplicated and demonstrated similar results. Data are presented in mean ± SD format. ^∗∗ P < 0.01 versus control group, ^∗∗∗ P < 0.001 versus control group.