AMD3100 and SDF‑1 regulate cellular functions of endothelial progenitor cells and accelerate endothelial regeneration in a rat carotid artery injury model

Abstract

The present study was conducted to assess the effects of AMD3100 and stromal cell-derived factor 1 (SDF-1) on cellular functions and endothelial regeneration of endothelial progenitor cells (EPCs). The cell proliferation and adhesion capacity of EPCs were evaluated in vitro following treatment with AMD3100 and SDF‑1 using a Cell Counting Kit‑8 assay. Furthermore, the expression levels of C‑X‑C motif chemokine receptor 4 (CXCR4) and C‑X‑C motif chemokine receptor 7 (CXCR7) were detected before and after treatment with AMD3100 and SDF‑1 to elucidate their possible role in regulating the cellular function of EPCs. A rat carotid artery injury model was established to assess the influences of AMD3100 and SDF‑1 on endothelial regeneration. AMD3100 reduced the proliferation and adhesion capacity of EPCs to fibronectin (FN), whereas it increased the adhesion capacity of EPCs to human umbilical vein endothelial cells (HUVECs). However, SDF‑1 stimulated the proliferation and cell adhesion capacity of EPCs to HUVECs and FN. Additionally, the expression levels of CXCR7 but not CXCR4 were upregulated following AMD3100 treatment, whereas the expression levels of both CXCR4 and CXCR7 were upregulated after SDF‑1 treatment. In vivo results demonstrated that AMD3100 increased the number of EPCs in the peripheral blood and facilitated endothelial repair at 7 days after treatment. However, local administration of SDF‑1 alone did not enhance reendothelialization 7 and 14 days after treatment. Importantly, the combination of AMD3100 with SDF‑1 exhibited superior therapeutic effects compared with AMD3100 treatment alone, accelerated reendothelialization 7 days after treatment, and attenuated neointimal hyperplasia at day 7 and 14 by recruiting more EPCs to the injury site. In conclusion, AMD3100 could positively regulate the adhesion capacity of EPCs to HUVECs via elevation of the expression levels of CXCR7 but not CXCR4, whereas SDF‑1 could stimulate the proliferation and adhesion capacity of EPCs to FN and HUVECs by elevating the expression levels of CXCR4 and CXCR7. AMD3100 combined with SDF‑1 outperformed AMD3100 alone, promoted early reendothelialization and inhibited neointimal hyperplasia, indicating that early reendothelialization attenuated neointimal hypoplasia following endothelial injury.

Figure 1.

Characteristics of EPCs. (A) Fluorescence-activated cell sorting revealed that EPCs used in the present study were positive for CD34 and KDR, whereas they were negative for CD45. (B) Typical features of EPCs at day 1, 4, 7 and 14. (C) EPCs could take up ac-LDL and bind to UEA-1. Scale bar, 150 µm. ac-LDL, acetylated low-density lipoprotein; EPCs, endothelial progenitor cells; KDR, kinase insert domain receptor; UEA-1, Ulex europaeus agglutinin I.

Figure 2.

Proliferation curve and EC₅₀ values are influenced by AMD3100 and SDF-1. (A) Proliferation curve of EPCs following treatment with AMD3100 at various concentrations. (B) EC₅₀ of AMD3100 was calculated based on the proliferation curve. (C) Proliferation curve of EPCs after treatment with SDF-1 at various concentrations. (D) EC₅₀ of SDF-1 was calculated based on the proliferation curve. EC₅₀, 50% effective concentration; EPCs, endothelial progenitor cells; OD, optical density; SDF-1, stromal cell-derived factor 1.

Figure 3.

Adhesion capacity, and the expression levels of CXCR4 and CXCR7 are affected by AMD3100 and SDF-1 treatment. (A) Adhesion capacity to FN was impaired by AMD3100 treatment, whereas AMD3100 treatment stimulated the adhesion capacity to HUVECs. A similar tendency was observed after treatment with AMD3100 combined with SDF-1. Scale bar, 25 µm. (B) Confocal immunofluorescence microscopy confirmed that both CXCR4 and CXCR7 were expressed in EPCs. Scale bar, 100 µm. (C) Western blotting revealed that treatment with AMD3100 upregulated the expression levels of CXCR7 but not CXCR4. However, SDF-1 or AMD3100 combined with SDF-1 upregulated the expression levels of CXCR4 and CXCR7. Furthermore, the effects of AMD3100 combined with SDF-1 on the expression levels of CXCR7 were the greatest among the four groups. n=5. *P<0.05; **P<0.001. C, control; A, AMD3100 alone; S, SDF-1 alone; AS, AMD3100 combined with SDF-1; CXCR, C-X-C motif chemokine receptor; EPCs, endothelial progenitor cells; FN, fibronectin; HUVECs, human umbilical vein endothelial cells; SDF-1, stromal cell-derived factor 1.

Figure 4.

Number of EPCs in circulation and at the injury site. (A) AMD3100 treatment could effectively mobilize circulating EPCs in a time-dependent manner. However, SDF-1 treatment did not increase the number of circulating EPCs at any time point. Additionally, AMD3100 combined with SDF-1 (group AS) increased the number of circulating EPCs. However, there was no significant difference in circulating EPCs observed between group A and group AS. (B) AMD3100 treatment recruited more EPCs to the injury site. SDF-1 treatment did not increase the number of EPCs at the injury site. However, pretreatment with AMD3100, followed by local administration of SDF-1 recruited more EPCs to the injury site. The effects of AMD3100 combined with SDF-1 (group AS) on recruitment of EPCs were stronger than those of AMD3100 alone. Scale bar, 50 µm. n=5. *P<0.05; **P<0.001. C, control; A, AMD3100 alone; S, SDF-1 alone; AS, AMD3100 combined with SDF-1; EPCs, endothelial progenitor cells; SDF-1, stromal cell-derived factor 1.

Figure 5.

Effects of AMD3100 and SDF-1 treatment on intimal repair. (A) AMD3100 and AMD3100 combined with SDF-1 promoted reendothelialization compared with the control treatment 7 days after treatment. Furthermore, the treatment effects of the combination of AMD3100 and SDF-1 on reendothelialization were greater than those of AMD3100 treatment alone. Scale bar, 500 µm. (B) Early reendothelialization reduced neointimal hyperplasia in groups A and AS. The treatment effects of the combination of AMD3100 and SDF-1 were the most prominent among the four groups. Scale bar, 100 µm. n=5. *P<0.05; **P<0.001. C, control; A, AMD3100 alone; S, SDF-1 alone; AS, AMD3100 combined with SDF-1; I/M ratio, intima/media ratio; SDF-1, stromal cell-derived factor 1.