Transplantation of vascular endothelial growth factor 165‑transfected endothelial progenitor cells for the treatment of limb ischemia

Abstract

The present study aimed to investigate the effects of neovascularization in rabbits with limb ischemia transplanted with vascular endothelial growth factor (VEGF)165‑transfected endothelial progenitor cells (EPC). Bone marrow mononuclear cells were isolated by gradient centrifugation, cultured in M199 culture medium and induced into EPCs using VEGF, basic fibroblast growth factor, and insulin‑like growth factor‑1, and subsequently identified. The EPCs were transfected with Adv‑green fluorescent protein‑VEGF165 and the proliferation potential of the cells was determined using an MTT assay. The protein expression levels of VEGF were measured by detecting its concentration levels in the supernatant using an ABC‑ELISA assay. A rabbit hind limb ischemic model was established and randomly divided into three groups: (A) Control group, (B) EPC‑transplanted group, and (C) Ad‑VEGF165/EPCs‑transplanted group. The effects of transplantation and the levels of recanalization were detected. Incorporation of the transplanted cells into the ischemic region was confirmed by 5‑bromodeoxyuridine staining, and the levels of recanalization were measured by computer tomography ateriography and immunohistochemical staining. Bone marrow‑derived EPCs were induced, cultivated, and successfully identified. The results of the present study determined the optimum transfection ratio that promoted the growth of EPCs. The EPCs were successfully transfected with VEGF165, and EPC proliferation was not affected by the transfection. The supernatant protein concentration levels of VEGF were markedly higher in the VEGF165‑transfected group, as compared with those of the control group. Introduction of the transplanted cells into the ischemic region of group C occurred more efficiently, as compared with groups A and B. The recanalization capillary density in group C was significantly higher, as compared with groups A and B. VEGF gene transfection was able to improve the quality of EPCs, and the response of rabbits with limb ischemia to transplantation with VEGF‑transfected EPCs was significantly better, as compared with transplantation with EPCs alone.

Figure 1

Culture of endothelial progenitor cells (EPCs). (A and B) EPCs formed spindles. Magnification, ×20. (C) Numerous spindles were observed following 8 days of culture. (D) Following 14 days of culture, the EPCs fused together. Both magnification, ×10.

Figure 2

Identification of endothelial progenitor cells (EPCs). (A) A small number of pinocytotic vesicles could be observed in the EPCs. The cells stained positively for endothelial markers Magnification, ×8,000. (B) Willebrand-factor (vWF) and (C) CD133, as determined by immunohistochemistry; magnification, ×10.

Figure 3

The 5-bromodeoxyuridine-labeled endothelial progenitor cells were observed by fluorescence microscopy, and the labeled cells emitted green fluorescence. Magnification, ×40.

Figure 4

(A) Three different multiplicities of infection (MOI: 10, 50, and 100) were used. (B) The transfected endothelial progenitor cells (EPCs) emitted green fluorescence. Magnification, ×10. (C) The proliferative activity levels of the non-transfected cells (non/EPCs), Ad-transfected EPCs (Ad/EPCs), and Ad-VEGF165-transfected EPCs (Ad-VEGF/EPCs) were similar. Data are presented as the mean ± standard deviation.

Figure 5

The protein expression levels of vascular endothelial growth factor (VEGF) in Ad-VEGF165-transfected EPCs (Ad-VEGF/EPCs) were higher, as compared with those of non-transfected cells (non/EPCs), and Ad-transfected EPCs (Ad/EPCs). ^*P<0.01, vs. the non/EPCs group; and ^#P<0.01, vs. the Ad/EPCs group. Data are presented as the mean ± standard deviation.

Figure 6

(A) Distal artery occlusion was observed in the ischemic limbs following ischemic experimentation. (B) Two New Zealand rabbits from each group were transplanted with 5-bromodeoxyuridine (Brdu)-labeled endothelial progenitor cells (EPCs), and the Brdu positive cells (brown) were observed. Magnification, ×200.

Figure 7

(A) Hematoxylin and eosin staining and (B) immunohistochemical staining using the von Willebrand-factor antibody. (C) The blood vessel densities were measured. ^*P<0.01, vs. group A; and ^#P<0.01, vs. group B. EPCs, endothelial progenitor cells; Ad-VEGF/EPCs, Ad-VEGF165-transfected EPCs.

Figure 8

Skin temperature variation following transplantation. The skin temperatures of the rabbits of groups B and C were markedly higher, as compared with those of group A on the days 21 and 28. ^*P<0.01, group B, vs. group A; and ^#P<0.01 group C, vs. group A. EPCs, endothelial progenitor cells; Ad-VEGF/EPCs, Ad-VEGF165-transfected EPCs.

Figure 9

Analysis of recanalization by computerizezd tomography ateriography (CTA). (A) The control group was treated with culture medium. (B) Ad-transfected EPCs (Ad/EPCs) transplantation group; (C) Ad-VEGF165-transfected EPCs (Ad-VEGF/EPCs) transplantation group.

Figure 10

Analysis of the establishment of collateral circulation in group C was observed following soft tissue removal.