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

Wang S, Chen Z, Tang X, Liu H, Yang L, Wang Y - Mol Med Rep (2015)

Bottom Line: Bone marrow‑derived EPCs were induced, cultivated, and successfully identified.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Vascular Surgery, Beijing Anzhen Hospital, Capital Medical University, Beijing 100029, P.R. China.

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.

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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.
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f2-mmr-12-04-4967: 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.

Mentions: Nuclei, mitochondria, and vacuoles were observed in the cells using TEM, along with a few pinocytotic vesicles, which were characteristic of endothelial cell structure. Microvilli were visible close to the cellular membrane (Fig. 2A). Using anti-vWF polyclonal antibody and FITC-labeled antibody as primary and secondary antibodies, respectively, the spindle cells emitted green fluorescence under the fluorescence microscope (Fig. 2B). Using anti-CD133 monoclonal antibody and TRITC-labeled antibody as primary and secondary antibodies, respectively, the spindle cells emitted red fluorescence under the fluorescence microscope (Fig. 2C). Since vWF and CD133 are components of endothelial cells, these results indicate that the BM-MNCs had successfully differentiated into EPCs.


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

Wang S, Chen Z, Tang X, Liu H, Yang L, Wang Y - Mol Med Rep (2015)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4581802&req=5

f2-mmr-12-04-4967: 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.
Mentions: Nuclei, mitochondria, and vacuoles were observed in the cells using TEM, along with a few pinocytotic vesicles, which were characteristic of endothelial cell structure. Microvilli were visible close to the cellular membrane (Fig. 2A). Using anti-vWF polyclonal antibody and FITC-labeled antibody as primary and secondary antibodies, respectively, the spindle cells emitted green fluorescence under the fluorescence microscope (Fig. 2B). Using anti-CD133 monoclonal antibody and TRITC-labeled antibody as primary and secondary antibodies, respectively, the spindle cells emitted red fluorescence under the fluorescence microscope (Fig. 2C). Since vWF and CD133 are components of endothelial cells, these results indicate that the BM-MNCs had successfully differentiated into EPCs.

Bottom Line: Bone marrow‑derived EPCs were induced, cultivated, and successfully identified.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Vascular Surgery, Beijing Anzhen Hospital, Capital Medical University, Beijing 100029, P.R. China.

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.

Show MeSH
Related in: MedlinePlus