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Transplantation of VEGF165-gene-transfected endothelial progenitor cells in the treatment of chronic venous thrombosis in rats.
Chin Med J (Engl). 2010 Feb 20; 123(4):471-7.CM

Abstract

BACKGROUND

The organization and recanalization of thrombi is a dynamic and complex process. The aim of this research was to study the cotherapeutic effect of stem cell transplantation and gene transfection on chronic venous thrombosis.

METHODS

We constructed a recombinant adenoviral vector carrying the vascular endothelial growth factor 165 (VEGF165) gene by using the pAdEasy system, which was subsequently identified and amplified. Simultaneously, endothelial progenitor cells (EPCs) were isolated from rat bone marrow using Ficoll, cultured in EBM-2MV medium, and identified. Then, the cells were transfected with the recombinant Ad-VEGF165. The EPCs were labeled with 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine (Dil) before transplantation. A rat model of chronic vein thrombosis was developed by partial ligation of the inferior vena cava. The rats were randomly divided into 4 groups (n = 25, each): A, Ad-VEGF165/EPC-transplantation group received 1 ml (10(6)) of Ad-VEGF165/EPCs; B, EPC-transplantation group received 1 ml (10(6)) of EPCs; C, Ad/EPC-transplantation group received 1 ml (10(6)) of Ad/EPCs; D, control group received 1 ml of the transplantation medium. The thrombi and adjacent caval walls were harvested 28 days after transplantation; real-time quantitative polymerase chain reaction was used to detect the expression level of vascular endothelial growth factor (VEGF) mRNA; and western blotting was used to measure changes in VEGF protein expression. Hematoxylin-eosin staining and immunohistochemical staining were performed to detect recanalization. Neovascularization was detected by immunohistochemical staining using the antibody for von Willebrand factor (vWF), which is a component of endothelial cells. The capillary density was quantitatively determined by counting the capillaries under a high-power microscope.

RESULTS

The Ad-VEGF165 was constructed, and bone-marrow-derived EPCs were cultivated and successfully identified. We determined the optimum transfection ratio that promoted the growth of EPCs. After transfection, the EPCs secreted the VEGF protein. After transplantation, the in vivo survival of EPCs and their differentiation into endothelial cells were determined by detecting the fluorescence associated with the Dil stain. VEGF mRNA was expressed in groups A, B, C and D after transplantation, and the VEGF mRNA level in group A was significantly higher than those in groups B, C and D (P < 0.05); the VEGF mRNA levels in groups B and C were significantly higher than those in group D (P < 0.05), and there was no statistical significance between the VEGF mRNA levels in groups B and C. The recanalization capillary density in group A was significantly higher than those in groups B, C (P < 0.05) and D (P < 0.01); the recanalization capillary densities in groups B and C were significantly higher than that in group D (P < 0.05). Moreover, there was no statistical significant difference between the values for groups B and C.

CONCLUSIONS

The EPCs were successfully transfected by Ad-VEGF165. A suitable transfection ratio can improve the efficiency of EPCs and the possibility of promotion of angiogenesis after transplantation. Transfected EPCs caused accelerated organization and recanalization of vein thrombi.

Authors+Show Affiliations

Department of Vascular Surgery, Second Affiliated Hospital of Suzhou University, Suzhou, Jiangsu 215004, China.No affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

20193489

Citation

Meng, Qing-you, et al. "Transplantation of VEGF165-gene-transfected Endothelial Progenitor Cells in the Treatment of Chronic Venous Thrombosis in Rats." Chinese Medical Journal, vol. 123, no. 4, 2010, pp. 471-7.
Meng QY, Li XQ, Yu XB, et al. Transplantation of VEGF165-gene-transfected endothelial progenitor cells in the treatment of chronic venous thrombosis in rats. Chin Med J (Engl). 2010;123(4):471-7.
Meng, Q. Y., Li, X. Q., Yu, X. B., Lei, F. R., Jiang, K., & Li, C. Y. (2010). Transplantation of VEGF165-gene-transfected endothelial progenitor cells in the treatment of chronic venous thrombosis in rats. Chinese Medical Journal, 123(4), 471-7.
Meng QY, et al. Transplantation of VEGF165-gene-transfected Endothelial Progenitor Cells in the Treatment of Chronic Venous Thrombosis in Rats. Chin Med J (Engl). 2010 Feb 20;123(4):471-7. PubMed PMID: 20193489.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Transplantation of VEGF165-gene-transfected endothelial progenitor cells in the treatment of chronic venous thrombosis in rats. AU - Meng,Qing-you, AU - Li,Xiao-qiang, AU - Yu,Xiao-bin, AU - Lei,Feng-rui, AU - Jiang,Kun, AU - Li,Chuan-yong, PY - 2010/3/3/entrez PY - 2010/3/3/pubmed PY - 2010/5/28/medline SP - 471 EP - 7 JF - Chinese medical journal JO - Chin Med J (Engl) VL - 123 IS - 4 N2 - BACKGROUND: The organization and recanalization of thrombi is a dynamic and complex process. The aim of this research was to study the cotherapeutic effect of stem cell transplantation and gene transfection on chronic venous thrombosis. METHODS: We constructed a recombinant adenoviral vector carrying the vascular endothelial growth factor 165 (VEGF165) gene by using the pAdEasy system, which was subsequently identified and amplified. Simultaneously, endothelial progenitor cells (EPCs) were isolated from rat bone marrow using Ficoll, cultured in EBM-2MV medium, and identified. Then, the cells were transfected with the recombinant Ad-VEGF165. The EPCs were labeled with 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine (Dil) before transplantation. A rat model of chronic vein thrombosis was developed by partial ligation of the inferior vena cava. The rats were randomly divided into 4 groups (n = 25, each): A, Ad-VEGF165/EPC-transplantation group received 1 ml (10(6)) of Ad-VEGF165/EPCs; B, EPC-transplantation group received 1 ml (10(6)) of EPCs; C, Ad/EPC-transplantation group received 1 ml (10(6)) of Ad/EPCs; D, control group received 1 ml of the transplantation medium. The thrombi and adjacent caval walls were harvested 28 days after transplantation; real-time quantitative polymerase chain reaction was used to detect the expression level of vascular endothelial growth factor (VEGF) mRNA; and western blotting was used to measure changes in VEGF protein expression. Hematoxylin-eosin staining and immunohistochemical staining were performed to detect recanalization. Neovascularization was detected by immunohistochemical staining using the antibody for von Willebrand factor (vWF), which is a component of endothelial cells. The capillary density was quantitatively determined by counting the capillaries under a high-power microscope. RESULTS: The Ad-VEGF165 was constructed, and bone-marrow-derived EPCs were cultivated and successfully identified. We determined the optimum transfection ratio that promoted the growth of EPCs. After transfection, the EPCs secreted the VEGF protein. After transplantation, the in vivo survival of EPCs and their differentiation into endothelial cells were determined by detecting the fluorescence associated with the Dil stain. VEGF mRNA was expressed in groups A, B, C and D after transplantation, and the VEGF mRNA level in group A was significantly higher than those in groups B, C and D (P < 0.05); the VEGF mRNA levels in groups B and C were significantly higher than those in group D (P < 0.05), and there was no statistical significance between the VEGF mRNA levels in groups B and C. The recanalization capillary density in group A was significantly higher than those in groups B, C (P < 0.05) and D (P < 0.01); the recanalization capillary densities in groups B and C were significantly higher than that in group D (P < 0.05). Moreover, there was no statistical significant difference between the values for groups B and C. CONCLUSIONS: The EPCs were successfully transfected by Ad-VEGF165. A suitable transfection ratio can improve the efficiency of EPCs and the possibility of promotion of angiogenesis after transplantation. Transfected EPCs caused accelerated organization and recanalization of vein thrombi. SN - 2542-5641 UR - https://www.unboundmedicine.com/medline/citation/20193489/Transplantation_of_VEGF165_gene_transfected_endothelial_progenitor_cells_in_the_treatment_of_chronic_venous_thrombosis_in_rats_ L2 - https://Insights.ovid.com/pubmed?pmid=20193489 DB - PRIME DP - Unbound Medicine ER -