Molecular Biology and Genetics
Electronic Journal of Biotechnology ISSN: 0717-3458 Vol. 6 No. 3, Issue of December 15, 2003
© 2003 by Pontificia Universidad Católica de Valparaíso -- Chile Received January 31, 2003 / Accepted July 18, 2003
RESEARCH ARTICLE

Therapeutic angiogenesis following intramuscular gene transfer of vascular endothelialgrowth factor 121 in a dog model of hindlimb ischemia 

Ariana G. Ojalvo*
Centro de Ingeniería Genética y Biotecnología
Ave. 31 e/ 158 y 190, Cubanacán
P.O. Box 6162, La Habana 10600, Cuba
Tel: 53 7 271 6022
Fax: 53 7 2714764
E-mail: ariana.garcia@cigb.edu.cu

Alina Seralena
 Centro de Ingeniería Genética y Biotecnología
Ave. 31 e/ 158 y 190, Cubanacán
P.O. Box 6162, La Habana 10600, Cuba
Tel: 53 7 271 6022
Fax: 53 7 2714764
E-mail: alina.seralena@cigb.edu.cu

Raysa Vázquez
 Centro de Ingeniería Genética y Biotecnología
Ave. 31 e/ 158 y 190, Cubanacán
P.O. Box 6162, La Habana 10600, Cuba
Tel: 53 7 271 6022
Fax: 53-7-271 4764
E-mail: raysa.vazquez@cigb.edu.cu

José F. Montequín
 Instituto de Angiología y Cirugía Vascular
Hospital Salvador Allende
Calzada del Cerro 1551, Cerro, La Habana, Cuba
Tel: 53 7 877 6493
E-mail: montequi@infomed.sld.cu

Nelson S. Vispo
 Centro de Ingeniería Genética y Biotecnología
Ave. 31 e/ 158 y 190, CubanacánP.O. Box 6162, La Habana 10600, Cuba
Tel: 53 7 271 6022
Fax: 53 7 271 4764
E-mail: nelson.santiago@cigb.edu.cu

Ricardo Silva
 Centro de Ingeniería Genética y Biotecnología
Ave. 31 e/ 158 y 190, Cubanacán
P.O. Box 6162, La Habana 10600, Cuba
Tel: 53 7 271 6022
Fax: 53 7 271 4764
E-mail: ricardo.silva@cigb.edu.cu 

Alfredo Aldama
 Instituto de Angiología y Cirugía Vascular
Hospital Salvador Allende
Calzada del Cerro 1551, Cerro, La Habana, Cuba
Tel: 53 7 877 6493

Yaquelin Puchades
 Centro de Ingeniería Genética y Biotecnología
Ave. 31 e/ 158 y 190, Cubanacán
P.O. Box 6162, La Habana 10600, Cuba
Tel: 53 7 271 6022
Fax: 53 7 271 4764
E-mail: yaquelin.puchades@cigb.edu.cu

Luis T. Sorell
 Instituto de Angiología y Cirugía Vascular
Hospital Salvador Allende
Calzada del Cerro 1551, Cerro, La Habana, Cuba
Tel: 53 7 877 6493 

Pedro Lopez-Saura
 Centro de Ingeniería Genética y Biotecnología
Ave. 31 e/ 158 y 190, Cubanacán
P.O. Box 6162, La Habana 10600, Cuba
Tel: 53 7 271 6022
Fax: 53 7 271 8070 

María A. Alfonso
 Instituto de Angiología y Cirugía Vascular
Hospital Salvador Allende
Calzada del Cerro 1551, Cerro, La Habana, Cuba
Tel: 53 7 877 6493

Rafael Simón (†)
 Instituto de Angiología y Cirugía Vascular
Hospital Salvador Allende
Calzada del Cerro 1551, Cerro, La Habana, Cuba

Alfonso Alí
 Centro de Ingeniería Genética y Biotecnología
Ave. 31 e/ 158 y 190, Cubanacán
P.O. Box 6162, La Habana 10600, Cuba
Tel: 53 7 271 6022
Fax: 53 7 271 4764 

Armando Seuc
 Instituto de Angiología y Cirugía Vascular
Hospital Salvador Allende
Calzada del Cerro 1551, Cerro, La Habana, Cuba
Tel: 53 7 877 6493

Luis Herrera
 Centro de Ingeniería Genética y Biotecnología
Ave. 31 e/ 158 y 190, Cubanacán
P.O. Box 6162, La Habana 10600, Cuba
Tel: 53 7 271 6022
Fax: 53 7 271 8070
E-mail: luis.herrera@cigb.edu.cu

*Corresponding author


Financial support: This work was supported by the Center for Genetic Engineering and Biotechnology, Havana, Cuba.

Keywords: collateral development, gene therapy, naked plasmid DNA, peripheral vascular disease, revascularization.

Abbreviations:

AP: alkaline phosphatase
ATCC: American tissue culture collection
CMV: cytomegalovirus
D-MEM: Dulbecco’s modified eagle medium
ELISA: enzyme-linked immunosorbent assay
FCS: fetal calf serum
GFP: green fluorescent protein
GOT: glutamic-oxaloacetic transaminase

GPT: glutamic-pyruvic transaminase
HMEC: human microvascular endothelial cells
PCR: polymerase chain reaction
PEI: polyethylenimine
SD: standard deviation
SV40t SS/pA: SV40t splicing/polyadenylation signals
TFF: tangential flow filtration
VEGF: vascular endothelial growth factor

Abstract
Full Text

Vascular endothelial growth factor (VEGF), an endothelial cell-specific mitogen, has been shown to promote therapeutic angiogenesis in animal models of critical limb ischemia. Ischemic skeletal muscle is advantageous for taking up and expressing foreign genes transferred as naked plasmid DNA. Accordingly, we investigated the hypothesis that intramuscular administration of naked plasmid DNA encoding the 121-amino acid isoform of VEGF could augment collateral development and tissue perfusion in a dog hindlimb ischemia model. Unilateral hindlimb ischemia was surgically induced in Beagle dogs. Ten days later, animals received intramuscular injections of pVEGF121 plasmid directly in the ischemic muscles. Angiogenic effects were evaluated by angiography, calf blood pressure ratio and vasomotor reserve analyses. Thirty days after gene transfer, angiographically recognizable collateral vessels were increased in pVEGF121-treated animals compared with controls. Improvement in perfusion to the ischemic limb was documented by a significantly higher calf blood pressure ratio for pVEGF121 (0.79 ± 0.05) versus controls (0.56 ± 0.14, P<0.01). Vasomotor reserve assay suggested amelioration in blood availability at the microcirculation level in pVEGF121-treated animals. Hematological variables showed no significant modification due to the treatment. Our results suggest that intramuscular gene transfer of VEGF121 may promote therapeutic angiogenesis in critical limb vascular insufficiency.
 
Supported by UNESCO / MIRCEN network 
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