Construction of recombinant adenovirus co-expression vector carrying the human transforming growth factor-beta1 and vascular endothelial growth factor genes and its effect on anterior cruciate ligament fibroblasts.
BACKGROUND
Remodeling of the anterior cruciate ligament (ACL) graft usually takes longer than expected. Gene therapy offers a radical different approach to remodeling of the graft. In this study, the internal ribosome entry site (IRES) sequence was used to construct a new recombinant adenovirus which permits co-expression of transforming growth factor-beta1 (TGFbeta1) and vascular endothelial growth factor 165 (VEGF165) genes (named Ad-VEGF165-IRES-TGFbeta1). We investigated the effects of the new adenovirus on the migration of and matrix synthesis by ACL fibroblasts.


METHODS
Adenoviral vector containing TGFbeta1 and VEGF165 genes was constructed. ACL fibroblasts were obtained from New Zealand white rabbits. After ACL fibroblasts were exposed to Ad-VEGF165-IRES-TGFbeta1, the expression of VEGF165 and TGFbeta1 proteins were assessed by enzyme-linked immunosorbent assay (ELISA) and Western blotting analysis. Bioassay of VEGF165 and TGFbeta1 proteins were assessed by Western blotting analysis. Proliferation and migration of ACL fibroblasts were assessed by in vitro wound closure assay. Gene expression of collagen type I, collagen type III, and fibronectin mRNA among matrix markers were assessed by real-time PCR.


RESULTS
The results showed the successful construction of a recombinant co-expression adenovirus vector containing TGFbeta1 and VEGF165 genes. Co-expression of TGFbeta1 and VEGF165 can induce relatively rapid and continuous proliferation of ACL fibroblasts and high gene expression of collagen type I, collagen type III, and fibronectin mRNA among matrix markers.


CONCLUSIONS
Co-expression of TGFbeta1 and VEGF165 genes has more powerful and efficient effects on the migration of and matrix synthesis by ACL fibroblasts.
Wei
Xue-Lei
XL
Institute of Sports Medicine, Peking University Third Hospital, Beijing, China.
Lin
Lin
L
Hou
Yu
Y
Fu
Xin
X
Zhang
Ji-Ying
JY
Mao
Ze-Bin
ZB
Yu
Chang-Long
CL
D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D016118:Anterior Cruciate Ligament / Q000166:cytology / Q000378:metabolism*; D002465:Cell Movement; D002478:Cells, Cultured; D003094:Collagen / Q000235:genetics; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D005347:Fibroblasts / Q000502:physiology; D005353:Fibronectins / Q000235:genetics; D015316:Genetic Therapy; D005822:Genetic Vectors; D006801:Humans; D011817:Rabbits; D053773:Transforming Growth Factor beta1 / Q000235:genetics*; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics*; D014945:Wound Healing
D005353:Fibronectins; D053773:Transforming Growth Factor beta1; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; D003094:Collagen; D048049:Extracellular Signal-Regulated MAP Kinases