Adenovirus-based vascular endothelial growth factor gene delivery to human pancreatic islets.
Islet transplantation is limited by islet graft failure due to poor revascularization, host immune rejection and nonspecific inflammatory response. Delivery of human vascular endothelial growth factor (hVEGF) gene to the islets is likely to promote islet revascularization and survival. We used a bicistronic adenoviral vector encoding hVEGF and CpG-free allele of green fluorescent protein (Adv-GFP-hVEGF) and introduced into human pancreatic islets by transfection. We found that transfection efficiency and apoptosis were dependent on the multiplicity of infection (MOI). Compared to Adv-GFP transfected and nontransfected islets, the levels of hVEGF secreted from Adv-GFP-hVEGF transfected islets were higher and exhibit a linear relationship between hVEGF expression and MOI (10-5000). Persistent, but low level expression of hVEGF from nontransfected islets was also observed. This may be due to expression of the endogenous hVEGF gene under hypoxic conditions. The levels of DNA fragmentation determined by ELISA of islet lysates were dependent on the MOI of Adv-GFP-hVEGF. On glucose challenge, insulin release from transfected islets was comparable to nontransfected islets. Immunohistochemical staining for hVEGF was very high in Adv-GFP-hVEGF transfected islets. Weak staining was also observed for hCD31 in both transfected and nontransfected islets. These findings suggest that Adv-GFP-hVEGF is a potential candidate for promoting islet revascularization.
Cheng
K
K
Department of Pharmaceutical Sciences, University of Tennessee Health Science Center, Memphis, TN, USA.
Fraga
D
D
Zhang
C
C
Kotb
M
M
Gaber
A O
AO
Guntaka
R V
RV
Mahato
R I
RI
D000256:Adenoviridae / Q000235:genetics*; D017209:Apoptosis; D002478:Cells, Cultured; D003920:Diabetes Mellitus / Q000628:therapy; D015870:Gene Expression; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors / Q000008:administration & dosage*; D005947:Glucose; D049452:Green Fluorescent Proteins; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods; D007328:Insulin / Q000378:metabolism; D000078790:Insulin Secretion; D007515:Islets of Langerhans / Q000378:metabolism* / Q000473:pathology; D008164:Luminescent Proteins / Q000235:genetics; D009336:Necrosis; D019408:Platelet Endothelial Cell Adhesion Molecule-1 / Q000276:immunology; D011993:Recombinant Fusion Proteins / Q000008:administration & dosage*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics*; D019562:Viral Load
D007328:Insulin; D008164:Luminescent Proteins; D019408:Platelet Endothelial Cell Adhesion Molecule-1; D011993:Recombinant Fusion Proteins; D042461:Vascular Endothelial Growth Factor A; D049452:Green Fluorescent Proteins; D005947:Glucose