Inhibition of ocular angiogenesis by an adenovirus carrying the human von Hippel-Lindau tumor-suppressor gene in vivo.
OBJECTIVE
The purpose of this study was to investigate the effect of the von Hippel-Lindau (VHL) protein on VEGF gene expression in vitro and to determine whether adenovirus-mediated VHL intraocular gene transfer inhibits the development of angiogenesis in a monkey model of multiple branch retinal vein occlusion (BRVO).


METHODS
A recombinant adenovirus vector adVHL was constructed to deliver the human VHL gene. Total RNA prepared from various kinds of cells transduced with adLacZ (control) or adVHL under normoxic or hypoxic conditions was subjected to Northern blot analyses. Either adLacZ or adVHL was delivered by preretinal injection in monkeys. The effects of adLacZ or adVHL on ocular neovascularization in laser-induced multiple BRVO was evaluated in color photographs and with fluorescein angiography (FA).


RESULTS
VHL expression in adVHL-transduced cells was confirmed at the transcript and protein levels. VHL overexpression significantly decreased the levels of VEGF transcripts in human aortic endothelial cells (HAECs); retinal pigment epithelium (RPE) cells; and RCC 786-O cells, renal carcinoma cells lacking VHL expression under normoxia. In contrast, VHL had no effect on the hypoxia-mediated increase in VEGF expression in these cells, although basal levels of VEGF expression were substantially reduced. Color photographs and FA revealed that retinal neovascularization and iris rubeosis accompanied by multiple BRVO in a monkey model were obviously suppressed by VHL overexpression. Northern blot analysis and immunostaining for VHL and VEGF indicated that VHL transfer obviously suppressed VEGF gene expression in VHL-transduced tissues such as retina or RPE.


CONCLUSIONS
The results showed that adenovirus expressing VHL led to a significant reduction in VEGF expression in vitro under normoxic or hypoxic conditions. adVHL effectively inhibited angiogenesis in retina and iris in laser-induced multiple BRVO in monkey eyes. These data suggest that gene therapy based on VHL gene delivery has potential in the treatment of human ocular neovascularization.
Akiyama
Hideo
H
Department of Ophthalmology, Gunma University School of Medicine, Gunma, Japan.
Tanaka
Toru
T
Itakura
Hirotaka
H
Kanai
Hiroyoshi
H
Maeno
Tositaka
T
Doi
Hiroshi
H
Yamazaki
Miki
M
Takahashi
Kyoichi
K
Kimura
Yasutaka
Y
Kishi
Shoji
S
Kurabayashi
Masahiko
M
D000260:Adenoviruses, Human / Q000235:genetics*; D000818:Animals; D015152:Blotting, Northern; D015153:Blotting, Western; D004195:Disease Models, Animal; D004730:Endothelium, Vascular / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D005451:Fluorescein Angiography; D019084:Fluorescent Antibody Technique, Indirect; D005786:Gene Expression Regulation; D016147:Genes, Tumor Suppressor; D015316:Genetic Therapy; D005822:Genetic Vectors; D006801:Humans; D008251:Macaca; D010857:Pigment Epithelium of Eye / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D015861:Retinal Neovascularization / Q000378:metabolism / Q000517:prevention & control*; D014162:Transfection; D014407:Tumor Cells, Cultured; D025521:Tumor Suppressor Proteins / Q000235:genetics* / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000235:genetics* / Q000378:metabolism; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics / Q000378:metabolism; D051794:Von Hippel-Lindau Tumor Suppressor Protein
D012333:RNA, Messenger; D025521:Tumor Suppressor Proteins; D042461:Vascular Endothelial Growth Factor A; D044767:Ubiquitin-Protein Ligases; D051794:Von Hippel-Lindau Tumor Suppressor Protein; C497446:VHL protein, human