Overexpression of thymosin beta-10 inhibits VEGF mRNA expression, autocrine VEGF protein production, and tube formation in hypoxia-induced monkey choroid-retinal endothelial cells.
OBJECTIVE
One purpose of this study was to examine if expression of the thymosin beta(10 )(TB10), thymosin beta(4) (TB4), and VEGF165 isoform genes would change in response to hypoxia in monkey choroidal retinal endothelial cells (RF/6A). We also sought to determine if overexpression of the TB10 gene could inhibit VEGF165 mRNA expression, autocrine VEGF production, and tube formation in hypoxic RF/6A cells.


METHODS
An adenovirus vector, Ad-TB10, was constructed to deliver the human TB10 gene. Under hypoxic conditions, TB10, TB4, and VEGF165 mRNA were measured using real-time PCR, and VEGF protein expression was evaluated by ELISA. For tube formation assays, the RF/6A cells were transfected with Ad-TB10 and exposed to hypoxia for 4 h.


RESULTS
VEGF165 mRNA was 2- and 5.8-fold increased when the RF/6A cells were exposed to hypoxia for 2 and 4 h, respectively. TB10 mRNA decreased at 2 h and increased to a baseline level at 4 h, while TB4 mRNA level increased slightly by 2 and 4 h. Increased VEGF165 mRNA was down-regulated by transfected Ad-TB10, and ELISA results showed that VEGF protein levels increased 2.4-fold after 4-hour hypoxia, and decreased 2-fold after transfection with Ad-TB10. Hypoxia also induced tube formation of the RF/6A cells. This was significantly reduced by Ad-TB10.


CONCLUSIONS
These data indicate that (1) TB4 and TB10 mRNA expression in RF/6A cells present different changes under 2- and 4-hour hypoxia, and (2) TB10 may be an effective inhibitor of angiogenesis by inhibiting VEGF expression and autocrine protein production.
Department of Ophthalmology, People's Hospital, Peking University, Beijing, China.
Tonghe
T
Zhang
Xiaoxin
X
Li
Wenzhen
W
Yu
Zheng
Z
Yan
He
H
Zou
Xiangjun
X
He
D000818:Animals; D015687:Cell Hypoxia / Q000235:genetics / Q000502:physiology*; D002461:Cell Line, Transformed; D002829:Choroid / Q000166:cytology; D042783:Endothelial Cells / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D005786:Gene Expression Regulation / Q000235:genetics*; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D008253:Macaca mulatta; D012333:RNA, Messenger / Q000378:metabolism; D012160:Retina; D013947:Thymosin / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics* / Q000378:metabolism*
D012333:RNA, Messenger; D042461:Vascular Endothelial Growth Factor A; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D013947:Thymosin; C039302:thymosin beta(10)