17 Beta-estradiol increases VEGF receptor-2 and promotes DNA synthesis in retinal microvascular endothelial cells.
OBJECTIVE
Estrogen is known to promote angiogenesis in gonads. The presence of estrogen receptors in the vascular endothelium of organs other than gonads has been reported. The goal of this study was to determine whether estrogen promotes the proliferation of retinal microvascular endothelial cells and to explore the mechanism of it.


METHODS
DNA was quantitated using primary cultures of bovine retinal endothelial cells that were incubated with different doses of 17 beta-estradiol (E2), VEGF, or both. The changes in expression level of VEGF and VEGF receptor-2 (VEGFR2) were measured using northern blot analysis after treatment with E2. The presence of estrogen receptors in the endothelial cells was studied by immunohistochemistry and western blot analysis.


RESULTS
17 Beta-estradiol (E2) increased the DNA level in bovine retinal capillary endothelial cells (BRECs) by 177% at 1 nM (P < 0.05) and 150% at 10 nM (P < 0.05) by comparison with unstimulated BREC. One hundred nanomole tamoxifen completely blocked the E2-induced DNA synthesis in BRECs. Ten nanomole E2 augmented vascular endothelial growth factor (VEGF)-induced DNA synthesis in BRECs significantly (160%, P < 0.01). Ten nanomole E2 also increased VEGF mRNA expression, which peaked after 24 hours (6.7 times, P < 0.05), and VEGF receptor-2 (VEGFR2) mRNA expression, which peaked after 9 hours (2.4 times, P < 0.05). The mRNA expression level of VEGFR2 peaked with 10 nM E2 (P < 0.05) and that of VEGF reached maximum with 1 nM E2 (15 times, P < 0.001). VEGFR2 and VEGF proteins increased in parallel with their mRNA levels. Immunocytochemistry showed estrogen receptor expression in BRECs, and western blot analysis indicated the presence of a 67-kDa protein that was compatible with the estrogen receptor.


CONCLUSIONS
These findings suggest that E2 may stimulate BREC growth by the receptor-mediated pathway and that E2 may augment the VEGF-dependent angiogenesis partly through the upregulation of VEGFR2.
Suzuma
I
I
Department of Ophthalmology and Visual Sciences, Kyoto University Graduate School of Medicine, Japan.
Mandai
M
M
Takagi
H
H
Suzuma
K
K
Otani
A
A
Oh
H
H
Kobayashi
K
K
Honda
Y
Y
D000818:Animals; D015152:Blotting, Northern; D015153:Blotting, Western; D002417:Cattle; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured; D004247:DNA / Q000096:biosynthesis*; D004261:DNA Replication / Q000187:drug effects; D016228:Endothelial Growth Factors / Q000235:genetics / Q000378:metabolism; D004730:Endothelium, Vascular / Q000187:drug effects* / Q000378:metabolism; D004958:Estradiol / Q000494:pharmacology*; D004965:Estrogen Antagonists / Q000494:pharmacology; D019084:Fluorescent Antibody Technique, Indirect; D008222:Lymphokines / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000096:biosynthesis; D020794:Receptor Protein-Tyrosine Kinases / Q000235:genetics / Q000378:metabolism*; D011960:Receptors, Estrogen / Q000378:metabolism; D017978:Receptors, Growth Factor / Q000235:genetics / Q000378:metabolism*; D040262:Receptors, Vascular Endothelial Growth Factor; D012171:Retinal Vessels / Q000187:drug effects* / Q000378:metabolism; D013629:Tamoxifen / Q000494:pharmacology; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors
D016228:Endothelial Growth Factors; D004965:Estrogen Antagonists; D008222:Lymphokines; D012333:RNA, Messenger; D011960:Receptors, Estrogen; D017978:Receptors, Growth Factor; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors; D013629:Tamoxifen; D004958:Estradiol; D004247:DNA; D020794:Receptor Protein-Tyrosine Kinases; D040262:Receptors, Vascular Endothelial Growth Factor