Angiopoietin-1 upregulation by vascular endothelial growth factor in human retinal pigment epithelial cells.
OBJECTIVE
To determine whether vascular endothelial growth factor (VEGF) regulates angiopoietin (Ang)-1 and -2 expression in retinal pigment epithelial (RPE) cells.


METHODS
Expression of VEGF, Ang1, and Ang2 in surgically removed human choroidal neovascular membranes (CNVMs) was analyzed by double-label confocal immunofluorescence microscopy. Total RNA was extracted from cultured human RPE cells treated with VEGF for mRNA analysis. Northern blot analysis was performed to examine the time course and dose response of Ang1 and Ang2 mRNA expression. mRNA stability and nuclear run-on analyses were performed. Secreted Ang1 and Ang2 protein levels in conditioned media from RPE cells were examined by Western blot analysis.


RESULTS
Ang1 and Ang2 immunostaining colocalized with VEGF-positive stromal cells in human CNVMS: Ang1 and Ang2 mRNAs were expressed by cultured serum-starved RPE cells. VEGF upregulated Ang1 mRNA in a time- and dose-dependent manner without a significant change in Ang2 mRNA. Ang1 and Ang2 mRNAs in RPE cells were as stable as that of S18. VEGF stimulation further increased the half-life of Ang1 mRNA, but did not alter its transcription rate. VEGF increased the amount of Ang1, but not Ang2, protein secreted into the medium.


CONCLUSIONS
The colocalization of Ang1 and Ang2 with VEGF in CNVM stromal cells and the upregulation of Ang1 expression by VEGF in cultured RPE cells suggest that VEGF may selectively modulate Ang expression during CNV.
Hangai
M
M
Department of Ophthalmology, Doheny Eye Institute, Keck School of Medicine at the University of Southern California, Los Angeles 90033, USA.
Murata
T
T
Miyawaki
N
N
Spee
C
C
Lim
J I
JI
He
S
S
Hinton
D R
DR
Ryan
S J
SJ
D042683:Angiopoietin-1; D042702:Angiopoietin-2; D015152:Blotting, Northern; D015153:Blotting, Western; D002478:Cells, Cultured; D020256:Choroidal Neovascularization / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D016228:Endothelial Growth Factors / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D004791:Enzyme Inhibitors / Q000378:metabolism; D019084:Fluorescent Antibody Technique, Indirect; D015870:Gene Expression; D006801:Humans; D008222:Lymphokines / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism*; D018613:Microscopy, Confocal; D008875:Middle Aged; D010857:Pigment Epithelium of Eye / Q000187:drug effects* / Q000378:metabolism; D011506:Proteins / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D013997:Time Factors; D015854:Up-Regulation / Q000187:drug effects; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors
C467630:ANGPT1 protein, human; D042683:Angiopoietin-1; D042702:Angiopoietin-2; D016228:Endothelial Growth Factors; D004791:Enzyme Inhibitors; D008222:Lymphokines; D008562:Membrane Glycoproteins; D011506:Proteins; D012333:RNA, Messenger; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors