Angiopoietin-1 upregulation by vascular endothelial growth factor in humanretinal pigment epithelial cells

PURPOSE. TO determine whether vascular endothelial growth factor (VEGF) reg ulates angiopoietin (Ang)-1 and -2 expression in retinal pigment epithelial (RPE) cells. METHODS. Expression of VEGF, Ang1, and Ang2 in surgically removed human cho roidal 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 p erformed to examine the time course and dose response of Ang1 and Ang2 mRNA expression. mRNA stability and nuclear nm-on analyses were performed. Secr eted Angl and Ang2 protein levels in conditioned media from RPE cells were examined by Western blot analysis. RESULTS. Angl and Ang2 immunostaining colocalized with VEGF-positive stroma l cells in human CNVMs. Angl and Ang2 mRNAs were expressed by cultured seru m-starved RPE cells. VEGF upregulated Angl mRNA in a time- and dose-depende nt manner without a significant change in Ang2 mRNA. Angl and Ang2 mRNAs in RPE cells were as stable as that of S18. VEGF stimulation further increase d the half-life of Ang1 mRNA, but did not alter its transcription rate. VEG F increased the amount of Ang1, but not Ang2, protein secreted into the med ium. CONCLUSION. The colocalization of Ang1 and Ang2 with VEGF in CNVM stromal c ells and the upregulation of Angl expression by VEGF in cultured RPE cells suggest that VEGF may selectively modulate Ang expression during CM.