Hypoxic conditions induce endogenous

CD13/APN expression and proximal promoter activity. (A) Endogenous CD13/APN mRNA in hypoxia-induced retinal neovessels is up-regulated. RNA isolated from retinas at 0, 12, and 24 hours in relative hypoxia was assayed for CD13/APN and control β-actin levels by RT-PCR. The Southern blot of RT-PCR products (middle panel) was quantitated by phosphorimager analysis. (B) Cobalt chloride treatment induces endogenous CD13/APN in endothelial cell lines. Serum-starved KS1767 cells were treated with cobalt chloride (100 μM) for 12, 24, and 48 hours, and total cellular CD13/APN RNA was assessed. (C) Hypoxia and cobalt chloride treatment induces CD13/APN cell-surface protein levels. Serum-starved KS1767 cells were treated with cobalt chloride (100 μM) or incubated in 1% or 10% oxygen for 24 hours and analyzed for CD13/APN expression with the MY7 monoclonal antibody or negative control immunoglobulin G (IgG) by flow cytometry. (D) Hypoxia induces the CD13/APN proximal promoter. Twenty-four hours after transient transfection of KS1767 cells in 1% serum with 3 μg of proximal promoter plasmid or pGL2basic vector alone and 1 μg of MAP1-SEAP control, cells were subjected to normoxia, hypoxia (1% oxygen), or cobalt chloride (100 μM) treatment for 24 hours before assay for luciferase activity. Results are shown as fold induction over identically treated cells transfected with the promoterless plasmid pGL2basic.