EFFECTS OF HYPOXIA ON GLIAL-CELL EXPRESSION OF ANGIOGENESIS-REGULATING FACTORS VEGF AND TGF-BETA

Perivascular glial cells are thought to be involved in physiologic vas cularization and also in pathologic angiogenesis in the central nervou s system. We have previously shown that astrocytes are a source of tra nsforming growth factor-beta (TGF-beta) and another inhibiting factor, which block endothelial cell growth and induce their apoptosis. Astro glia are also known to express vascular endothelial growth factor (VEG F), which is up-regulated during hypoxia. Here we demonstrate the effe cts of hypoxia on the expression of both TGF-beta and VEGF by retinal glial cells. Muller cells isolated from rat retina were incubated unde r hypoxia or normoxia and the resulting conditioned media (H-MCM and N -MCM) were assayed for their effects on growth of bovine retinal capil lary endothelial (BRE) and the TGF-beta-sensitive mink lung epithelial CCL cells. The expression and quantities of VEGF and TGF-beta (active vs, latent form) were determined by immune-adsorption, Western or Nor thern blotting, and ELISA. N-MCM stimulated BRE cell growth by twofold but inhibited CCL cells under similar assay conditions,whereas H-MCM had a weak stimulating effect on BRE and substantial inhibitory activi ty on CCL cells. Adsorption of MCM by specific antibodies as well as W estern and Northern blot analysis indicated that stimulating and inhib itory activities of MCM are due to the presence of VEGF and TGF-beta, respectively. ELISA revealed that the hypoxia condition converts laten t TGF-beta into its active form. In N-MCM, TGF-beta is found predomina ntly in the latent form, but in hypoxia MCM it is mainly active. Furth ermore, it was found that treatment of Muller cells with exogenous TGF -beta under either hypoxia or normoxia increases VEGF expression in a time- and dose-dependent fashion. TGF-beta activation may, therefore, be prerequisite for hypoxia-induced upregulation of VEGF and stimulati on of angiogenesis in vivo. (C) 1998 Wiley-Liss, Inc.