TGF-beta increases retinal endothelial cell permeability by increasing MMP-9: Possible role of glial cells in endothelial barrier function

PURPOSE. TO determine transforming growth factor (TGF) beta effects on matr ix metalloproteinases (MMPs) as a potential cause of the blood-retinal barr ier breakdown at the onset of angiogenesis. Previously, glial cells were sh own to play a role in the angiogenesis process and to express the angiogeni c regulating factor TGF-beta, which becomes active under hypoxia conditions . Here, the authors demonstrate that retinal endothelial cells express MMP- 9 when treated with TGF-beta or cocultured with glial cells and that both T GF-beta and MMP-9 increase endothelial cell permeability. METHODS. Primary cultures of bovine retinal endothelial (BRE) cells grown o n porous membranes were treated with TGF-beta or purified MMP-9, and permea bility changes were assayed. The amount and distribution of the tight junct ion protein occludin also was analyzed by immunocytochemistry and Western b lotting. Cell extracts or conditioned media from TGF-beta -treated BRE cell s and from glial cell-BRE cocultures were analyzed for MMP-9 content by sub strate gel electrophoresis (zymography) or Western blotting. RESULTS. Both TGF-beta and MMP-9 increased the permeability of BRE monolaye rs and reduced the levels of the junction protein occludin. The effect of M MP-9 on permeability was rapid, but the TGF-beta -induced permeability requ ired longer incubation and was blocked by anti-TGF-beta and anti-MMP-9 anti bodies as well as by TGF-beta latency-associated peptide. Zymography showed that MMP-9 activity, which was very low or absent in untreated BRE culture s, was dramatically increased by TGF-beta as well as by coculturing with ei ther astrocytes or Muller glial cells. Anti-TGF-beta antibody blocked the T GF-beta effect, but not the coculture effect on MMP-9 production. CONCLUSIONS. These data indicate a direct correlation between TGF-beta -ind uced MMP-9 activity and increased endothelial cell permeability. Moreover, endothelial cell production of MMP-9 is regulated by glial cells through ex pression of TGF-beta or by direct cell-to-cell contact. During retinal dise ase, glial cell production of active TGF-beta may contribute to breakdown o f the brood-retinal barrier by stimulating endothelial cell MMP-9 productio n.