TGF-β 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) β effects on matrix metalloproteinases (MMPs) as a potential cause of the blood-retinal barrier breakdown at the onset of angiogenesis. Previously, glial cells were shown to play a role in the angiogenesis process and to express the angiogenic regulating factor TGF-β, which becomes active under hypoxia conditions. Here, the authors demonstrate that retinal endothelial cells express MMP-9 when treated with TGF-β or cocultured with glial cells and that both TGF-β and MMP-9 increase endothelial cell permeability. Methods. Primary cultures of bovine retinal endothelial (BRE) cells grown on porous membranes were treated with TGF-β or purified MMP-9, and permeability changes were assayed. The amount and distribution of the tight junction protein occludin also was analyzed by immunocytochemistry and Western blotting. Cell extracts or conditioned media from TGF-β-treated BRE cells and from glial cell-BRE cocultures were analyzed for MMP-9 content by substrate gel electrophoresis (zymography) or Western blotting. Results. Both TGF-β and MMP-9 increased the permeability of BRE monolayers and reduced the levels of the junction protein occludin. The effect of MMP-9 on permeability was rapid, but the TGF-β-induced permeability required longer incubation and was blocked by anti-TGF-β and anti-MMP-9 antibodies as well as by TGF-β latency-associated peptide. Zymography showed that MMP-9 activity, which was very low or absent in untreated BRE cultures, was dramatically increased by TGF-β as well as by coculturing with either astrocytes or Müller glial cells, Anti-TGF-β antibody blocked the TGF-β effect, but not the coculture effect on MMP-9 production. Conclusions. These data indicate a direct correlation between TGF-β-induced MMP-9 activity and increased endothelial cell permeability, Moreover, endothelial cell production of MMP-9 is regulated by glial cells through expression of TGF-β or by direct cell-to-cell contact. During retinal disease, glial cell production of active TGF-β may contribute to breakdown of the blood-retinal barrier by stimulating endothelial cell MMP-9 production.