TGF-beta increases retinal endothelial cell permeability by increasing MMP-9: possible role of glial cells in endothelial barrier function.
OBJECTIVE
To determine transforming growth factor (TGF) beta 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-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 TGF-beta and MMP-9 increase endothelial cell permeability.


METHODS
Primary cultures of bovine retinal endothelial (BRE) cells grown on porous membranes were treated with TGF-beta 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-beta-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-beta 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-beta-induced permeability required longer incubation and was blocked by anti-TGF-beta and anti-MMP-9 antibodies as well as by TGF-beta latency-associated peptide. Zymography showed that MMP-9 activity, which was very low or absent in untreated BRE cultures, was dramatically increased by TGF-beta as well as by coculturing with either astrocytes or Müller glial cells. Anti-TGF-beta antibody blocked the TGF-beta effect, but not the coculture effect on MMP-9 production.


CONCLUSIONS
These data indicate a direct correlation between TGF-beta-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-beta or by direct cell-to-cell contact. During retinal disease, glial cell production of active TGF-beta may contribute to breakdown of the blood-retinal barrier by stimulating endothelial cell MMP-9 production.
Vascular Biology Center, Medical College of Georgia, Augusta, GA 30912, USA.
M A
MA
Behzadian
X L
XL
Wang
L J
LJ
Windsor
N
N
Ghaly
R B
RB
Caldwell
D000818:Animals; D001813:Blood-Retinal Barrier / Q000502:physiology*; D015153:Blotting, Western; D002417:Cattle; D002463:Cell Membrane Permeability / Q000187:drug effects; D002478:Cells, Cultured; D018920:Coculture Techniques; D004730:Endothelium, Vascular / Q000187:drug effects* / Q000201:enzymology; D019084:Fluorescent Antibody Technique, Indirect; D020780:Matrix Metalloproteinase 9 / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism; D009457:Neuroglia / Q000502:physiology*; D062793:Occludin; D051381:Rats; D012171:Retinal Vessels / Q000187:drug effects* / Q000201:enzymology; D016212:Transforming Growth Factor beta / Q000494:pharmacology*
D008565:Membrane Proteins; D062793:Occludin; C568987:Ocln protein, rat; D016212:Transforming Growth Factor beta; D020780:Matrix Metalloproteinase 9