Abstract
Purpose: :
To determine the effect of matrix metalloproteinase (MMP)–mediated proteolysis on the anti–angiogenic activity of pigment epithelium–derived factor (PEDF) in vitro and determine the MMP–mediated cleavage sites within the primary sequence of PEDF. Furthermore, we have determined the effect of inhibiting MMPs on the intraocular levels of PEDF protein expressed from an adenoviral expression system in a mouse eye.
Methods: :
To determine the site at which PEDF is cleaved by MMP–9, purified PEDF was incubated with and without MMP–9 (1:200 mass ratio MMP to PEDF) at 37°C for 24h then with trypsin (1:40 trypsin to PEDF) overnight. The peptides were separated by reverse–phase HPLC on a C–18 column, collected, treated with 2–sulfobenzoic acid anhydride, and sequenced by MALDI–MS. The anti–angiogenic activity of MMP–treated PEDF was quantified using the mouse aortic ring bioassay and a standard invasion assay. The effect of MMP inhibition on PEDF levels was evaluated by immunoassay 1 day after intravitreal injection of 1x109 particles of adenovector carrying the gene for human PEDF into the right eyes of C57–Bl6 mice that had been pretreated with or without 2 ng of the MMP–2 and –9 inhibitor, SB–3CT.
Results: :
We have found proteolytic digestion of PEDF by the pro–angiogenic proteinase MMP–9 eliminates the anti–angiogenic activity of PEDF as measured in the mouse aortic ring assay, and converts PEDF into an endothelial chemoattractant factor, as the products formed from the digestion of PEDF with MMP–9 increase the invasiveness of cultured endothelial cells in vitro. PEDF is proteolytically processed by MMP–9 at the serpin reactive center loop of PEDF as well as near the N–terminal, anti–angiogenic region of the protein. Finally, pretreatment of mouse eyes with intravitreal injection of the MMP–2 and –9 inhibitor, SB–3CT, tripled the observed amount of expressed PEDF in mouse eyes intravitreally transduced with an adenovector containing the gene for human PEDF.
Conclusions: :
PEDF levels are decreased in the human eye during proliferative disease, while MMP activity is increased. Cleavage of PEDF by MMP–9 eliminates the anti–angiogenic activity of PEDF, while the inhibition of MMP within the mouse eye correlates with higher levels of expressed PEDF. Our results suggest that pro–angiogenic MMPs modulate PEDF activity by proteolysis at the serpin reactive center loop or the N–terminal, anti–angiogenic region of the protein and this may be important in ocular disease progression.
Keywords: neovascularization • protein structure/function • proteolysis