Cleavage of structural components of mammalian vitreous by endogenous matrix metalloproteinase-2
Our goal was to determine if the major endogenous vitreous matrix metalloproteinase (MMP-2) could digest known collagenous components of the vitreous body. Matrix metalloproteinase-2 and its associated inhibitors were isolated from porcine vitreous by affinity column chromatography. The inhibitors were inactivated by chemical modification with dithiothreitol and iodoacetamide. The latent MMP-2 was then activated with the organo-mercurial, p-aminophenyl mercuric acetate (APMA). Bovine vitreous fibrillar collagens (types II, V/XI and IX) were isolated by pepsin extraction and differential salt precipitation. Intact type IX collagen was purified by selective salt precipitation followed by ion exchange and size exclusion chromatography. These isolated collagens were incubated for 6 to 24 h with different concentrations of activated MMP-2, and the extent of collagen degradation was analyzed. Activated MMP-2 was also introduced into freshly isolated vitreous gels and the degree of liquefaction was determined. Our results showed that the activated MMP-2 has no apparent effect upon type II collagen but can degrade type V/XI collagen and type IX collagen fragments (COL2 and COL2 + COL3). In addition, when the type IX collagen was in the intact helical form, MMP-2 appeared to selectively digest alpha3 (IX) chains. This suggested that vitreous MMP-2 preferentially cleaved certain vitreous collagen chains into large fragments than small peptides. MMP-2 also disrupted the vitreous gel in vitro, releasing proteins but not hexuronic acid or sulfated glycosaminoglycans into the liquefied supernatant. We conclude that MMP-2 activity should be considered as a potential mechanism of vitreous liquefaction that is seen in aging and various pathological states. Keywords: matrix metalloproteinase; type II collagen; type IX collagen; type V/XI collagen; vitreous; pig
Brown
DJ
Ophthalmology Research Laboratories, Department of Surgery, Cedars-Sinai Research Institute, Cedars-Sinai Medical Center, University of California at Los Angeles Medical School Affiliate, Davis Building, Room 5069, 8700 Beverly Blvd, Los Angeles, CA 90048, USA
Bishop
P
Hamdi
H
Kenney
MC