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Cleavage of structural components of mammalian vitreous by endogenous matrix metalloproteinase-2
1Ophthalmology 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
Abstract:
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
Insights
Matrix metalloproteinase-2 (MMP-2) in the vitreous body can degrade specific collagen types, contributing to vitreous liquefaction observed in aging and disease.
Area of Science:
- Ophthalmology
- Biochemistry
- Molecular Biology
Background:
- The vitreous body's structural integrity relies on collagenous components.
- Matrix metalloproteinases (MMPs) are implicated in tissue remodeling and degradation.
- Matrix metalloproteinase-2 (MMP-2) is a major endogenous protease found in the vitreous.
Purpose of the Study:
- To investigate the capacity of endogenous vitreous matrix metalloproteinase-2 (MMP-2) to degrade key collagenous components of the vitreous body.
- To determine if MMP-2 activity contributes to vitreous liquefaction.
Main Methods:
- Isolation and purification of active MMP-2 and specific collagen types (II, V/XI, IX) from porcine and bovine vitreous.
- Enzymatic assays involving incubation of purified collagens with activated MMP-2.
- Analysis of collagen degradation products and assessment of vitreous gel liquefaction in vitro.
Main Results:
- Activated MMP-2 degraded type V/XI collagen and fragments of type IX collagen (COL2, COL2 + COL3).
- MMP-2 selectively digested alpha3 (IX) chains of intact type IX collagen.
- MMP-2 disrupted vitreous gels, releasing proteins but not glycosaminoglycans.
Conclusions:
- Vitreous MMP-2 preferentially cleaves specific collagen chains, generating large fragments.
- MMP-2 activity is a potential mechanism driving vitreous liquefaction.
- This process is relevant to age-related and pathological changes in the vitreous.