October 2000
Volume 41, Issue 11
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Biochemistry and Molecular Biology  |   October 2000
Elevated Levels of Proteolytic Enzymes in the Aging Human Vitreous
Author Affiliations
  • Anne Vaughan–Thomas
    From the Connective Tissue Biology Laboratories, School of Biosciences, Cardiff University, Wales, United Kingdom.
  • Sophie J. Gilbert
    From the Connective Tissue Biology Laboratories, School of Biosciences, Cardiff University, Wales, United Kingdom.
  • Victor C. Duance
    From the Connective Tissue Biology Laboratories, School of Biosciences, Cardiff University, Wales, United Kingdom.
Investigative Ophthalmology & Visual Science October 2000, Vol.41, 3299-3304. doi:https://doi.org/
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      Anne Vaughan–Thomas, Sophie J. Gilbert, Victor C. Duance; Elevated Levels of Proteolytic Enzymes in the Aging Human Vitreous. Invest. Ophthalmol. Vis. Sci. 2000;41(11):3299-3304. doi: https://doi.org/.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

purpose. To identify whether aging of human vitreous is accompanied by an elevation in degradative enzymes within the tissue. methods. Human vitreous samples from donors aged 10 to 88 years were placed in two groups based on donor age of less than or more than 50 years. Homogenized samples were analyzed by gelatin substrate zymography for matrix metalloproteinases (MMP). Serine proteinases were detected by casein substrate zymography, and a specific antibody was used to confirm the identity of, and to quantify, the serine proteinase, plasmin. results. Progelatinase A (ProMMP-2) was present in all the vitreous samples but did not show an age-related increase. MMP-2 was also present at low levels. Progelatinase B (ProMMP-9) was found in approximately 80% of samples analyzed, but neither its presence nor level of activity was age dependent. Of the serine proteinase activities detected, an enzyme of approximately 80 kDa was identified by Western blot analysis as plasmin(ogen). Quantitative analysis revealed a significant increase in plasmin(ogen) with age. conclusions. This study shows there is an age-related increase in potential degradative activity in human vitreous that may be responsible for degenerative changes such as vitreous liquefaction. The data suggest that increased levels of these enzymes precede, or are indicative of, underlying ocular disease in some individuals.

The mammalian vitreous is an avascular connective tissue that is composed of very low concentrations of extracellular matrix components contained within a highly hydrated structure. The structural components of the vitreous gel include fibrillar networks, hyaluronan, and noncollagenous proteins. 1 The collagen fibrillar network is composed mainly of type II collagen, with collagen types IX and V/XI being present as quantitatively minor components. Type VI collagen is also present and has been proposed to interact with both collagen and hyaluronan. The vitreous undergoes liquefaction with age, and a decrease in gel volume is particularly evident after the fourth decade. 2 Liquefaction is associated with the formation of lacunae within the vitreous body that contain little or no collagen, the collagen fibrils being aggregated in what remains of the surrounding gel-like vitreous. 3  
Age-related vitreous liquefaction is thought to contribute to posterior vitreous detachment (PVD) which, if incomplete, may lead to retinal detachment or formation of macular hole and blindness. PVD is associated with separation of the vitreous cortex from the inner limiting lamina (ILL), suggesting that as well as extracellular matrix degradation within the vitreous body, there may be significant degradation at the periphery resulting in weakened interactions between the vitreous and ILL. Studies have shown that there is loss of adherence of the vitreous cortex to the ILL with increased age. 4 Because the vitreous is relatively acellular, degradative enzymes must be produced either by the few hyalocytes found within the vitreous cortex or, alternatively, by exogenous sources, such as the cells of the vitreous base and retina or from compromised vasculature in surrounding tissues. 
Enzymes involved in the degradation and remodeling of connective tissues include matrix metalloproteinases (MMPs), cysteine proteases, and serine proteases. 5 MMPs are involved in physiological and pathologic extracellular matrix remodeling and both progelatinase A (proMMP-2) and gelatinase B (MMP-9) have been identified previously in human vitreous. 6 7 MMP-2 cleaves denatured collagen and a number of native collagens present in the vitreous or surrounding tissues, including types IV, V, VII, and XI. The possible role of MMP-2 in vitreous liquefaction is supported by recent findings that it can cleave the hybrid type V/XI collagen and, in vitro, is capable of liquefying the vitreous gel. 8  
The concentration of serum proteins in vitreous, 9 which may include serine proteases such as plasmin, increases with age. Plasmin can degrade fibronectin, type IV collagen, proteoglycan core protein, and fibrin, 5 and evidence for its role in MMP-2 activation has been reported recently. 10 It may therefore be speculated that increased levels of plasmin leads to increased activation of endogenous proMMP-2 in vitreous. 
In this study, we measured the levels of several proteases in vitreous samples from individuals of less than or more than 50 years of age to test the hypothesis that liquefaction and increased susceptibility to PVD are associated with increased degradative capacity of the vitreous. These findings show increased vitreous proteolytic activity in individuals of more than 50 years of age, suggesting a correlation with the increase in vitreous liquefaction known to occur within this age group. 
Methods
Preparation of Human Vitreous Samples
Human eyes, collected within 30 hours of death from donors aged between 10 and 88 years, were obtained from the Corneal Transplantation Service Eye Bank at Bristol Eye Hospital (UK) after removal of corneas for transplantation. Exclusion criteria included history of intraocular surgery, human immunodeficiency virus (HIV), hepatitis, and systemic diseases with ocular manifestations. Eyes were also excluded if any signs of gross hemorrhage or tissue abnormality were observed. Each sample, which consisted of vitreous dissected from both eyes of individuals, was liquefied by centrifugation at 60,000 rpm for 1 hour at 4(C (Optima TLX; Beckman, Berkeley CA) and the sedimented insoluble material removed. Liquefied vitreous supernatants were homogenized by mixing and diluted with equal volumes of 2 × electrophoresis sample buffer (0.12 M Tris/HCl, [pH 6.8], containing 4% [wt/vol] sodium dodecyl sulfate [SDS], 20% [vol/vol] glycerol, and 0.01%[ wt/vol] bromophenol blue). 
By the methods to be described, samples were analyzed in two groups, one of less than and one of more than 50 years of age. These groupings were based on the assumption that increased levels of degradative activity would be associated with the increased levels of vitreous liquefaction observed in older people. 2 For all analyses, values obtained were related to unit volumes of vitreous sample, as opposed to vitreal protein concentrations, because the latter may be subject to selective filtration of proteins on the basis of molecular weight, if derived from extravitreal sources. 
SDS–Polyacrylamide Gel Electrophoresis
Liquefied vitreous samples (15 μl), prepared as described, were resolved by SDS–polyacrylamide gel electrophoresis (SDS-PAGE) 11 on 10% (wt/vol) polyacrylamide gels. After electrophoresis, the gels were stained with Coomassie Brilliant Blue R250 (0.005% wt/vol; Sigma, Poole, UK) in 7.5% (vol/vol) acetic acid, and 10% (vol/vol) methanol, and subsequently destained in 7.5% (vol/vol) acetic acid and 10% (vol/vol) methanol. To ascertain the molecular weights of protein species detected after Coomassie blue staining or substrate gel zymography, a range of prestained molecular weight standards (7.2–205 kDa; BioRad Laboratories, Herts, UK) were applied to the gels (5 μl per lane). 
Gelatin and Casein Substrate Zymography for Protease Activity
MMP and serine proteinase activities were detected by gelatin and casein zymography respectively, as described previously. 12 Gelatin (bovine skin; Sigma) or casein (casein Hammarsten; BDH Chemicals, Poole, UK), at a final substrate concentration of 0.5 mg/ml, were incorporated into SDS-polyacrylamide gels containing 10% or 12% (wt/vol) acrylamide respectively. 
Standard volumes of vitreous samples (15 μl), bovine fibroblast–conditioned medium (10 μl) known to contain MMP activity, and protein molecular weight standards were loaded onto the gels and resolved by electrophoresis. The gels were agitated in 2.5% (vol/vol) Triton X-100 for 1 hour and subsequently incubated for 16 to 20 hours at 37°C in buffers optimal for proteolysis. Gelatin substrate gels were incubated in 50 mM Tris/HCl (pH 7.8), containing 50 mM CaCl2, 0.5 M NaCl, and 1 mM aminophenylmercuric acetate (APMA). Although SDS activates proMMP-2 and proMMP-9, APMA was included to ensure that they were activated fully. Casein substrate gels were incubated in 100 mM sodium phosphate buffer (pH 6.8), containing 8 mM EDTA and 0.2% (vol/vol) Triton X-100. 
Gels were stained with Coomassie blue as described and areas of proteolysis corresponding to degradative activity were observed as clear, unstained bands. Serine protease activities were identified by their sensitivity to inhibition by 1 mM phenylmethylsulfonyl fluoride (PMSF) and soybean trypsin inhibitor (1 μg/ml). MMP activities were subjected to inhibition by 10 mM EDTA. 
Quantification of MMP-2 Activity by Gelatin Zymography
The relative MMP-2 activity in a standard volume (15 μl) of each vitreous sample was quantified using a linear response curve. Different loadings of the MMP-2 standard (fibroblast conditioned medium) were processed by zymography, as described, and the substrate cleared was measured by scanning densitometry (Umax color scanner [Umax Systems GMbH, Germany] and Photoshop [Adobe, San Jose, CA] and NIH Image [National Institutes of Health, Bethesda, MD] imaging software). The range of enzyme activity that produced a linear densitometric response as detected by the color scanner, was used to ascertain the limits of sensitivity of the assay. Vitreous samples, or dilutions thereof, which produced levels of proteolysis within this linear range were measured. The absorbance of each gel in areas devoid of any sample and in areas of proteolysis produced by constant amount of an internal standard (fibroblast-conditioned medium) were measured to allow for gel-to-gel variation. The relative activity present within each undiluted sample was expressed as a percentage of the internal standard included on each gel. 
Detection of Plasmin(ogen) in Vitreous by Western Blot Analysis
Western blot analysis 13 was used to confirm the findings of the casein substrate zymography. Samples (10 μl), protein molecular weight standards, and plasmin (Sigma) and plasminogen standards (not shown; Sigma) were resolved on 7.5% (wt/vol) SDS-polyacrylamide gels and transferred subsequently to polyvinylidene difluoride membrane (PVDF, Immobilon; Millipore, Bedford, MA). 
Nonspecific binding sites on the membrane were blocked by incubation in 3% (wt/vol) skimmed milk powder in Tris-buffered saline (TBS; 0.05 M Tris-HCl, [pH 8.0], containing 0.15 M NaCl). The membrane was incubated sequentially with polyclonal goat antiserum to human plasmin(ogen) and horseradish peroxidase (HRP)–conjugated anti-goat IgG (both from Sigma), both diluted in TBS containing 0.2% (vol/vol) Tween 20 (TBS-Tween). The membrane was washed extensively in TBS-Tween between incubations. Specific binding of the anti-plasmin (ogen) was detected using enhanced chemiluminescence reagents (Amersham, Amersham, UK) on film (Hyperfilm-ECL; Amersham). 
Quantitative Analysis of Plasmin(ogen) Protein in Vitreous
Because standard preparations of human plasmin containing known amounts of the protein were available, values for the plasmin(ogen) protein present in vitreous could be obtained, irrespective of measurement of activity. A range of plasmin standards (0.135–2.125 ng) and plasminogen (0.135–2.125 ng, not shown) were resolved by electrophoresis and detected by Western blot analysis as described to obtain a linear densitometric response curve. This curve, which was obtained after optimization of amounts loaded, film exposure time, and scanning parameters, was used to set the limits of detection for the blot analysis of vitreous samples. Because all samples were detected on a single film, the baseline values were constant. To account for variation between gels, a plasmin standard curve was included on each gel along with the samples. This method for detecting human plasmin(ogen) levels was further validated using human serum samples (unrelated to the vitreous samples; not shown). 
Statistical Analysis
To compare the proteolytic activities in vitreous samples obtained from individuals less than and more than 50 years of age, data are presented as means ± SEM and have been analyzed for significance (Mann–Whitney test). Correlation of the proteolytic activities of individuals with their age was tested using Spearman’s rank analysis. Differences shown by both statistical analyses were considered significant at P < 0.05. 
Results
Identification of Vitreous MMP Activities by Gelatin Substrate Zymography
MMP species were identified as bands of substrate cleared within gelatin zymography gels. Bands of gelatinolytic activities of 92, 72, and 65 kDa were identified in vitreous, all of which were susceptible to inhibition by 10 mM EDTA (Fig. 1A ). These bands corresponded to the activities of proMMP-9, proMMP-2, and the active form of MMP-2, respectively. In the absence of gelatin, several protein species were observed in all vitreous samples (Fig. 1A) . One species, at a molecular weight of approximately 67 kDa, comigrated with active MMP-2 and interfered with its detection by zymography. ProMMP-2 and proMMP-9 activities were unaffected by the presence of vitreous protein. Preliminary analysis of individual samples revealed that proMMP-2 and active MMP-2 were present in all samples, with an apparent increase in proMMP-2 activity with age but no change in the level of active MMP-2. ProMMP-9 was present in 79% of the samples, but neither its presence nor the level of activity present appeared to be dependent on the age of the individual donor. 
Quantitative Analysis of ProMMP-2 in Human Vitreous
The relative quantity of proMMP-2 in each vitreous sample was determined by scanning densitometry. The linear relationship obtained between the area of substrate cleared, measured in absorbance units, and the amount of enzyme loaded was limited to proMMP-2 concentrations within one order of magnitude (Fig. 1B , inset). Therefore, the vitreous sample volumes loaded were adjusted, by dilution, to fall within this linear range (Fig. 1B , main picture). Although there was a trend toward higher activity of proMMP-2 (percentage of internal standard) in the group aged more than 50 years (72.0% ± 6.0%) compared with the group aged less than 50 years (61.8% ± 7.3%), the increase was not significant (P = 0.392). The spread of data obtained from the older group was much greater, with some individuals having particularly high levels of proMMP-2. 
Identification of Vitreous Serine Protease Activities by Casein Substrate Zymography
With the use of casein substrate zymography (Fig. 2A ), three additional bands of proteolytic activity were identified. These proteolytic activities were not inhibited by 10 mM EDTA but were inhibited by 1 mM PMSF or soybean trypsin inhibitor (1 μg/ml), confirming their identities as serine proteases. 
Detection of Plasmin in Human Vitreous by Western Blot Analysis
Western blot analysis of vitreous samples using an antibody specific to human plasmin(ogen) identified the major serine protease activity of approximately 80 kDa as plasmin (Fig. 2B) . The molecular weight of the protein present in human vitreous and serum (not shown) varied somewhat, relative to the commercial human serum-derived plasmin and plasminogen standards. This discrepancy could be due to retarded mobility of the vitreous proteins in the presence of other matrix components, such as hyaluronan. The immunoreactive band could be resolved into a doublet or triplet, which, because the electrophoresis was performed under nonreducing conditions, may represent plasmin and plasminogen. Other weak reactivities within the vitreous samples corresponding to higher molecular weight species were shown to be due to nonspecific binding of the secondary antibody. 
Quantitative Measurement of Plasmin in Vitreous by Western Blot Analysis
Western blot analysis and detection of bound antibody by enhanced chemiluminescence and scanning is linear over only a narrow range. The range of standard plasmin(ogen) that gave a linear densitometric response curve was determined to be 0.1 to 2.2 ng (Fig. 2C , inset). For analysis of the vitreous samples, the volumes loaded were adjusted, by dilution, to fall within this linear range (Fig. 2C , main picture). Plasmin(ogen) concentrations (in nanograms per unit volume of vitreous) from the older age group (3.12 ± 0.42 ng) were calculated to be significantly higher (P = 0.001) than concentrations in the younger age group (1.179 ± 0.13 ng). As for the proMMP-2 data, the values obtained for plasmin concentrations were spread over a much greater range in the older group, with some individuals again expressing very high levels. 
Correlation Analysis
To determine whether enzyme levels correlated linearly to the age of individual donors, Spearman’s rank correlation analysis was performed (Fig. 3) . Plasmin was found to be correlated both to the age and to the proMMP-2 levels obtained for each individual. However, there was no linear correlation between age and proMMP-2 levels. It is interesting to note that the individuals expressing high levels of proMMP-2 were also expressing high levels of plasmin and therefore had an increased degradative capacity overall. 
Discussion
Liquefaction, or the decrease in the volume of vitreous gel, increases with age and is particularly evident after the fourth decade. 2 Age-related changes also occur at the vitreoretinal interface, resulting in decreased adhesion between the ILL and vitreous cortex. 4 The mechanisms by which these changes occur may be mediated by matrix-degrading enzymes. 7 8 14 Assuming that the decrease in gel vitreous volume observed after the fourth decade is a result of increased degradative activity, we measured MMP-2, MMP-9, and plasmin in vitreous samples obtained from two groups of donors, aged less than or more than 50 years. 
MMP-9 has been identified in vitreous from patients with diabetes 6 or after rhegmatogenous retinal detachment. 15 Our data, and that of Plantner et al. 7 suggest that MMP-9 is also a component of vitreous in the absence of ocular disease. The presence of this enzyme suggests potential degradative capacity but, because neither the presence nor level of MMP-9 expression is age dependent, it is unlikely that MMP-9 contributes to age-related liquefaction. Nevertheless, regional variation in the expression of MMP-9 around the periphery of the vitreous cortex could be a predisposing factor in nonuniform detachment and vitreoretinal pathogenesis in some individuals. 
Although proMMP-2 did not increase significantly with age, the data showed a trend toward increased spread between individuals with age, with some individuals in the older group expressing particularly high levels. A study of MMP activity in Bruch’s membrane and choroid revealed a similar trend 16 indicating generalized accumulation of proteolytic activities within ocular tissues with age. The increase in plasmin(ogen) concentrations with age is particularly interesting, because this enzyme is probably derived from surrounding vasculature. Vitreous plasmin(ogen) levels may reflect degeneration of vasculature in surrounding tissues such as the retina. Plasmin cooperates with the membrane type (MT) MMP-1 in the activation of proMMP-2. 10 Because the present study identified a trend toward increased proMMP-2 and a significant increase in plasmin with age, we speculate that degradative cascades underlie age-related liquefaction of the vitreous. 
PVD may also be driven by enzymes derived from tissues surrounding the vitreous. Degradation of vitreous and ILL components, such as type V/XI collagen and type IV collagen, would lead to loss of interactions at the vitreoretinal interface. As for MMP-9, regional variations in the expression or release of these enzymes in the vitreous cortex could lead to disease. Coexpression of proMMPs and plasmin may result in very little increase of active enzyme within the vitreous overall, as our data suggest, whereas locally, in the vicinity of cells expressing membrane-bound MT-MMP-1, increase of active MMP-2 may be substantial. The interactions between extracellular matrix, MT-MMP-1, and MMPs may result in temporal and spatial extracellular matrix degradation, even in the presence of high concentrations of inhibitors. 17  
In conclusion, we have identified age-related changes in the expression of degradative enzymes within the vitreous and variations between individuals. In studies of pathologic vitreous, it is therefore important that these variations be taken into account. However, these changes may also be indicative of underlying degeneration of ocular tissues and susceptibility to age-related diseases in a large proportion of the population. Further study is needed to elucidate whether such elevations of proteolytic activities in individuals precede and result in disease. 
 
Figure 1.
 
Identification and quantitative analysis of proMMP-2 in human vitreous. (A) Vitreous samples (15 μl) were resolved on 10% (wt/vol) SDS-polyacrylamide gels containing 0.5 mg/ml gelatin as substrate. Subsequently, the gels were incubated in the absence (left) or presence (middle) of 10 mM EDTA. The vitreous samples were also resolved on 10%(vol/vol) SDS-polyacrylamide gels in the absence of gelatin (right) stained with Coomassie blue. The positions to which MMP activities in a characterized fibroblast conditioned medium standard migrated within the same gels are indicated. Each panel shows five unrelated vitreous samples chosen at random. (B) A proMMP-2 standard (fibroblast-conditioned medium) was resolved on 10% (wt/vol) SDS-polyacrylamide gels containing 0.5 mg/ml gelatin. The area (absorbance units) of substrate cleared by the 72-kDa enzyme was measured by scanning densitometry. The range of proMMP-2 activity that gave a linear densitometric response (r2 = 0.977) was established (inset). Vitreous samples were diluted to contain proMMP-2 activities within this linear densitometric range. Values obtained were corrected for dilution and expressed as a percentage of the activity of an internal standard included on each gel. The data represent individual values obtained for donors less than (n = 11) and more than (n = 22) 50 years of age. The bars represent the mean of each group. Higher activity of proMMP-2 (percentage of internal standard) was observed in the group aged more than 50 years (72.0% ± 6.0%) compared with the group aged less than 50 years (61.8% ± 7.3%), but this was not significant (P = 0.392).
Figure 1.
 
Identification and quantitative analysis of proMMP-2 in human vitreous. (A) Vitreous samples (15 μl) were resolved on 10% (wt/vol) SDS-polyacrylamide gels containing 0.5 mg/ml gelatin as substrate. Subsequently, the gels were incubated in the absence (left) or presence (middle) of 10 mM EDTA. The vitreous samples were also resolved on 10%(vol/vol) SDS-polyacrylamide gels in the absence of gelatin (right) stained with Coomassie blue. The positions to which MMP activities in a characterized fibroblast conditioned medium standard migrated within the same gels are indicated. Each panel shows five unrelated vitreous samples chosen at random. (B) A proMMP-2 standard (fibroblast-conditioned medium) was resolved on 10% (wt/vol) SDS-polyacrylamide gels containing 0.5 mg/ml gelatin. The area (absorbance units) of substrate cleared by the 72-kDa enzyme was measured by scanning densitometry. The range of proMMP-2 activity that gave a linear densitometric response (r2 = 0.977) was established (inset). Vitreous samples were diluted to contain proMMP-2 activities within this linear densitometric range. Values obtained were corrected for dilution and expressed as a percentage of the activity of an internal standard included on each gel. The data represent individual values obtained for donors less than (n = 11) and more than (n = 22) 50 years of age. The bars represent the mean of each group. Higher activity of proMMP-2 (percentage of internal standard) was observed in the group aged more than 50 years (72.0% ± 6.0%) compared with the group aged less than 50 years (61.8% ± 7.3%), but this was not significant (P = 0.392).
Figure 2.
 
Analysis of serine proteases activity in human vitreous using casein substrate zymography. (A) The figure shows unrelated, random samples incubated in the absence (left) or presence (right) of serine protease inhibitors (1 mM PMSF and 1μ g/ml soybean trypsin inhibitor). Three bands of proteolytic activity of approximately 70 and 80 kDa and more than 120 kDa were observed that were absent in the presence of serine protease inhibitors. (B) Human plasmin standards (2.125, 1.06, 0.53, 0.27, and 0.135 ng; lanes 1 through 5, respectively) and vitreous samples (lanes 7 through 15) were analyzed by Western blot analysis. Plasmin(ogen) at a molecular weight of approximately 80 kDa was detected in the vitreous. Higher molecular weight bands of 120 and 180 kDa were due to nonspecific binding of the secondary antibody alone. (C) The plasmin standards (2.125, 1.06, 0.53, 0.27, and 0.135 ng) detected by Western blot analysis and enhanced chemiluminescence were quantified by scanning densitometry to obtain a linear relationship (r2 = 0.990; inset). Vitreous samples from donors aged less than (n = 13) and more than (n = 20) 50 years, were analyzed. The data plotted represent values obtained for individual samples after correction for dilution. Bars indicate the mean of each group. Plasmin(ogen) concentrations from the older age group (3.12 ± 0.42 ng) were calculated to be significantly higher (P = 0.001) than concentrations in the younger age group (1.179 ± 0.13 ng).
Figure 2.
 
Analysis of serine proteases activity in human vitreous using casein substrate zymography. (A) The figure shows unrelated, random samples incubated in the absence (left) or presence (right) of serine protease inhibitors (1 mM PMSF and 1μ g/ml soybean trypsin inhibitor). Three bands of proteolytic activity of approximately 70 and 80 kDa and more than 120 kDa were observed that were absent in the presence of serine protease inhibitors. (B) Human plasmin standards (2.125, 1.06, 0.53, 0.27, and 0.135 ng; lanes 1 through 5, respectively) and vitreous samples (lanes 7 through 15) were analyzed by Western blot analysis. Plasmin(ogen) at a molecular weight of approximately 80 kDa was detected in the vitreous. Higher molecular weight bands of 120 and 180 kDa were due to nonspecific binding of the secondary antibody alone. (C) The plasmin standards (2.125, 1.06, 0.53, 0.27, and 0.135 ng) detected by Western blot analysis and enhanced chemiluminescence were quantified by scanning densitometry to obtain a linear relationship (r2 = 0.990; inset). Vitreous samples from donors aged less than (n = 13) and more than (n = 20) 50 years, were analyzed. The data plotted represent values obtained for individual samples after correction for dilution. Bars indicate the mean of each group. Plasmin(ogen) concentrations from the older age group (3.12 ± 0.42 ng) were calculated to be significantly higher (P = 0.001) than concentrations in the younger age group (1.179 ± 0.13 ng).
Figure 3.
 
Correlation between vitreous plasmin concentration, proMMP-2 activity, and age. Vitreous proMMP-2 activity and age of donors (A) were not significantly correlated (Spearman’s coefficient = 0.09; P = 0.619). A significant correlation (Spearman’s coefficient = 0.449; P = 0.008) was observed between plasmin protein concentration in vitreous and age of donor (B). A significant correlation (Spearman’s coefficient = 0.681; P < 0.001) was also observed between vitreous proMMP-2 activity and plasmin concentration, showing an association between elevated levels of both enzymes (C).
Figure 3.
 
Correlation between vitreous plasmin concentration, proMMP-2 activity, and age. Vitreous proMMP-2 activity and age of donors (A) were not significantly correlated (Spearman’s coefficient = 0.09; P = 0.619). A significant correlation (Spearman’s coefficient = 0.449; P = 0.008) was observed between plasmin protein concentration in vitreous and age of donor (B). A significant correlation (Spearman’s coefficient = 0.681; P < 0.001) was also observed between vitreous proMMP-2 activity and plasmin concentration, showing an association between elevated levels of both enzymes (C).
The authors thank the Bristol Eye Bank for providing samples and Emma Ovenstone for technical assistance. 
Bishop P. The biochemical structure of mammalian vitreous. Eye. 1996;10:664–670. [CrossRef] [PubMed]
Balazs EA, Denlinger JL. Aging changes in the vitreous. Sekular R Kline D Dismukes N eds. Aging and Human Visual Function. 1982;45–57. Alan R Liss New York.
Sebag J, Balazs EA. Morphology and ultrastructure of human vitreous fibres. Invest Ophthalmol Vis Sci. 1989;30:1867–1871. [PubMed]
Sebag J. Age-related differences in the human vitreoretinal interface. Arch Ophthalmol. 1991;109:966–971. [CrossRef] [PubMed]
Murphy G, Reynolds JJ. Extracellular matrix degradation. Royce PM Steinmann B eds. Connective Tissue and Its Heritable Disorders. 1993;287–316. Wiley-Liss New York.
Brown DJ, Hamdi H, Bahri S, Kenney MC. Characterisation of an endogenous metalloproteinase in human vitreous. Curr Eye Res. 1994;13:639–647. [CrossRef] [PubMed]
Plantner JJ, Smine A, Quinn TA. Matrix metalloproteinase inhibitors in human interphotoreceptor matrix and vitreous. Curr Eye Res. 1998;17:132–140. [CrossRef] [PubMed]
Brown DJ, Bishop P, Hamdi H, Kenney MC. Cleavage of structural components of mammalian vitreous by endogenous matrix metalloproteinase-2. Curr Eye Res. 1996;15:439–445. [CrossRef] [PubMed]
Balazs EA, Delinger JL. The Vitreous. Davson H eds. The Eye. 1978;533–587. Academic Press New York.
Baramova EN, Bajou K, Remacle A, et al. Involvement of the PA/plasmin system in the processing of pro-MMP-9 and in the second step of pro-MMP-2 activation. FEBS Lett. 1997;405:157–162. [CrossRef] [PubMed]
Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970;277:680–685.
Gilbert SJ, Wotton PR, Tarlton JF, Duance VC, Bailey AJ. Increased expression of promatrix metalloproteinase-9 and neutrophil elastase in canine dilated cardiomyopathy. Cardiovasc Res. 1997;34:377–383. [CrossRef] [PubMed]
Towbin H, Stachelin T, Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA. 1979;76:4350–4354. [CrossRef] [PubMed]
DelaPaz MA, Itoh Y, Toth CA, Nagase H. Matrix metalloproteinases and their inhibitors in human vitreous. Invest Ophthalmol Vis Sci. 1998;39:1256–1260. [PubMed]
Kon CH, Occleston NL, Chateris D, Daniels J, Aylward GW, Khaw PT. A prospective study of matrix metalloproteinases in proliferative vitreoretinopathy. Invest Ophthalmol Vis Sci. 1998;39:1524–1529. [PubMed]
Guo L, Hussain G, Limb A, Marshall J. Age-dependent variation in metalloproteinase activity of isolated human BruchÆs membrane and choroid. Invest Ophthalmol Vis Sci. 1999;40:2676–2682. [PubMed]
Basbaum CB, Werb Z. Focalized proteolysis: spatial and temporal regulation of extracellular matrix degradation at the cell surface. Curr Opin Cell Biol. 1996;8:731–738. [CrossRef] [PubMed]
Figure 1.
 
Identification and quantitative analysis of proMMP-2 in human vitreous. (A) Vitreous samples (15 μl) were resolved on 10% (wt/vol) SDS-polyacrylamide gels containing 0.5 mg/ml gelatin as substrate. Subsequently, the gels were incubated in the absence (left) or presence (middle) of 10 mM EDTA. The vitreous samples were also resolved on 10%(vol/vol) SDS-polyacrylamide gels in the absence of gelatin (right) stained with Coomassie blue. The positions to which MMP activities in a characterized fibroblast conditioned medium standard migrated within the same gels are indicated. Each panel shows five unrelated vitreous samples chosen at random. (B) A proMMP-2 standard (fibroblast-conditioned medium) was resolved on 10% (wt/vol) SDS-polyacrylamide gels containing 0.5 mg/ml gelatin. The area (absorbance units) of substrate cleared by the 72-kDa enzyme was measured by scanning densitometry. The range of proMMP-2 activity that gave a linear densitometric response (r2 = 0.977) was established (inset). Vitreous samples were diluted to contain proMMP-2 activities within this linear densitometric range. Values obtained were corrected for dilution and expressed as a percentage of the activity of an internal standard included on each gel. The data represent individual values obtained for donors less than (n = 11) and more than (n = 22) 50 years of age. The bars represent the mean of each group. Higher activity of proMMP-2 (percentage of internal standard) was observed in the group aged more than 50 years (72.0% ± 6.0%) compared with the group aged less than 50 years (61.8% ± 7.3%), but this was not significant (P = 0.392).
Figure 1.
 
Identification and quantitative analysis of proMMP-2 in human vitreous. (A) Vitreous samples (15 μl) were resolved on 10% (wt/vol) SDS-polyacrylamide gels containing 0.5 mg/ml gelatin as substrate. Subsequently, the gels were incubated in the absence (left) or presence (middle) of 10 mM EDTA. The vitreous samples were also resolved on 10%(vol/vol) SDS-polyacrylamide gels in the absence of gelatin (right) stained with Coomassie blue. The positions to which MMP activities in a characterized fibroblast conditioned medium standard migrated within the same gels are indicated. Each panel shows five unrelated vitreous samples chosen at random. (B) A proMMP-2 standard (fibroblast-conditioned medium) was resolved on 10% (wt/vol) SDS-polyacrylamide gels containing 0.5 mg/ml gelatin. The area (absorbance units) of substrate cleared by the 72-kDa enzyme was measured by scanning densitometry. The range of proMMP-2 activity that gave a linear densitometric response (r2 = 0.977) was established (inset). Vitreous samples were diluted to contain proMMP-2 activities within this linear densitometric range. Values obtained were corrected for dilution and expressed as a percentage of the activity of an internal standard included on each gel. The data represent individual values obtained for donors less than (n = 11) and more than (n = 22) 50 years of age. The bars represent the mean of each group. Higher activity of proMMP-2 (percentage of internal standard) was observed in the group aged more than 50 years (72.0% ± 6.0%) compared with the group aged less than 50 years (61.8% ± 7.3%), but this was not significant (P = 0.392).
Figure 2.
 
Analysis of serine proteases activity in human vitreous using casein substrate zymography. (A) The figure shows unrelated, random samples incubated in the absence (left) or presence (right) of serine protease inhibitors (1 mM PMSF and 1μ g/ml soybean trypsin inhibitor). Three bands of proteolytic activity of approximately 70 and 80 kDa and more than 120 kDa were observed that were absent in the presence of serine protease inhibitors. (B) Human plasmin standards (2.125, 1.06, 0.53, 0.27, and 0.135 ng; lanes 1 through 5, respectively) and vitreous samples (lanes 7 through 15) were analyzed by Western blot analysis. Plasmin(ogen) at a molecular weight of approximately 80 kDa was detected in the vitreous. Higher molecular weight bands of 120 and 180 kDa were due to nonspecific binding of the secondary antibody alone. (C) The plasmin standards (2.125, 1.06, 0.53, 0.27, and 0.135 ng) detected by Western blot analysis and enhanced chemiluminescence were quantified by scanning densitometry to obtain a linear relationship (r2 = 0.990; inset). Vitreous samples from donors aged less than (n = 13) and more than (n = 20) 50 years, were analyzed. The data plotted represent values obtained for individual samples after correction for dilution. Bars indicate the mean of each group. Plasmin(ogen) concentrations from the older age group (3.12 ± 0.42 ng) were calculated to be significantly higher (P = 0.001) than concentrations in the younger age group (1.179 ± 0.13 ng).
Figure 2.
 
Analysis of serine proteases activity in human vitreous using casein substrate zymography. (A) The figure shows unrelated, random samples incubated in the absence (left) or presence (right) of serine protease inhibitors (1 mM PMSF and 1μ g/ml soybean trypsin inhibitor). Three bands of proteolytic activity of approximately 70 and 80 kDa and more than 120 kDa were observed that were absent in the presence of serine protease inhibitors. (B) Human plasmin standards (2.125, 1.06, 0.53, 0.27, and 0.135 ng; lanes 1 through 5, respectively) and vitreous samples (lanes 7 through 15) were analyzed by Western blot analysis. Plasmin(ogen) at a molecular weight of approximately 80 kDa was detected in the vitreous. Higher molecular weight bands of 120 and 180 kDa were due to nonspecific binding of the secondary antibody alone. (C) The plasmin standards (2.125, 1.06, 0.53, 0.27, and 0.135 ng) detected by Western blot analysis and enhanced chemiluminescence were quantified by scanning densitometry to obtain a linear relationship (r2 = 0.990; inset). Vitreous samples from donors aged less than (n = 13) and more than (n = 20) 50 years, were analyzed. The data plotted represent values obtained for individual samples after correction for dilution. Bars indicate the mean of each group. Plasmin(ogen) concentrations from the older age group (3.12 ± 0.42 ng) were calculated to be significantly higher (P = 0.001) than concentrations in the younger age group (1.179 ± 0.13 ng).
Figure 3.
 
Correlation between vitreous plasmin concentration, proMMP-2 activity, and age. Vitreous proMMP-2 activity and age of donors (A) were not significantly correlated (Spearman’s coefficient = 0.09; P = 0.619). A significant correlation (Spearman’s coefficient = 0.449; P = 0.008) was observed between plasmin protein concentration in vitreous and age of donor (B). A significant correlation (Spearman’s coefficient = 0.681; P < 0.001) was also observed between vitreous proMMP-2 activity and plasmin concentration, showing an association between elevated levels of both enzymes (C).
Figure 3.
 
Correlation between vitreous plasmin concentration, proMMP-2 activity, and age. Vitreous proMMP-2 activity and age of donors (A) were not significantly correlated (Spearman’s coefficient = 0.09; P = 0.619). A significant correlation (Spearman’s coefficient = 0.449; P = 0.008) was observed between plasmin protein concentration in vitreous and age of donor (B). A significant correlation (Spearman’s coefficient = 0.681; P < 0.001) was also observed between vitreous proMMP-2 activity and plasmin concentration, showing an association between elevated levels of both enzymes (C).
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