Proteoglycan Composition in the Human Sclera During Growth and Aging

Abstract

purpose. Scleral proteoglycans were characterized from human donor eyes aged 2 months to 94 years to identify age-related changes in the synthesis and/or accumulation of these extracellular matrix components.

methods. Newly synthesized proteoglycans (previously radiolabeled with ³⁵SO₄) and total accumulated scleral proteoglycans were extracted with 4 M guanidine hydrochloride and separated by molecular sieve chromatography on a Sepharose CL-4B column. The elution positions of newly synthesized and total accumulated proteoglycans were determined by assaying each fraction for radioactivity and glycosaminoglycans, respectively. Regression analyses were performed on the three major proteoglycan peaks to identify age-related changes in scleral proteoglycan composition. Scleral proteoglycans were further purified by anion-exchange chromatography and characterized by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and Western blot analyses.

results. Human scleral proteoglycans were apparent as three major peaks after chromatography on Sepharose CL-4B. The two faster eluting peaks contained alternative forms of the cartilage proteoglycan, aggrecan, whereas the third peak contained the small proteoglycans biglycan and decorin. The relative percentage of newly synthesized and total accumulated aggrecan increased approximately two- to sixfold from infancy to 94 years. In contrast, the relative percentage of newly synthesized and total accumulated biglycan and decorin decreased by approximately 25%. Chromatography and Western blot results indicated that the absolute amounts of all three proteoglycans significantly increased in concentration within the sclera from birth to the fourth decade. Beyond the fourth decade, decorin and biglycan decreased in all scleral regions and were present in lowest concentrations by the ninth decade. In contrast, aggrecan, which was present in highest concentration in the posterior sclera, was not significantly reduced with increasing age.

conclusions. The age-related changes in scleral proteoglycan composition observed in the present study are likely to contribute to the regional alterations in biomechanical properties of the sclera associated with growth and aging.

The sclera is a specialized connective tissue that provides the structural framework that defines the shape and axial length of the eye. The scleral extracellular matrix has been shown to contain collagens type I, III, ^{1 2 3V VI, 4} and VIII ⁵ ; elastic fibers ⁶ ; and the proteoglycans aggrecan, biglycan, and decorin. ⁷ It is accepted that the biomechanical properties of the sclera depend largely on the interactions of these macromolecular components. 

Recent studies indicate that the synthesis and turnover of scleral proteoglycans is a dynamic process that is dramatically influenced by the visual environment. ^{8 9 10 11 12} Changes in proteoglycan synthesis in the posterior sclera are closely correlated with changes in ocular size and refraction, suggesting a close relationship between scleral proteoglycan composition and the growth state of the sclera. ^{13 14} Aggrecan, a proteoglycan typically found in cartilage, is composed of a large (∼220 kDa) core protein and functions to provide tissue with resilience due to the water-binding capacity of its chondroitin and keratan sulfate glycosaminoglycan side chains. ¹⁵ Biglycan and decorin, relatively small chondroitin-dermatan sulfate proteoglycans, are present in many connective tissues but differ in distribution and function. Decorin is present in close association with collagen fibrils of many if not all connective tissues, where it regulates collagen fibril formation ¹⁶ and organization in the extracellular matrix. ¹⁷ Decorin has also been shown to suppress cell growth by upregulating the cell cycle inhibitory protein p21, ¹⁸ as well as by binding to transforming growth factor (TGF)-β, thereby neutralizing its growth-promoting activity in Chinese hamster ovary cells. ¹⁹ Biglycan has been found on the surfaces of differentiating cells and is occasionally present in tissue locations in which decorin is completely absent. ²⁰ Although no exact role for biglycan has been confirmed, the core protein of biglycan has been shown to bind TGF-β ²¹ and to collagen type I, ²² suggesting some functional similarities to decorin. 

As with most tissues, the sclera exhibits regional variation in structure and function. The human sclera is thickest at the posterior pole and thinnest at the equator; it thickens at the corneal limbus. ²³ The posterior sclera contains the scleral canal and the lamina cribrosa for passage of the optic nerve and contains the sites of perforation by the long and short posterior ciliary arteries, the short ciliary veins, and the vortex veins. ⁴ The anterior sclera is adjacent to the cornea at the corneal limbus and demonstrates a significantly higher modulus of elasticity than the posterior sclera, resulting in significantly greater stiffness in anterior sclera. ²⁴ Regional analysis of scleral glycosaminoglycans indicates that the sclera around the optic nerve is the area richest in dermatan sulfate, the sclera around the equator is the richest in hyaluronic acid, and the sclera around the fovea is richest in chondroitin sulfate. ²⁵  

With increasing age, the sclera undergoes a progressive degeneration of collagen and elastic fibers, ²⁶ a loss of mucopolysaccharides (glycosaminoglycans), scleral dehydration, ²⁷ and an accumulation of lipids and calcium salts. ^{27 28} These changes are associated with increases in tissue density, scleral thinning, yellowing, and decreases in scleral elasticity. ²⁹ Often superimposed on the aging process are a variety of disorders associated with alterations in scleral structure, including hyperopia, myopia, and age-related macular degeneration. ³⁰  

Because the sclera undergoes significant structural and functional changes throughout life, it is likely that the composition of scleral proteoglycans is modified during the course of development, maturation, and aging, and is responsible for some of the age-related changes previously reported to occur in the human sclera. It was therefore the purpose of the present study to compare scleral proteoglycan synthesis and accumulation profiles from donors of a variety of ages to identify age-related changes in proteoglycan composition. The identification of age-related changes in proteoglycan synthesis and accumulation will not only help to elucidate the mechanisms leading to structural changes in the aging sclera, but will also identify changes in proteoglycan metabolism important during the growth and development of the human sclera. 

Materials and Methods

Extraction of Proteoglycans

Sclera from human donor eyes, aged 2 months to 94 years, were received within 48 hours after death from the National Disease Research Interchange. Human tissue was handled according to the tenets of the Declaration of Helsinki, and the research was approved by the University of North Dakota’s institutional review board. On receipt, sclera were immediately frozen at −80°C or placed in organ culture for radiolabeling (described later). For biosynthesis experiments, one or both sclera from each donor was radiolabeled and chromatographed separately as described later in the article. To extract proteoglycans, adherent muscle, fat, lamina cribrosa and optic nerve head were removed from the sclera and the entire sclera (anterior, equatorial, and posterior regions) was minced into small (<2 mm³) pieces with a razor blade. The minced tissue was extracted in 4 M guanidine-HCl containing 0.01 M sodium acetate, 0.01 M sodium EDTA, 0.005 M benzamidine-HCl, and 0.1 Mε -amino-n-caproic acid at 4°C as described previously. ⁷  

Organ Culture and Radiolabeling

Sclera used for proteoglycan biosynthesis experiments were cleaned of adherent adnexa and cut into approximately 11-mm-diameter buttons with the aid of a surgical trephine. Each button was radiolabeled in organ culture with 0.5 ml Dulbecco’s modified Eagle’s medium containing 15% fetal bovine serum, streptomycin (10 μg/ml), penicillin G (100 U/ml), amphotericin B (0.25 μg/ml), and ³⁵SO₄ (250 μCi/ml) for 24 hours, to label proteoglycans. After they were radiolabeled, the scleral punches were minced into small pieces and extracted with 4 M guanidine-HCl, as described earlier. The radiolabeled scleral extracts were dialyzed exhaustively in 0.01 M Na₂SO₄ followed by dialysis in distilled water and lyophilized. 

Chromatography

Lyophilized scleral extracts that were not radiolabeled or were radiolabeled with ³⁵SO₄ were reconstituted into column buffer (4 M guanidine-HCl, containing 0.02 M Tris [pH 6.8] and 0.1% CHAPS) and applied to a Sepharose CL-4B column (100 × 1.6 cm) and eluted with the same buffer at a flow rate of 0.2 ml/min. ³¹ These column conditions were optimized to resolve the fastest migrating proteoglycans that eluted near the void volume. An aliquot from each fraction was measured for glycosaminoglycan content using the dimethylmethylene blue (DMMB) assay ³² or for the presence of radioactivity by liquid scintillation counting, and tubes containing the peak fractions were pooled, dialyzed, and lyophilized. Measurements of the areas under each peak of each chromatographic profile were used to calculate the relative and absolute amounts of proteoglycans present in scleral extracts. 

Pooled samples from CL-4B chromatography were reconstituted in 6 M urea containing 0.05 M Tris (pH 6.8), 0.1% CHAPS, and 0.15 M NaCl and applied to diethylaminoethyl (DEAE) Sepharose step columns (3.7 × 1.5 cm) equilibrated in the same solvent to separate glycoproteins from proteoglycans. ⁷  

Electrophoretic Techniques

Proteoglycans separated by Sepharose CL-4B chromatography and isolated by DEAE chromatography were characterized by Western blot analyses using antisera generated against human aggrecan (generously provided by Robin Poole, Joint Diseases Laboratory, McGill University, Montreal, Quebec, Canada), antisera against a peptide of human biglycan (generously supplied by Peter Roughly, Joint Diseases Laboratory, McGill University), antisera against a synthetic peptide containing the exon 5 sequence of human decorin (generously supplied by David McQuillan, Center for Extracellular Matrix Biology, Texas A&M University, Houston), and antibodies against a synthetic peptide in the G1 region of aggrecan (generously supplied by John Sandy, Biochemistry Section, Shriner’s Hospital for Children, Tampa, FL). Proteoglycan fractions were digested with chondroitinase ABC, endo-β-galactosidase, and/or keratanase II (Seikagaku America, Ijamsville, MD), ⁷ electrophoresed on 5% or 10% sodium dodecyl sulfate (SDS)–polyacrylamide gels, transferred to nitrocellulose, reacted with antibodies to the proteoglycan core proteins, and detected with chemiluminescent substrate (Western Star; Tropix, Bedford, MA). Digitized images of the Western blot analysis were obtained using a flatbed scanner, and densitometry was performed by computer (Image ver. 1.61; National Institutes of Health, Bethesda, MD). ³⁵SO₄-labeled proteoglycan core proteins were visualized after digestion with chondroitinase ABC and endo-β-galactosidase, separation on 5% SDS-polyacrylamide gels, and fluorography to detect the presence of ³⁵SO₄ on the residual glycosaminoglycan stubs that remained attached to the proteoglycan core proteins. 

Extraction of Proteoglycans from Different Scleral Regions

Human sclera were cleaned of extraocular muscle, vitreous, retina, and choroid and dissected into anterior, equatorial, and posterior regions. The anterior region was defined as a circumferential belt approximately 5 mm wide, immediately adjacent to the cornea–scleral junction. The equatorial region was defined as a circumferential belt approximately 5 mm wide located midway between the cornea–scleral junction and the posterior pole. The posterior scleral region was located at the posterior pole of the eye, excluding the lamina cribrosa and optic nerve head. A sample of each scleral region (0.1 g) was extracted in 1 ml GuHCl, as described earlier. Aliquots of each extract were measured directly for glycosaminoglycan content by using dimethylmethylene blue ³² or dialyzed exhaustively against distilled deionized water and lyophilized. Lyophilized samples were digested with chondroitinase ABC, endo-β-galactosidase, and/or keratanase II and subjected to Western blot analyses. 

Statistical Analyses

Regression lines were constructed for the data by using a simple linear model (Statview Student; Abacus Concepts, Berkeley, CA). Comparisons between groups were made using analysis of variance. 

Results

Separation of Proteoglycans Extracted from Sclera

The elution positions of newly synthesized and accumulated scleral proteoglycans could be identified after chromatography on Sepharose CL-4B by measuring the amount of radioactivity in fractions from sclera that had been previously radiolabeled with ³⁵SO₄ or by measuring the concentration of total accumulated sulfated glycosaminoglycans in each fraction, respectively. In most chromatographic profiles, variable amounts of three peaks were identified; peak A, a small peak representing the largest scleral proteoglycan; peak B, a slightly smaller sized population of proteoglycans, present in an amount similar to that in peak A; and peak C, the most abundant proteoglycan species, representing the smallest scleral proteoglycans (Fig. 1) . 

When expressed relative to total newly synthesized proteoglycans, the sclera of young donors primarily synthesized small proteoglycans that eluted last from the column (peak C). With advancing age, a relative increase was seen in the amount of larger proteoglycans that eluted earlier from the column (+172% and +618% for peaks A and B, respectively). A similar age-related increase was observed in the amount of total accumulated proteoglycans contained in peak B, when expressed relative to total glycosaminoglycan content (+100.18%). The relative synthesis and accumulation of peak C proteoglycans decreased by approximately 25% in samples from birth to 94 years of age. Linear regression analyses were used to measure the correlation of the relative percentages of peaks A, B, and C with advancing age (Fig. 2) . Newly synthesized peak A and B proteoglycans were positively correlated with age (r = 0.651, P = 0.012; r = 0.758, P = 0.002, respectively), although the percentage of newly synthesized peak B proteoglycans appeared to hold fairly constant through age 68 and thereafter increased sharply through age 94. The relative accumulation of peak A proteoglycans was not significantly correlated with age (r = 0.449, P = 0.093), whereas total accumulated peak B proteoglycans increased significantly with age (r = 0.691, P = 0.004). In contrast, newly synthesized and total accumulated peak C proteoglycans were negatively correlated with age (r = 0.816, P = 0.0004; r = 0.774, P = 0.0007, respectively). 

As a proportion of wet weight of tissue, peaks A and B were positively correlated with age only up to 53 years (r = 0.747, P = 0.033; r = 0.867, P = 0.004, respectively), after which, no significant correlation could be detected. The amount of peak C, however, showed an increase from infancy to age 37 (r = 0.868, P = 0.011) and then decreased from age 37 to 94 (r = 0.786, P = 0.012). 

Characterization of Proteoglycan Core Proteins

Western blot analyses indicated that peak A was composed of aggrecan, consisting of a large core protein (∼300 kDa) and attached chondroitin sulfate and keratan sulfate glycosaminoglycan side chains. When present, the core protein of peak A proteoglycans was similar in size (∼300 kDa) for sclera of all ages, with an additional smaller core (∼290 kDa) also present in the 83-year-old sclera (Fig. 3) . Western blot analyses of peak B proteoglycans indicated that peak B was composed of two proteoglycan species that reacted with the anti-aggrecan antisera; one proteoglycan had a core protein similar in size to that of peak A proteoglycans but contained only chondroitin sulfate side chains. The second proteoglycan contained a slightly smaller core protein (∼270 kDa) but contained both chondroitin and keratan sulfate side chains (Fig. 4) . Analysis of ³⁵SO₄-labeled proteoglycans indicated that two large core proteins of slightly different molecular weight were synthesized within the 24-hour labeling period (Fig. 5) , suggesting that either a second product of different size is synthesized by the scleral fibroblasts, or alternatively, that intracellular or extracellular processing pathways rapidly modulate some of the newly synthesized proteoglycan molecules. ³³  

Peak C proteoglycans from sclera of different aged donors were subjected to Western blot analyses using antibodies specific to the core proteins of biglycan and decorin. Anti-biglycan antisera detected the 45-kDa biglycan core protein in chondroitinase ABC digests of peak C (Fig. 6A ). No bands could be detected in the undigested peak C samples, because the proteoglycan form of biglycan is nonreactive with this antisera in Western blot analyses. Peak C proteoglycans were also subjected to Western blot analyses with antibodies against a synthetic peptide containing the exon 5 sequence of human decorin. Chondroitinase ABC digestion of peak C produced a doublet of core proteins at 43 and 47 kDa (Fig. 6B) in all the scleral extracts examined. 

Regional Analysis of Total Glycosaminoglycans

Total sulfated glycosaminoglycans were measured in anterior, equatorial, and posterior scleral regions from donors aged 10 months to 93 years (Fig. 7) . Total sulfated glycosaminoglycans present in the anterior sclera were found to decrease as a function of age (r = 0.655, P = 0.006). In equatorial and posterior sclera, a subset of older eyes retained high levels of glycosaminoglycans resulting in considerable variation in the glycosaminoglycan content within equatorial and posterior sclera of donors aged 80 to 93 years. Therefore, no significant correlation could be detected between age and total sulfated glycosaminoglycans in these regions. A decrease in scleral hydration has been shown to occur in the aging human sclera and is associated with the loss of dermatan sulfate. ³⁴ Because highly sulfated glycosaminoglycans contribute substantially to the degree of hydration (and therefore the weight) of the sclera, our estimates of total sulfated glycosaminoglycan concentration expressed relative to wet weight may underestimate the actual loss of glycosaminoglycans in the aging sclera. 

Regional Analysis of Proteoglycan Core Proteins

Proteoglycans were extracted from equal wet weights of anterior, equatorial, and posterior sclera and subjected to Western blot analyses using anti-aggrecan, anti-biglycan, and anti-decorin antisera. After digestion with chondroitinase ABC and keratanase, three major core proteins, migrating at approximately 300, 270, and 190 kDa, were detected, which reacted with anti-aggrecan antibodies (Figs. 8A , 8B ). The approximate 190-kDa band was not previously detected in the DEAE-purified column fractions but was confirmed to be a G1-containing fragment of the aggrecan core (data not shown). We speculate that the 190-kDa band was a fragment of the aggrecan core protein that may have been lost during DEAE purification or did not resolve into a visible peak after chromatography on Sepharose CL-4B because of low levels of sulfation. Aggrecan was undetectable in all scleral regions of the 6-year-old donor but appeared to increase in equatorial and anterior regions up to ages 36 and 67, respectively, and then decrease thereafter. In most donor eyes, the posterior sclera contained the highest concentration of aggrecan of the three scleral regions, and although in varying amounts, aggrecan was retained in the posterior sclera throughout the aging process. Comparison of the percentage of aggrecan present in the three scleral regions indicated that significantly higher percentage of aggrecan accumulated in the posterior sclera (68.41% ± 7.35% [SEM]) compared with the anterior sclera (17.07% ± 6.80%, P = 0.0003) or equatorial sclera (14.49% ± 4.98%, P = 0.001; Fig. 8C ). 

The amount of biglycan in anterior and equatorial scleral regions increased up to age 35 and then appeared to decrease to lowest concentrations by age 89 (Figs. 9A , 9B ). The amount of biglycan present in the posterior human sclera appeared to remain fairly constant from 19 to 68 years and then demonstrated a decrease by age 89 years. A small amount of biglycan core could be visualized in the undigested (U) lanes from the 35-year-old anterior and equatorial samples, indicating that in this donor, a portion of biglycan core was unglycosylated. When the same Western blot was stripped and reprobed with anti-decorin antibodies, age-related changes were observed that were similar to those for biglycan, with the exception that decorin accumulated to a relatively greater extent in the 19-year-old anterior sclera and was reduced in the 35-year-old equatorial sclera (Figs. 10A , 10B ). Although biglycan and decorin could be detected in the sclera of the 10-week-old infant when the Western blot analyses were overexposed, undetectable levels were present, compared with levels seen in the other age groups. The Western blot data for biglycan and decorin support the results obtained from the analyses of the chromatographic profiles of peak C (expressed as micrograms glycosaminoglycan per gram scleral wet weight), which indicate that biglycan and decorin rapidly accumulate in the sclera in the first four decades of life and then progressively decrease in concentration in subsequent years. 

Discussion

The results of the present study show that the population of scleral proteoglycans changes during the normal growth and aging processes. The concentration of all three scleral proteoglycans increased in the sclera from infancy up to the fourth decade. Aging was associated with a relative increase in the synthesis and accumulation of large chondroitin-keratan sulfate proteoglycans, with core proteins of more than 200 kDa (peaks A and B) and a relative decrease in the synthesis and accumulation of the small chondroitin-dermatan sulfate proteoglycans, with core proteins of approximately 45 kDa (peak C). Western blot analyses of scleral proteoglycans indicated that the large chondroitin-keratan sulfate proteoglycans identified in peaks A and B after chromatography on Sepharose CL-4B are different populations of aggrecan; aggrecan in peak A contained the largest core protein (∼300 kDa), containing both keratan and chondroitin sulfate side chains. Aggrecan in peak B contained two populations: one contained the full-length core protein (∼300 kDa), but contained only chondroitin sulfate, and the other population contained both chondroitin and keratan sulfate but had a smaller core protein (∼270 kDa). Peak C proteoglycans made up most of the scleral proteoglycans (ranging from∼ 43% to 79% in old and young sclera, respectively) and were shown by Western blot analyses to include the small proteoglycans biglycan and decorin. 

The relative increase in aggrecan synthesis and accumulation observed with increasing age is due in part to age-related decreases in the absolute amounts of biglycan and decorin, observed to occur after age 37. Western blot and chromatographic results from two different series of eyes (ranging from infant to elderly) indicated that aggrecan, biglycan, and decorin increased in concentration within the sclera during the adolescent and early adult years, but only biglycan and decorin appeared to decline after the fourth decade and were present at lower concentrations in older sclera (>53 years). As a result, the small proteoglycans represented a higher proportion of total proteoglycans in young than in older donors. A remarkably similar pattern of age-related proteoglycan changes was obtained for human articular cartilage using a competitive radioimmunoassay for decorin (DSPG-2). ³⁵ The rapid accumulation of decorin and biglycan observed in the sclera in the first four decades of life and their subsequent decrease in older tissue suggest that the matrix functions of these proteoglycans are more important in the juvenile and adolescent growth periods of the human sclera. It can be inferred that defects in the scleral synthesis of decorin and biglycan would most likely become apparent in childhood and adolescence, because the disordered scleral matrix would result in abnormalities in scleral growth and overall eye length. 

Proteoglycan compositions were compared between three scleral regions from donors of a wide variety of ages. Total glycosaminoglycan content was shown to decrease significantly with age only in the anterior sclera when expressed relative to wet weight. Western blot analyses indicated that decorin and biglycan were decreased in all three scleral regions by 89 years of age, compared with that of the 19-, 37-, and 68-year-old scleras. In contrast, aggrecan accumulated in the posterior sclera to significantly higher levels than in the anterior or equatorial sclera and was retained in the posterior sclera throughout the aging process. Taken together, the results of the present study indicate that the reduction in glycosaminoglycans observed in the anterior sclera with increasing age results from reduced amounts of decorin and biglycan. Although age-related decreases in decorin and biglycan were observed in the posterior sclera as well, total glycosaminoglycan content was not significantly reduced in this region because of the retention of aggrecan and its associated glycosaminoglycans in the posterior sclera of aging eyes. 

The retention of aggrecan in the posterior sclera of aging eyes may be related to the observation that the posterior sclera is less rigid than the anterior sclera. In cartilage, aggrecan has been shown to bind large amounts of water and provide cartilage with properties of resiliency and the ability to withstand compressive forces. ¹⁵ If aggrecan has a similar function in the sclera, its presence may allow the posterior sclera to remain pliable, thereby sparing the circulation to the choroid and retina through the posterior ciliary blood vessels. Decreases in aggrecan concentration would significantly reduce the glycosaminoglycan concentration in the posterior sclera and may lead to increased scleral rigidity, which has been associated with hyperopia and high myopia ³⁰ and is a possible cause of age-related macular degeneration due to impaired choroidal perfusion. ³⁶  

The mechanisms responsible for the regional differences in proteoglycan composition and the age-related decreases in the synthesis and accumulation of decorin and biglycan after the fourth decade are unclear. The posterior pole is subjected to significantly higher degrees of mechanical stress due to the presence of the scleral canal and the insertions of the oblique muscles. ³⁷ We hypothesize that these stresses, together with those associated with intraocular pressure, may play a role in regulating the synthesis and accumulation of aggrecan observed in the sclera at the posterior pole. 

Several growth factors, such as TGF-β1 ^{38 39 40 41} and TGF-β2, ⁴² IL-1β, ³⁸ insulin-like growth factor-1, ^{42 43} retinoic acid, ⁴⁴ and dexamethasone ⁴⁵ have been shown to regulate proteoglycan synthesis under a variety of conditions by a variety of cells. However, the cellular responses to these growth factors vary with the cell type, the tissue of origin, and the anatomic site within the tissue. ⁴³ Considering the unusual phenotype of the human scleral fibroblast, ⁷ which exhibits characteristics of chondrocytes (i.e., aggrecan synthesis) as well as those of typical fibroblasts, additional experiments are needed to identify the mechanisms that regulate proteoglycan synthesis by these cells. 

 Supported by National Institutes of Health Grant EY09391 (JAR), The Macula Vision Research Foundation (JAR), and Howard Hughes Undergraduate Research Program Grant HHMI 71195-539401 (SP, RWS, BAM).

 Submitted for publication September 15, 1999; revised December 20, 1999; accepted January 11, 2000.

 Commercial relationships policy: N.

 

Figure 1.

 

Separation of newly synthesized (³⁵SO₄-labeled) and total accumulated scleral proteoglycans by molecular sieve chromatography. Proteoglycans were extracted from the sclera of donors aged 7 months to 94 years. (The elution profiles of ages 5 years, 37 years, 63 years, and 83 years are shown as examples.)

Figure 2.

 

Age-related changes in the proteoglycan content of human sclera. Left: percentage of total newly synthesized proteoglycans in peaks A, B, and C, relative to total proteoglycan content (n = 14). Middle: percentage of total accumulated proteoglycans in peaks A, B, and C, relative to total proteoglycan content (n = 15). Right: total accumulated proteoglycans in peaks A, B, and C, expressed as micrograms per gram wet weight sclera (n = 15).

Figure 3.

 

Western blot analyses of peak A proteoglycans from donors aged 5, 37, 63, and 83 years with anti-aggrecan. Peak A proteoglycans were further purified by DEAE chromatography, and a portion of the samples were treated with chondroitinase ABC, endo-β-galactosidase, and keratanase II before electrophoresis on 5% polyacrylamide gels (Digested). Enz, enzyme alone.

Figure 4.

 

Comparison of peak A proteoglycans with peak B proteoglycans by Western blot analysis with anti-aggrecan. Peak A and B proteoglycans, purified by Sepharose CL-4B and DEAE chromatography, were digested (+) with chondroitinase ABC (C’ase), endo-β-galactosidase (Endo-β), and/or keratanase II (K’ase II) before electrophoresis on 5% polyacrylamide gels.

Figure 5.

 

Determination of newly synthesized aggrecan core protein sizes from peak A and peak B proteoglycans. ³⁵SO₄-labeled proteoglycans, isolated from the leading edge of peak A and from the trailing edge of peak B were electrophoresed on 5% polyacrylamide gels, dried, and visualized by fluorography. Digested samples (D) were treated with chondroitinase ABC and endo-β-galactosidase before electrophoresis. U, undigested.

Figure 6.

 

Western blot analyses of peak C proteoglycans from donors aged 5, 37, 63, and 83 years with anti-biglycan (A) and anti-decorin (B) antisera. Peak C proteoglycans were further purified by DEAE chromatography, and a portion of the samples were treated with chondroitinase ABC before electrophoresis on 10% polyacrylamide gels (+ C’ase ABC). Enz, enzyme alone.

Figure 7.

 

Total sulfated glycosaminoglycan concentration in different regions of the sclera as a function of age. Proteoglycans were extracted from equal wet weights of anterior, equatorial, and posterior sclera with 4 M guanidine HCl, and total sulfated glycosaminoglycans were measured in the extracts using dimethylmethylene blue.

Figure 8.

 

Aggrecan accumulation in different scleral regions from donors of various ages detected by Western blot analysis. (A) 4 M guanidine HCl extracts from anterior (A), equatorial (E), and posterior (P) sclera were dialyzed, lyophilized, and digested with chondroitinase ABC, endo-β-galactosidase, and keratanase II before electrophoreses on 5% polyacrylamide gels. Several bands, migrating at approximately 300, 270, and 190 kDa, representing aggrecan core proteins, were detected on the immunoblots. (B) The 300-, 270-, and 190-kDa aggrecan core protein bands were quantified by densitometry and summed for each sample. (C) Total accumulated aggrecan was compared among the three scleral regions and expressed as a percentage of the total scleral aggrecan for all eyes aged 6 to 90 years.*** P ≤ 0.0003, for posterior sclera compared with either anterior sclera or equatorial sclera (analysis of variance; n = 8). Enz, enzyme alone.

Figure 9.

 

Biglycan accumulation in different scleral regions from donors of various ages detected by Western blot analysis. Guanidine HCl (4 M) extracts from anterior (Ant), equatorial (Eq), and posterior (Post) sclera were dialyzed and lyophilized, and an aliquot was digested with chondroitinase ABC before electrophoresis on 10% polyacrylamide gels. (A) The biglycan core protein could be visualized as a band migrating at approximately 45 kDa in the digested (D) samples. Enz, enzyme alone. (B) The 45-kDa biglycan core protein was quantified by densitometry as described in the Methods section.

Figure 10.

 

Decorin accumulation in anterior (Ant), equatorial (Eq) and posterior (Post) scleral regions from donors of various ages detected by Western blot analysis. The blot from Figure 9 was stripped and reprobed with anti-decorin antisera. (A) The decorin core protein could be visualized as a doublet migrating at approximately 43 to 48 kDa in the digested (D) samples. Enz, enzyme alone. (B) The 43- to 48-kDa decorin core protein doublet was quantified by densitometry as described in the Methods section.