Organization of ground substance proteoglycans in normal and osteoarthritic knee cartilage.
код для вставкиСкачать209 ORGANIZATION OF GROUND SUBSTANCE PROTEOGLYCANS IN NORMAL AND OSTEOARTHRITIC KNEE CARTILAGE KENNETH D. BRANDT and MARSHALL PALMOSKI A study of the organization of proteoglycans in articular cartilage indicates that nonaggregated proteoglycans existed in larger numbers in osteoarthritic than in normal cartilage and that proteoglycan aggregates in arthritic cartilage were smaller than normal. After dissociation from hyaluronic acid and tissue glycoproteins, no difference in hydrodynamic size of disaggregated proteoglycans was noted, but chondroitin sulfate chains of those from diseased cartilage were shorter than normal. The data suggest that there is a defect in proteoglycan aggregation in osteoarthritic cartilage which could be of pathogenetic significance. The mechanical properties (eg stiffness on compression) that permit articular cartilage to subserve its From the Arthritis and Connective Tissue Disease Section, Evans Department of Clinical Research, University Hospital, and the Thorndike Memorial Laboratory and Division of Medicine, Boston City Hospital, Boston, Massachusetts. Supported by grants from the USPHS National Institute of Arthritis and Metabolic Diseases (AM-17215. AM-04599. and TIAM-5285) and from the N I H General Clinical Research Centers Branch of the Division of Research Resources (RR-533). Kenneth D. Brandt, M.D.: Special Research Fellow, National Institutes of Health, and Associate Professor of Medicine, Boston University School of Medicine; Marshall Palmoski, Ph.D.: Thorndike Memorial Laboratory and Division of Medicine, Boston City Hospital, and Recipient of a Postdoctoral Fellowship from The Arthritis Foundation. Address reprint requests t o Kenneth D. Brandt, M.D., Indiana University Medical Center, 1100 West Michigan Street, Indianapolis, Indiana 46202. Submitted for publication March 29, 1975; accepted June 30, 1975. Arthritis and Rheumatism, Vol. 19, No. 2 (March-April 1976) weight-bearing function depend on the presence of proteoglycans (PGs) in the tissue ground substance ( I ) . In vitro some, but not all, cartilage PGs can be shown to aggregate after addition of hyaluronic acid (HA) (2), and it is probable that in vivo the PGs exist as very large aggregates (3) with many PG molecules noncovalently linked to HA (4). A tissue glycoprotein (GPL) (3) is also present in these complexes and appears to stabilize the PG-HA interaction (5). I n osteoarthritis (OA) the fundamental defect, although still unknown, seems to reside in the articular cartilage, where loss of ground substance occurs. Recent studies have shown that some PGs of OA cartilage are more readily extracted by neutral isoosmotic salt than are those of normal cartilage ( 6 ) , but whether this difference reflects an abnormality in PG aggregation in OA remains to be determined. Although few comparisons of intact PGs of normal and OA cartilage have been reported (7-9), their glycosaminoglycans (GAGs) have been compared more extensively. The diseased tissue may contain less chondroitin sulfate and keratan sulfate (7,lO-13) and shorter chains of chondroitin sulfate than normal (l0,14). GAGs do not exist free in vivo, however, but rather as constituents of PGs, so that analyses of total tissue GAGs reflect the composition of PGs that were not aggregated as well as of those that were. Taking into account the possibility that differences existed in the degree of their aggregation in the tissues, the present study compares PGs of normal and OA cartilage before and after density gradient centrifugation 210 in 4 M guanidinium chloride, a procedure k n o w n t o dissociate them f r o m complexes with H A and GPL (3, 15). In addition a subpopulation o f PGs extracted f r o m t h e same tissues merely by brief stirring in neutral isoosmotic sodium acetate, and presumably n o t aggregated in vivo, was also studied. T h e results indicate t h a t in OA some PGs are held i n t h e cartilage less firmly t h a n normal a n d t h a t t h e PG aggregates in OA cartilage tend t o b e smaller t h a n normal. T h e s e results may be o f pathogenetic significance. MATERIALS AND METHODS Tissues Knee joints from 6 steers 6-9 years of age were obtained at the abattoir and stored at -20°C. After thawing to 4°C the articular cartilage was removed from the distal femurs with a scalpel. Small samples were taken for histologic study and the cartilage from a ) normal and b) softened, fibrillated areas was pooled separately and then diced. Portions of approximately 150 mg were weighed and placed in acetone for 24 hours and then dried to constant weight in vacuo at 80°C. Samples of the acetone-dried cartilage were taken for determination of hydroxyproline, while the remainder was digested with pronase at 55°C for 24 hours in 0.1 M borate buffer, pH 8.2, containing 0.2 M CaCI,. For each gram of tissue, 10 mg of enzyme in 50 ml of buffer were employed. The small amount of undigested material remaining after incubation was removed by centrifugation, following which the G A G S in the supernatant were isolated by precipitation with 9-aminoacridine hydrochloride, as described below. The remainder of the pooled cartilage was divided into two portions: one was frozen in liquid nitrogen, pulverized in a steel die cooled in liquid nitrogen, and extracted with sodium acetate. The other, unpulverized portion was extracted directly with guanidinium chloride, as follows. Extraction and Isolation of Proteoglycans Sodium Acetate Extract. The pulverized cartilage was suspended (15 g/lOO ml) in cold 0.15 M sodium acetate, pH 6.8, and agitated for 7 minutes at the lowest speed of a Virtis homogenizer, following which the suspension was filtered through lint. The residue was washed with sodium acetate, the clear filtrate and washings were combined, and the PGs were isolated by precipitation with 9-aminoacridine hydrochloride (16) and converted to their sodium salts by ion exchange with Bio-Rad-AG-50 (Na+). The resin was removed by filtration, the filtrate was concentrated by rotary evaporation, and the PGs were further purified by a second precipitation with 9aminoacridine. After removal of the acridine as above, the PGs were precipitated with 80% (v/v) ethanol, washed with 80% (v/v) ethanol, absolute ethanol, and acetone, and dried in vacuo over P,O,. I n each case the amount of uronic acid remaining in the supernatant after precipitation with 9-aminoacridine was determined after the excess acridine had been removed with additional resin. BRANDT AND PALMOSKI Guanidinium Chloride Extract. A standard procedure (3) was used to prepare a ) aggregated PGs and b) purified disaggregated PGs, dissociated from HA and GPL, from the unpulverized cartilage: The tissue was stirred for 48 hours at 20°C in 4 M guanidinium chloride in 0.05 M sodium acetate, pH 5.8, after which the suspension was centrifuged and the supernatant removed by decantation. The cartilage residue was washed twice with fresh guanidinium chloride and supernatant and washings were combined and made 0.5 M in guanidinium chloride by dialysis, after which the retentate was removed and the sacs were rinsed with additional guanidinium. Cesium chloride was added to a density of 1.69 g/ml, and the solution was centrifuged at 4 ° C for 48 hours in the No. 40 angle rotor of a Beckman ultracentrifuge (average speed: 95,000 X g). Following centrifugation the gradient was fractionated into five equal parts and the fractions corresponding to the bottom two-fifths (density L 1.73 g/ml) were pooled. After exhaustive dialysis against distilled water the retentate was removed and the sacs were rinsed with water. Retentate and rinses were combined and lyophilized to yield a fraction containing the aggregated PGs. To obtain dissociated PGs, free of HA and GPL, portions of the lyophilized material were dissolved in 4 M guanidinium chloride, pH 5.8. The density was adjusted with cesium chloride to I .SO g/ml and the samples were centrifuged for 42 hours as described above. The bottom 2/5 of the gradient (density L 1.53 g/ml) was then recovered and, after dialysis as above, the PGs were lyophilized. Gel Chromatography. Columns of Sepharose 6B* (40 X 0.8 cm) and Sepharose 2B* (45 X 0.8 cm) were employed; their void volumes had been previously determined with Blue Dextran*. With both, samples of 1.5 mg were applied in 0.5 ml of 0.5 M sodium acetate, pH 6.5, and eluted with the acetate solution at a flow rate of 2 ml/hour. Fractions of 1 ml were collected and the uronic acid content of each fraction was determined. Average molecular size of chondroitin sulfate chains was assessed by chromatography on Sephadex G-200* (17). Approximately 10 mg of PGs were dissolved in 2 ml of 0.1 M sodium acetate buffer, pH 5.5, containing 2 mg EDTA and 0.6 mg cysteine hydrochloride, to which crude papain? was added. After digestion under toluene for 24 hours at 65"C, GAGS were precipitated with 80% (v/v) ethanol, washed with 80% (v/v) ethanol, absolute ethanol, and acetone, and dried in vacuo. Samples (approximately 3 mg) were applied to a column ( 1 15 X 0.8 cm) and eluted with 0.2 M .sodium acetate, pH 6.5. One-milliliter fractions were collected at a rate of 4 ml/hour and their uronic acid contents determined. Analytical Methods. Hexuronic acid, protein, hexose, and xylose were all determined as described previously (16). Hexosamine was determined by the method of Kraan and Muir (18), with glucosamine hydrochloride employed as standard. Samples were hydrolyzed for 3 hours in 8 M HCI under N2 at 95°C (19), following which the acid was rapidly removed by boiling under N,. Molar ratios of glucosamine:galactosamine were determined on a JEOL-SAH automated analyzer with samples hydrolyzed as they would be * Pharmacia Fine Chemicals, Piscataway, New Jersey. t Sigma Chemical Company, St. Louis, Missouri. G R O U N D SUBSTANCE PROTEOGLYCANS IN O A CARTILAGE 21 1 Table 1. Composition of Normal and Osteoarthritic Bovine Knee Cartilage ~ ~~ ~~ Composition (percent o f tissue dry weight) Tissue Dry Weight (percent of wet weight) Hydroxyproline Uronic Acid Hexosaniine Glucosamine: Galac(osaniine (molar ratio of total glycosaminoplycans) 24.5 28.0 7.9 8.3 2.67 2.3 I 2.38 2.10 I : 1.5 I : 1.3 Normal OA for total hexosamine. Amino acid analyses were performed on the same instrument with samples hydrolyzed in 6 M HCI under N, for 24 hours at 105"C, following which the acid was removed by boiling under N,. Hydroxyproline contents of acetone-dried cartilage were determined on the amino acid analyzer after hydrolysis of the tissue for 24 hours at 105°C in 6 M HCI and removal of the acid as above. Histochemistry. Samples of cartilage from each joint were stained with Safranin-0 and examined histologically. The severity of osteoarthritis was graded in each specimen according to the criteria of Mankin et a1 (20). RESULTS Cartilage that was grossly normal was also histologically normal and, after examination of Safranin-0 stained sections, was graded 0, whereas the grossly osteoarthritic cartilage ranged between Grades 5 a n d 8 (20). Whole normal a n d OA cartilage were similar with respect to dry weight per gram of fresh tissue (24.5% and 28.0% respectively) and hydroxyproline content (approximately 8% of dry weight). Consistent with the observed loss of Safranin-0 staining, the diseased cartilage contained less uronic acid a n d hexosamine than normal, as indicated by analysis of the G A G s isolated after pronase digestion of the tissue. T h e proportion of glucosamine relative to galactosamine in the total G A G s , however, was essentially the same (Table I ) . Uronic acid was not found in any of the supernatants after precipitation with 9-aminoacridine, a fact indicating that all t h e compounds containing uronic acid were precipitated. PGs were not quantitatively extracted by either of the procedures employed. Those in normal cartilage were less readily extracted by sodium acetate than those in OA cartilage, although there was no appreciable difference in yields of the guanidinium extracts, which contained much more of the tissue PGs (Table 2). Thus the sodium acetate extract of the OA cartilage contained seven times as much of the total tissue uronic acid (9%) as the sodium acetate extract of the normal cartilage (1.3%), whereas the guanidinium extracts of normal and O A cartilage contained 60% a n d 62% respectively of the tissue uronic acid (Table 2). Losses of uronic acid after dialysis of the guanidinium extracts of the normal a n d arthritic cartilage were minimal (5% a n d 6% respectively). Sixty-three percent of the uronic acid extracted from normal and 77% of that extracted from the diseased tissue were recovered Table 2 . Comparison of Proteoglycans Extracted from Normal and Osteoarthritic Bovine Knee Cartilage by 0.15 M Sodium Acetate, pH 6.8, and by 4 M Guanidinium Chloride. pH 5.8* Composition of Purified Proteoglycans* Percent Dry Weight Tissue Normal OA Norm al OA Extract Sodium acetate Sodium acetate Gu an id i n i urn chloride Guanidiniurn chloride Percent of Total Tissue Uronic Acid Uronic Acid Hexosamine Molar Ratio Protein Hexose Xylose Percent Glucosamine: Xylose: Retarded by Galactosaniine Uronic Acid S e p h a r o e 6 8 1.3 271 32.5 7.9 10.8 0.14 I :3.3 I :29 51 9.0 25.3 32.0 8. I 11.3 0.85 I :2.9 1:23 47 60 23.5 34.0 13.4 14. I 0.61 I : 1.8 I :30 8 62 16.8 33.1 13.0 15.5 0.62 I : 1.4 I :20 II * Proteoglycans extracted by 4 M guanidiniurn chloride were purified by equilibrium density gradient centrifugation in cesium chloride. then in cesium chloride in 4 M guanidinium chloride (see text). B R A N D T A N D PALMOSKI 212 from the bottom two-fifths of the initial cesium chloride gradient. Based on elution profiles of uronic acid after Sepharose 2B chromatography of the material in the bottom two-fifths of the initial cesium chloride gradient, PG aggregates of normal cartilage tended to be larger than those of OA cartilage. Thus 70% of the sample from normal, but only 40% of that from OA cartilage, eluted with a K,, 5 0.5 (21). In contrast the disaggregated PGs of normal and OA cartilage, which represented about 40% of the total uronic acid in each case, were essentially the same in average hydrodynamic size. Both eluted with a rather broad unimodal peak, somewhat after the void volume of the Sepharose 2B column. Over 9070 of the uronic acid in the aggregates of both normal and diseased cartilage was recovered as disaggregated proteoglycans after the second density gradient centrifugation. Whereas over 90% of the disaggregated PGs in the guanidinium extracts were excluded from Sepharose 6B, 50% of the PGs extracted by sodium acetate from normal as well as from OA cartilage were small enough to be retarded. In addition to their differences in hydrodynamic size, there were apparent differences in composition between the PGs exTable 3. Amino Acid Composition, Residues/1000 Residues, of Purified Proteoglycans+ of Normal and Osteoarthritic Bovine Knee Cartilage and Bovine Nasal Cartilage Normal Knee HYPro GlY Pro Leu Ala Val I le Phe ASP Arg LYS Glu Ser Thr TY r His CYS M el 0 1 on I12 ni 79 65 35 29 74 37 23 I33 I12 65 in 20 5 4 OA Knee Nasal Cartilage? 0 I14 I17 78 69 68 36 32 70 34 25 I 38 99 60 25 25 7 3 0 I00 99 86 79 64 33 40 67 38 23 141 I12 62 21 23 9 3 * Proteoglycans were extracted in 4 M guanidinium chloride and purified by equilibrium density gradient centrifugation in cesium chloride, then in cesium chloride in 4 M guanidinium chloride (see text). t Reference (3). tracted in sodium acetate and the purified disaggregated PGs prepared from the guanidinium extracts (Table 2). Thus PGs obtained by sodium acetate extraction contained more uronic acid and xylose, less protein and hexose, and approximately twice as much galactosamine, relative to glucosamine, as the disaggregated PGs in the guanidinium extracts. Although their protein contents were similar (Table 2) and the amino acid analyses of normal and OA PGs in the guanidinium extracts revealed no major differences between the two (Table 3), PGs from normal tissue contained more uronic acid and a higher proportion of galactosamine, relative to glucosamine, than did OA PGs. Based o'n Sephadex (3-200 chromatography of the GAGS after papain digestion, OA PGs in both extracts had shorter chondroitin sulfate chains than normal (Figure 1). In addition buoyant densities of the disaggregated PGs from normal cartilage tended to be greater than those of the disaggregated PGs from OA cartilage, as indicated by the distribution of uronic acid within the second cesium chloride gradient. Thus 94% of the PGs from normal, but only 82%of those from OA cartilage, had a density L 1.59 g/ml. DISCUSSION These data indicate a striking difference i n the ease with which PGs were extracted from knee cartilage with isoosmotic sodium acetate and with 4 M guanidinium chloride. Indeed, unless the surface area is greatly increased by pulverization, as in the present study, only negligible amounts of PGs can be extracted from articular cartilage with sodium acetate (22). In contrast considerably more PGs can be liberated from laryngeal cartilage by this solvent even without prior pulverization. Because the collagen content of articular cartilage is approximately twice that of laryngeal cartilage, the limited extractability of articular cartilage PGs may be due to their greater entrapment by collagen. However, because hydroxyproline contents of the normal and OA cartilage were the same, the several-fold greater yield of PGs from OA than from normal knee cartilage by limited sodium acetate extraction (Table 2) must be due to other factors. The effectiveness of 4 M guanidinium in extracting PGs from cartilage was presumably related to the fact that it dissociated PGs that existed in noncovalent linkage with HA and GPL (3,4). Nonetheless 4 M guanidinium does not extract PGs quantitatively, and PGs in the guanidinium extracts of both normal and OA cartilage in the present study represented only 60% of GROUND SUBSTANCE PROTEOGLYCANS IN OA CARTILAGE 10 + v~ 20 40 30 -Volume, 50 60 213 70 ml+ Fig 1. Gel chromatography of chondroitin suljhte chains on a column ( 1 15 X 0.8 cm) of Sephadex G-200. eluted with 0.2 M sodium acetate, pH 6.5. Fractions of I ml were collected and their uronic acid contents determined. The chains were isolated after papain digestion of proteoglycans from normal (0-0 ) and osteoarthritic (0-0) cartilage. the total tissue uronic acid. Thus, although a difference in the ease of extractability of PGs from normal and OA cartilage with sodium acetate was readily apparent (Table 2), this difference was totally obscured by the much greater efficiency of 4 M guanidinium as an extracting solvent. The PGs of cartilage are a heterogeneous population and vary among themselves in size and composition (16). It is therefore not surprising that there were qualitative as well as quantitative differences between the PGs in the sodium acetate and guanidinium chloride extracts. Because, in general, protein and keratan sulfate contents of PGs are directly related to their size (22), the analytical data are consistent with the interpretation that the PGs extracted with sodium acetate tended to be smaller than those extracted with guanidinium. This study, as well as previous work (22), indicates that sodium acetate extracts of articular cartilage contain significant amounts of smaller PGs, retarded on Sepharose 6B, which contain less protein and keratan sulfate than the larger PGs (16). These smaller PGs are also extracted by 4 M guanidine, although they represent only a small proportion of the total PGs extracted by that solvent (23), and PGs in the sodium extracts thus may be viewed as a subpopulation of those in the guanidinium extracts. The bulk of the PGs extracted by 4 M guanidinium have a buoyant density 2 1.50 g/ml (3) and are dissociated from HA and GPL, whose buoyant densities are 5 1.46 g/ml, by equilibrium density gradient centrifugation in cesium chloride in the presence of 4 M guanidinium (3,15). In the present study a significant proportion of the PGs isolated after the first density gradient centrifugation were eluted in the void volume on Sepharose 2B chromatography whereas those recovered from the bottom of the second gradient were essentially wholly retarded; these findings provide evidence that the PGs in 0.5 M guanidinium were indeed aggregated and that they were disaggregated by 4 M guanidinium. Not all PGs extracted from cartilage by 4 M guanidinium will aggregate in vitro in the presence of HA (2,4). PGs that are extracted by low-speed, brief homogenization in sodium acetate, as in the present study, are presumably not aggregated in vivo because these extraction conditions are unlikely to dissociate them from H A . Furthermore, when PGs extracted under these conditions were mixed in vitro with HA, no evidence of aggregation could be found, although aggregation of PGs that had been extracted with 4 M guanidinium chloride was readily demonstrated (24). On the other hand, after repeated, high speed homogenization of BRANDT AND PALMOSKI 2 14 articular cartilage in neutral 0.15 M sodium acetate, during which the tissue was subjected to considerable shear forces, PGs were eventually liberated whose hydrodynamic size was reversibly diminished by treatment with 8 M urea, without apparent change in their composition (16). These P G s were similar therefore to those extracted with 4 M guanidinium. The present study indicates that nonaggregated PGs (eg those readily extracted by sodium acetate) exist in considerably larger numbers in OA than in normal bovine knee cartilage. Recent studies have shown that PGs may also be liberated from O A bovine hip cartilage by isoosmotic neutral sodium acetate in much greater amounts than normal (6). In agreement with these results is the fact that, when diseased cartilage from dogs with naturally occurring or experimentally induced O A was extracted with 2 M calcium chloride, yields of P G s were approximately twice those obtained from normal cartilage (8). Comparable results have also been obtained with cartilage from lame pigs that had been reared intensively with restricted activity but that did not have OA (25). These data all suggest a n abnormality of PG aggregation, a possibiiity that is supported by comparison of the aggregated P G s of normal a n d O A cartilage in the present study, in which aggregates from the diseased cartilage tended to be smaller than normal. After disaggregation, however, P G s of normal and O A c a r t i l a g e were essentially t h e s a m e in a v e r a g e hydrodynamic size and had similar protein contents. Furthermore no appreciable difference was noted between the core proteins of normal a n d O A PGs on amino acid analysis, or between these and the core proteins of bovine nasal cartilage P G s (3) (Table 3). Even though they did not differ on Sepharose 2B gel chromatography, PGs from O A cartilage contained shorter chondroitin sulfate chains than those from normal. Moreover this difference in chain length was the same in sodium acetate and guanidinium chloride extracts. Chondroitin sulfate chain lengths of the PGs of normal cartilage, calculated from molar ratios of xy1ose:uronic acid under the assumption that all the xylose is engaged in linking chondroitin sulfate t o protein (26), averaged 30 repeating disaccharide units. Those o n PGs of O A cartilage averaged approximately 20 repeating units. Elution profiles of t h e GAGs on Sephadex G-200 chromatography (Figure 1 ) reflect the difference in chondroitin sulfate chain length indicated by the analytical data. T h e tendency toward lower buoyant density of O A PGs, in comparison with those of normal cartilage, may also be attributed to a smaller amount of polysaccharide, relative to protein, o n O A PGs. These data therefore agree with studies that reported a decrease in chondroitin sulfate chain length in O A on the basis of reducing end group analysis (10) or fractionation of cetylpyridinium chloride complexes of the G A G s (14). They are consistent also with data showing that PGs of human OA cartilage have a lower uronic acid:protein ratio than normal (9). T h e present study did not indicate that the keratan sulfate content of O A PGs was diminished, although such diminution has been noted by others (27). Whether the observed decrease in chondroitin sulfate chain length represents an abnormality in G A G synthesis or increased degradation of chondroitin sulfate in O A is not presently established. Hyaluronidase, which degrades chondroitin sulfate, is present in synovial fluid (28) although it has not been demonstrated in articular cartilage. Decreased aggregation of PGs in O A cartilage, as indicated by the present study, could facilitate penetration of synovial fluid hyaluronidase into the cartilage matrix and thus result in some degradation of chondroitin sulfate chains a n d possibly also of H A in the cartilage. REFERENCES I . Freeman MAR, Kempson GE: Load carriage, Adult 2. 3. 4. 5. 6. 7. 8. 9. Articular Cartilage. Edited by MAR Freeman. New York, Grune & Stratton, Inc, 1973, pp 228-246 Hardingham TE, Muir H: The specific interaction of hyaluronic acid with cartilage proteoglycans. Biochim Biophys Acta 279:401-405, 1972 Hascall VC, Sajdera SW: Proteinpolysaccharide complex from bovine nasal cartilage. The function of glycoprotein i n the formation of aggregates. J Biol Chem 24412384-2396, 1969 Hascall VC, Heinegard D: Aggregation of cartilage proteoglycans. 1. 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