Proteoglycan depletion rather than fibrillation determines the effects of salicylate and indomethacin on osteoarthritic cartilage.
код для вставкиСкачать548 PROTEOGLYCAN DEPLETION, RATHER THAN FIBRILLATION, DETERMINES THE EFFECTS O F SALICYLATE AND INDOMETHACIN ON OSTEOARTHRITIC CARTILAGE MARSHALL J . PALMOSKI and KENNETH D. BRANDT The effects of salicylate and indomethacin on glycosaminoglycan (GAG) synthesis by atrophic and osteoarthritic (OA) canine cartilage were examined in vitro by transecting distal femora at the metaphysis and incubating the knuckle, with its overlying cap of articular cartilage, in medium containing sodium salicylate or indomethacin, and 35S04. Atrophic cartilage had an intact articular surface, but its uronic acid content averaged 65% of the control level, and GAG synthesis was decreased to 50% of control values. Both salicylate and indomethacin decreased net GAG synthesis in the atrophic cartilage by an additional 10%. OA cartilage showed surface disruption, a uronic acid content 49% of the control value, and a 49% increase in net GAG synthesis. Salicylate and indomethacin profoundly decreased GAG synthesis in the OA cartilage. However, GAG synthesis and uronic acid content of cartilage which had been lacerated in vitro immediately prior to culture (to stimulate fibrillation) were normal and not affected by either drug. The data emphasize the importance of matrix proteoglycan content in protecting the _____ From the Rheumatology Division, Indiana University School of Medicine, Indianapolis. Supported in part by grants from the National Institute of Arthritis, Metabolism and Digestive Diseases (AM-20582 and AM-7448) and awards from the Arthritis Foundation and the Grace M. Showalter Trust. Marshall J . Palmoski, PhD: Associate Professor of Medicine and Anatomy (current address: Department of Pharmacology, Bristol-Myers Co., PO Box 4755, Syracuse, NY 13221); Kenneth D. Brandt, MD: Professor of Medicine and Head, Rheumatology Division, Indiana University School of Medicine. Address reprint requests to Kenneth D. Brandt, MD, Rheumatology Division, Indiana University School of Medicine, 541 Clinical Drive, Indianapolis, IN 46223. Submitted for publication August 13, 1984; accepted in revised form November 9, 1984. Arthritis and Rheumatism, Vol. 28, No. 5 (May 1985) chondrocyte from the suppressive effects of salicylate and indomethacin on GAG metabolism, and suggest that the lower proteoglycan content of OA cartilage may be more important than fibrillation in rendering it vulnerable to the metabolic effects of these drugs. We have previously reported that aspirin, when fed to dogs in quantities sufficient to achieve a serum salicylate concentration of 20-25 mg%, aggravated the degeneration of articular cartilage in osteoarthritic (OA) joints, although it had no effect on normal cartilage from the contralateral knees (1). In vitro studies have shown that the marked augmentation of glycosaminoglycan (GAG) synthesis in slices of cartilage from osteoarthritic joints was suppressed by salicylate to a much greater extent than GAG synthesis in normal cartilage, and that this difference was associated with greater uptake of the drug by fibrillated cartilage than by normal cartilage (2). Other data are consistent with the concept that the suppressive effects of salicylate and also of indomethacin on GAG synthesis in normal cartilage are inversely related to the proteoglycan content of the extracellular matrix, and indicate that the proteoglycan content of cartilage may affect its permeability to these drugs (3,4). To determine whether the augmented effect of salicylate on osteoarthritic cartilage was due to decreased proteoglycan content andlor to disruption of surface integrity, providing increased access of the drug to the chondrocytes, we examined the effects of salicylate on GAG synthesis in normal cartilage which had been lacerated in vitro to simulate fibrillation, as well as on osteoarthritic cartilage and atrophic cartilage, which had an intact surface but a reduced proteoglycan content. 549 SALICYLATE AND INDOMETHACIN IN OA MATERIALS AND METHODS Experimental groups and procedures. Fourteen adult mongrel dogs, whose weights ranged from 23-30 kg, were used. Four animals (the Cru-X group) underwent transection of the right anterior cruciate ligament, as previously described (1). All Cru-X animals bore weight on the operated limb by the third day after surgery and were maintained in pens (8 x 10') for 16 weeks, at which time they were killed with an overdose of sodium pentothal. The right hind legs of 4 additional dogs (the IK group) were immobilized in a cast with approximately 90" of hip and knee flexion, to induce articular cartilage atrophy (5). These animals were killed after 6 weeks of confinement in pens, as described above. A third group of 4 dogs (the Lac group) was killed with an overdose of sodium pentothal within 5 days after procurement. At the time of death, both knees of each animal were opened aseptically and the distal femurs were removed at the metaphysis with a bone saw. After the femurs were cleaned of adhering soft tissue, the articular cartilage of the central weight-bearing regions of the medial and lateral condyles of the right knee of each dog in the Lac group was lacerated with a scalpel at approximately 1-mm intervals, with care taken not to penetrate the subchondral bone. Culture technique. The medial and lateral condyles of both knees of each dog in the above 3 experimental groups were separated through the intercondylar groove with a bone saw, and were placed separately in breakers containing 15 ml of Ham's F-12 nutrient mixture, pH 7.4, supplemented with 10% newborn calf serum, streptomycin (50 pg/ml), penicillin (50 units/ml) (all obtained from Gibco, Grand Island, NY), and ascorbic acid (50 pg/ml). In half of the experiments, the medial condyles were incubated with gentle rocking for 24 hours at 37°C in 5% COz, 95% air in medium containing sodium salicylate (10-3M) or indomethacin (4 x 10-6M), with Na2-35S04(10 pCi/ml) added for the final 8 hours of incubation, while the lateral condyles were cultured in an identical fashion, but without the drug added. In the remaining experiments, the conditions were reversed, i.e., the lateral condyles were incubated in medium which contained drug, while the medial condyles were cultured in the absence of drug. Characterization of medial and lateral condyles. The remaining 2 dogs were killed, and cartilage from both knees was obtained and was used to provide comparisons between medial and lateral femoral condyles with respect to uronic acid content and GAG biosynthesis in the absence of drug. Determination of net glycosaminoglycan synthesis. Following incubation of the tissues as described above, the medium was decanted and the condyles were rinsed with 0.9% saline. The spent medium and rinse were combined and dialyzed against 200 volumes of 0.05M sodium acetate, pH 6.8, for 48 hours at 4°C in Spectrapor No. 3 dialysis tubing (approximate molecular weight cutoff = 3,500 daltons; Spectrum Medical Industries, Los Angeles, CA). The articular cartilage was then shaved with a scalpel from the central, habitually-loaded surface of each condyle. Cartilage from each condyle was pooled separately. Portions (approximately 15 mg) of the shaved cartilage were weighed and used for determination of uronic acid content. Representative full-thickness samples of the cartilage from the central weight-bearing regions of each condyle were obtained with a Craig biopsy needle and were placed in 10% buffered formalin for histologic study, while the remainder was divided into 3 approximately equal portions (20-30 mg wet weight each) which were used to assay net GAG synthesis. 'The triplicate samples were suspended in 3 ml of 0.1M borate buffer, pH 8.0, containing 0.02M calcium chloride, and were digested under toluene for 24 hours at 55°C with pronase (200 pg/lO mg cartilage wet weight; Calbiochem-Behring, San Diego, CA). The digests were placed in Spectrapor No. 3 membrane tubing and dialyzed overnight at 4°C against running tap water, following which aliquots (0.1 ml) were added to 10 ml of Ready-Solv HP 60 (Beckman Instruments, Irvine, CA) and counted in a Beckman liquid scintillation spectrometer. Results (nondialyzable counts per minute) were adjusted for differences in wet weight of the cartilage. Determination of uronic acid content. Portions (approximately 15 mg) of cartilage were placed in 2 changes of acetone for 24 hours, dried to constant weight in vacuo at 8O"C, and digested with pronase as above. The GAGS were isolated from the pronase digests by precipitation with 9-aminoacridine hydrochloride, and were converted to their sodium salts with Bio-Rad AG-50 (Na') (6). After the resin was removed by filtration, the uronic acid content of the filtrate was determined (7). Histologic examination. Histologic sections (6p) of full-thickness samples of the articular cartilage were examined microscopically after staining with Safranin 0-fast green. RESULTS Gross observations. None of the knee joints contained an effusion. At the time of death, articular cartilage from the control knees of the Cru-X and IK dogs, and from both knees of t he Lac dogs, was grossly normal. In all 4 Cru-X dogs, marginal osteophytes were present on medial and lateral femoral condyles of the unstable knees and the cartilage surface was dull and rough. In contrast, the unstable knees of IK dogs had no osteophytes and the cartilage was smooth, although it appeared thinner than in the control knees. Histology and histochemistry. In all control samples, the cartilage surface and tidemark were intact, and Safranin 0 staining was normal. All samples of Cartilage from the unstable knees of dogs from the Cru-X group showed a reduction in Safranin 0 staining in comparison with the contralateral control cartilage. Horizontal flaking of the cartilage surface was noted in all samples, but no vertical fibrillation was seen. T h e cartilage thickness was comparable with that of the PALMOSKI AND BRANDT 550 Table 1. Comparison of articular cartilage from medial and lateral femoral condyles of knees of normal dogs Dog Source of cartilage* Uronic acid content, % of tissue dry weight Net glycosaminoglycan synthesist RMC RLC LMC LLC RMC RLC LMC LLC 4.1 4.0 4.2 4.1 3.9 3.8 4.0 3.8 12,432 +. 1,021 12,805 2 858 13,301 t 1,231 13,110 +- 1,410 18,567 2 823 17,492 t 1,210 17,890 t 1,390 18,420 t 1,521 1 2 * RMC = right medial condyle; RLC = right lateral condyle; LMC = left medial condyle; LLC = left lateral condyle. t Nondialyzable "S cpm/lO mg wet weight of cartilage. Values represent mean & SEM of triplicate samples. contralateral control knees, and the tidemark was intact in all cases. No brood capsules were observed. Cartilage from the scored condyles of dogs from the Lac group showed cuts extending to varying depths below the surface. None penetrated the tidemark. Safranin 0 staining adjacent to the cuts and throughout the matrix was normal. In dogs from the IK group, the thickness of cartilage from the immobilized leg was decreased to about 70% of that of cartilage from the contralateral knee, and Safranin 0 staining was reduced. The articular surface, however, was intact in all samples of atrophic cartilage. Comparison of cartilage from medial and lateral femoral condyles of normal dogs. Studies utilizing 2 normal dogs indicated that there was no difference between the medial and lateral femoral condyles with respect to uronic acid content or net glycosaminoglycan synthesis (Table 1). In all cases, the uronic acid content was about 4% of the tissue dry weight, while net glycosaminoglycan synthesis in the medial condyles averaged 103 f 2% of that in the lateral conTable 2. Uronic acid content of osteoarthritic, lacerated, and atrophic articular cartilage Uronic acid content Group (no. of dogs)* Cru-X (4) Lac (4) IK (4) Source of cartilage Control knee Experimental knee Control knee Experimental knee Control knee Experimental knee % of tissue dry weight 2 SEM 4.1 2.0 4.3 % of control 0.2 - f 0.5 49 2 0.1 - 4.5 +. 0.2 96 65 2 4.0 2 0.1 2.6 t 0.2 * Cru-X = anterior cruciate ligament transection (osteoarthritic); Lac = cartilage lacerated in vitro; IK = immobilized knee (atrophic); control = cartilage from contralateral knee. dyles. Cartilage from the contralateral (control) knees of dogs in all 3 experimental groups exhibited normal uronic acid content (4.1 2 0.1% of the tissue dry weight) (Table 2). Uronic acid content and GAG synthesis in the experimental groups, and drug effects. Cartilage from dogs in the Cru-X group. The uronic acid content of osteoarthritic cartilage from the knees of dogs in the Cru-X group averaged 49% of that of cartilage from the contralateral knees of the same animals, while glycosaminoglycan synthesiswas increased. In the 4 dogs in this group, net GAG synthesis in the osteoarthritic cartilage was 49 & 5% greater than that in the corresponding control knee (P < 0.01). Both salicylate and indomethacin strikingly decreased glycosaminoglycan synthesis in OA cartilage (to 9% and 22%, respectively, of that in osteoarthritic samples cultured in the absence of the drugs) (Tables 3 and 4). Cartilage from dogs in the Lac group. Uronic acid content and net GAG synthesis in experimental knees of the dogs with lacerated cartilage were similar to that of control knees (Table 2). GAG synthesis was not affected by either salicylate or indomethacin (Tables 3 and 4). Cartilage from dogs in the ZK group. The uronic acid content of the atrophic cartilage in immobilized knees was reduced to 65% of that in the contralateral knees of dogs in the IK group (Table 2). In contrast to the osteoarthritic samples, in which GAG synthesis was increased, in the IK cartilage GAG synthesis was only 50 ? 4% of control values ( P < 0.01) (Tables 3 and 4). Both salicylate and indomethacin caused a further decrease in GAG synthesis in the atrophic cartilage, but this decrease was less than that seen when the osteoarthritic cartilage was incubated with the drugs (P < 0.01). Thus, in the absence of salicylate, GAG synthesis in IK cartilage averaged 48% of that in control cartilage, while in the presence of salicylate it averaged 37% of that in control cartilage (Table 3). Indomethacin, too, further reduced GAG synthesis in the atrophic cartilage by an additional 10% over the reduction seen without the drug (Table 4). Neither salicylate nor indomethacin affected net GAG synthesis in control cartilage of the contralatera1 knees of dogs from any of the experimental groups. DISCUSSION The results of this study confirm our previous finding that the proteoglycan content of the matrix SALICYLATE AND INDOMETHACIN IN OA Table 3. 55 1 Effect of salicylate on glycosaminoglycan synthesis by osteoarthritic, lacerated, and atrophic articular cartilage Net ;glycosaminoglycan synthesist Dog 3 4 5 6 7 8 Source of cartilage* Uronic acid content, % of tissue dry weight Control knee Cru-X knee Control knee Cru-X knee Control knee Lac knee Control knee Lac knee Control knee IK knee Control knee IK knee 4.1 1.5 3.9 1.8 4.2 4.4 4.5 4.7 4. I 2.8 4.0 2.5 No salicylate added Salicyiate, iO-3M ' 3 cpm/iO mg wet weight of cartilage 3JScpmilo mg % of control wet weight of cartilage - 16,989 f 1,342 2,209 t 275 11,560 t 748 1,695 2 350 23,576 f 1,032 22,149 f 760 16,783 t 1,213 16,191 t 683 30,047 t 3,083 12,420 2 1,501 29,685 t 2,450 9,836 f 1,021 17,414 & 1,542 26,295 f 2,742 12,630 f 1,421 17,452 f 982 22,560 5 2,404 23,070 ? 962 17,983 5 842 16,366 -C 1,732 28,345 5 2,978 15,196 f 1,543 31,224 5 3,211 13,114 5 942 151 138 102 91 54 42 % of control Salicylate No salicylate 98 8 92 10 105 96 93 99 I06 82 95 75 ~~ * Cru-X = anterior cruciate ligament transection (osteoarthritic); Lac = cartilage lacerated in vitro; IK = t = ~~ immobilized knee (atrophic); control cartilage from contralateral knee. Values represent mean 2 SEM of triplicate samples. affects the susceptibility of articular cartilage to the effects of nonsteroidal antiinflammatory drugs on GAG biosynthesis (3,4). In addition, our present findings suggest strongly that disruption of the cartilage surface (fibrillation) in osteoarthritis is less important than loss of proteoglycans in determining the effects of salicylate and indomethacin on cartilage GAG metabolism. Thus, GAG synthesis in cartilage which had been lacerated in vitro to simulate fibrillation, but whose uronic acid content was normal, was not affected by either salicylate or indomethacin. In contrast, GAG synthesis in atrophic cartilage, whose uronic acid content was diminished even though the articular surface was intact, was decreased by both drugs. We recognize that laceration of normal cartilage with a scalpel only crudely simulates osteoarthritic fibrillation. However, both conditions cause an increase in the surface area:volume ratio of the carti- Table 4. Effect of indomethacin on glycosaminoglycan synthesis by osteoarthritic, lacerated, and atrophic articular cartilage Net glycosaminoglycan synthesist 10 I1 12 13 14 Source of cartilage* Uronic acid content, % of tissue dry weight Control knee Cru-X knee Control knee Cru-X knee Control knee Lac knee Control knee Lac knee Control knee IK knee Control knee IK knee 4.3 2.6 4.2 2.0 4. I 4.3 4.3 4.5 3.8 2.1 4.2 2.8 No indomethacin added Indomethacin, 4 x 10-6M ' 3 cpm/lO mg '3c p d 1 0 mg wet weight of cartilage % of control wet weight of cartilage % of control 8,064 t 951 1 1,802 2 1,432 146 8,231 t 1,021 2,351 t 121 16,253 5 2,340 6,401 t 324 8,519 5 449 8,494 f 782 20,622 2 1,604 20,289 t 1,747 28,788 5 505 10,086 t 1,492 33,230 t 3,141 15,490 2 1,238 29 39 100 98 35 17,458 5 1,234 28,457 f 3,213 8,609 2 897 9,162 f 749 20,285 5 1,317 21,348 t 1,027 27,672 2 1,725 12,452 2 1,432 34,525 2 3,021 20,066 f 1,901 - 162 - 106 - 105 - 45 - 58 * Cru-X = anterior cruciate ligament transection (osteoarthritic); Lac = cartilage lacerated in vitro; IK from contralateral knee. t Values represent mean 2 SEM of triplicate samples. = cartilage - 47 = Indomethacin No indomethacin I02 20 93 23 99 93 102 95 I04 81 96 71 immobilized knee (atrophic); control PALMOSKI AND BRANDT 552 Figure 1. Canine knee cartilage, stained with Safranin 0-fast green. A , 16 weeks after anterior cruciate ligament transection. B, Articular surface lacerated at time of killing. C , Ipsilateral leg irnmmobilized for 6 weeks. D, Representative control knee. lage, which may affect ,uptake of drug from the culture medium. It is particularly notable, therefore, that the above differences in GAG synthesis occurred despite the fact that this ratio was increased to a much greater extent by laceration than by cruciate ligament transection, which produced only mild surface disruption (Figure I). The data in this study are consistent with our previous findings that aspirin, when fed to dogs in quantities sufficient to achieve a serum salicylate concentration of approximately 10-3M, aggravated the degeneration of articular cartilage from habitually loaded areas of immobilized (8) or unstable joints ( I ) , although it had no effect on normal articular cartilage from analogous sites in the contralateral knees of the same animal. It should be pointed out that the culture system employed herein, using whole femoral condyles, more closely resembles the in vivo conditions than do tissue slices in organ culture, since salicylate has been reported to suppress GAG synthesis in vitro in slices of cartilage from the weight-bearing surfaces of normal canine joints (9). The in vitro laceration technique used here is similar to that described by Meachim (lo), in which multiple superficial slices are made across the articular surface at close intervals. Notably, the laceration procedure itself had no effect on the uronic acid content of the cartilage or on GAG synthesis (Tables 2-4). Mankin and Boyle ( 1 1 ) reported an enhanced rate of GAG synthesis 1 day after scarification of rabbit femoral cartilage, with a return to control levels of synthesis within another 24 hours. Their study, however, utilized immature rabbits, and GAG synthesis was measured in tissue slices, which may account for the difference between their results and ours. In this study, salicylate and indomethacin caused significantly more marked suppression of GAG synthesis in OA cartilage than in atrophic cartilage (Tables 3 and 4). It seems unlikely that this was due only to the small difference in the uronic acid content of the tissues (2.0 and 2.6% of the tissue dry weight, for osteoarthritic and atrophic cartilage, respectively). The marked effect of these drugs on osteoarthritic SALICYLATE AND INDOMETHACIN IN OA cartilage thus suggests that the OA chondrocyte itself may be more vulnerable to the effects of these agents than the normal chondrocyte. ACKNOWLEDGMENTS We are grateful to James Bean for technical support and to Roberta Fehrman for secretarial assistance. REFERENCES Palmoski MJ, Brandt KD: In vivo effect of aspirin on canine osteoarthritic cartilage. Arthritis Rheum 26:9941001, 1983 Palmoski MJ, Colyer RA, Brandt KD: Marked suppression by salicylate of the augmented proteoglycan synthesis in osteoarthritic cartilage. Arthritis Rheum 23:83-91, I980 Palmoski MJ, Brandt KD: Relationship between matrix proteoglycan content and the effects of salicylate and indomethacin on articular cartilage. Arthritis Rheum 26528-53 I , 1983 Palmoski MJ, Brandt KD: Effects of salicylate and indomethacin on glycosaminoglycan and prostaglandin 553 E2 synthesis in intact canine knee cartilage ex vivo. Arthritis Rheum 27:398-403, 1984 5. Palrnoski MJ, Perricone E, Brandt KD: Development and reversal of a proteoglycan aggregation defect in normal canine knee cartilage after immobilization. Arthritis Rheum 22:508-517, 1979 6. Tsiganos CP, Muir H: Studies on protein-polysaccharides from pig laryngeal cartilage: extraction and purification. Biochem J 113:879-884, 1969 7. Bitter T, Muir H: A modified uronic acid carbazole reaction. Anal Biochem 4:330-334, 1962 8. Palrnoski MJ, Brandt KD: Aspirin aggravates the degeneration of canine joint cartilage caused by immobilization. Arthritis Rheum 25: 1333-1342, 1982 9. Palmoski MJ, Brandt KD: Effects of salicylate on proteoglycan metabolism in normal canine articular cartilage: in vitro. Arthritis Rheum 22:746-754, 1979 10. Meachim G: Effect of scarification on articular cartilage in the rabbit. J Bone Joint Surg 45B:150-161, 1963 11. Mankin HJ, Boyle CJ: The acute effects of lacerative injury on DNA and protein synthesis in articular cartilage, Cartilage, Degradation and Repair. Edited by CAC Bassett. National Research Council, National Academy of Sciences, 1967, pp 185-199
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