Protein and ribonucleic acid synthesis in articular cartilage of osteoarthritic dogs.код для вставкиСкачать
Protein and Ribonucleic Acid Synthesis in Articular Cartilage of Osteoarthritic Dogs By HENRY J. MANKINAND PATRICK G. LAING T IIE HISTOLOGICAL alterations in articuproach from those previously reported, and lar cartilage from osteoarthritic joints measures the rates of synthesis of protein and ribonucleic acid (RNA) as metabolic have been well known for many year~,lJ-.",~ h i t the biochemical abnormalities are less indicators. Since the protein core of the well defined, and most of the knowledge protein-polysaccharide molecule is syntheof the metabolism is speculative. Early ob- sized at the ribosome, and presumably unservations of changes in the metachromatic der control of genetic information transstaining3g4of the matrix were corroborated mitted by messenger RNA, assay of this by studies demonstrating a decrease in would seem a more direct method of dechondroitin sulfate content with little al- fining the metabolic state of the cell. In a teration in the ~ o l l a g e n . ~ , Collins ~.' and prior study from this laboratory, assays of his co-workers studied S'500,z incorpora- incorporated glycine-H3 and cytidine-H3 tion into osteoarthritic human cartilage, into cartilage were used to measure protein and showed an increase in rate approxi- and RNA synthesis and were shown to be mately proportional to the severity of the an excellent indicator of cellular injury."' process.8 These 2 sets of experimental data Over the past 5 years, the authors have led to the theory that there was an in- studied 9 German Shepherd dogs which creased turnover of protein-polysaccharide developed osteoarthritic changes in both in osteoarthritic cartilage, and recent re- femoral heads as a result of dysplasia of the ports regarding the presence of catheptic hips. Recently this study was terminated, enzymes in cartilage have added consider- and one phase of the final examination of ably to the credibility of this concept.9 the mechanical and biochemical abnormalThe radiosulfate technic is an excellent ities associated with the degenerative procmethod for the quantitative study of ess was the determination of the in vitro the metabolism of protein-polysaccharates of incorporation of glycine-H7 and ride,1°~11~12J" but it is not a direct indicytidine-H? into the cartilage from several cator, since the sulfation of the macromolecule is known to occur as a separate step areas of the damaged joint surfaces. The data obtained demonstrated a sigand is probably not directly related to nificant decrease in the rates of synthesis either messenger RNA or protein synthesis at the r i b o ~ o m e . ~Recent ~ . * ~ studies have of RNA and protein in the weight bearing indicated that there may be a temporal area of the femoral heads of these osteolag between synthesis of the complex and arthritic dogs. In the non-weight bearing its ~ u l f a t i o n . ' ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ areas, which were less severely involved, This study of the metabolic activity of the rates of synthetic activity did not differ osteoarthritic cartilage uses a different ap- significantly from those of normal cartilage. From the Department of Orthopaedic Surgery, Uninersity of Pittsburgh School of Medicine, Pittsburgh, Pennsyli,ania 1521 3. Supported in part by N.I.H. Service Grant AM 01863-03. HENRYJ. MANKIN,M.D.: Director of Ortho- paedics, The Hoepita1 for Joint Diseases; Professor of Orthopaedics, The Mount Sinai Medical School. Current address: The Hospital for Joint Diseases, New York, N.Y. PATRICKG. LAING,M.D.: Associate Professor of Orthopaedic Surgery, University of Pittshurglz School of A4edicine. 444 ARTHR:-rIS AND RHEUMATISM, V O L . 10, NO. 5 (October 1967) PROTEIN AND RIBONUCLEIC SYNTHESIS Fig. 1.-Photographs of the femoral head from a German Shepherd dog with osteoarthritis. The “weight bearing area” is indicated. Note the erosive changes and irregdarity of surface. Osteophytes a r e present on t h e inferior posterior margin. MATERIALSAND METHODS The 9 German Shepherd dogs used for this study were donated by a number of dog owners in western Pennsylvania who found the animals nnsatisfactory for breeding purposes because of “congenital” dysplasia of the hips. In every case the condition had progressed to frank osteoarthritis by the time they were first seen at the Orthop e d i c Laboratories of the University of Pittsburgh. ‘Three additional large ( b u t not thoroughbred) animals were obtained to serve as controls. None of these hacl either gross or microscopic evidence of osteoarthritis. All the osteoarthritic animals were caged in standard dog pens over a 5-year period and fed ad libitum with Purina Dog Chow. Basal Eagle’s medium was obtained from the Baltimore Biological Corporation, Baltimore, Maryland and glycine-H3 (Sp. Ac.200 mc./mM.) and cytidine-HS (Sp. Ac.7 mc./mM. ) were obtained from the New England Nuclear Corporation, Boston, Mass. The assay of the in vitro rates of incorporation of glycine-H3 and cytidine-H3 into the articular cartilage of the femoral heads was performed at the time of sacrifice. Since the animals varied in age, size and possibly purity of breed, it was necessary to have an “internal” control. This was achieved by measuring the rate of isotope incor- 445 poration into the cartilage from the knees of the osteoarthritic animals and from both hips and knees of the control animals, thus providing a ratio of rates of incorporation for hips and knees from both groups. Since cell population varied considerably depending on the area of study, DNA content was also determined, so that these ratios could be expressed on the basis of rates of incorporation per pgm of DNA. When the animal was killed, the hip and knee joints were rapidly exposed, examined grossly and photographed. It was apparent that the degree of osteoarthritic change varied considerably in different areas of the femoral head, so the cartilage surface was arbitrarily divided into 2 zones: the weight bearing area, which lies in the anterior superior quadrant in the dog; and the non-weight bearing area, consisting of the remainder of the femoral head (Fig. 1 ) . The cartilage covering the distal femur showed no abnormality in any of the animals. From these 3 areas-weight-bearing area, non-weight bearing area, and distal femoraslices of cartilage were resected (taking care to exclude the underlying bone) and the following studies performed. In vitro incorporation rates for glycine-H.’ and cytidine-H”. Ten cartilage slices from each area were separately incubated at 37 C in Eagle’s medium containing glycine-H3 or cytidine-H? (125 pc/ml.). After 1 hour the slices were harvested, fixed in formalin and dried and defatted in absolute alcohol. The dried samples were weighed to the nearest ,001 mgm. on a Mettler Micro Gramatic Balance, and the trichloracetic acid-insoluble fraction was assayed for incorporation radioactivity by a method previously described.“ Values obtained were expressed as counts per mgm. (dry weight) per minute. DNA determination and histological controls. Another 10 samples of the cartilage from each site were separately dried, weighed and assayed for DNA content by the indole microcolorimetric method described by Bonting and Jones.= Values obtained were expressed as micrograms of DNA per mgm. of dry weight. Standard hematoxylin and eosin sections were made of representative tissue from the same area (Fig. 2 ) . I t was thus possible to obtain expressions for the incorporation rates of glycine-H3 and cytidineH* per microgram of DNA for the 2 zones of the femoral head and the knees of both osteoarthritic and control animals. The incorporation rates for the hips were then expressed as multiples of those for the knees of the same animal for both osteoarthritic and control dogs, and thus allowed critical comparison. 446 MANKIN AND LAING Fig. 2.-Photomicrographs of the articular surfaces from the femoral head of a dog with osteoarthritis. A: weight bearing area. This shows severe changes with marked fibrillization of the surfaces. Cells are relatively well preserved ( x 100). B: non weight bearing area. There is mild hypercellularity noted but the chief finding is vascular buds crossing the "tideinark"-the basophilic line separating the zone of calcified cartilage (x 100). the rate, as measured in counts per kgm Figs. 3 and 4 are graphic representa- DNA per minute, appears to be approxtions of the data obtained in this study. Fig. imately the same for hip and knee. The 3 shows the rates of incorporation of gly- rate of incorporation is moderately recine-H3 and Fig. 4 the rates of cytidine-H3 duced in the cartilage of the weight bearinto articular cartilage of normal hips ing area of the arthritic hips, and essen(from control animals), and the weight tially unchanged or possibly slightly inbearing area and the non-weight bearing creased in the non-weight bearing area. areas of the osteoarthritic hips. Each value DISCUSSION is expressed as a multiple of the values From the date described above it is apfor the rates of isotope incorporation per microgram of DNA for the knee of the parent that the in vitro incorporation rates same animal, and each point on the graph of glycine-H3 and cytidine-H3 are considrepresents the mean of 10 such separate erably reduced in the cartilage from the determinations. weight bearing areas of osteoarthritic hips Upon analysis of Fig. 3, it is evident that of dogs and, if not reduced, at Ieast are not glycine-H3 incorporation is normally ap- increased in the non-weight bearing areas. proximately 1.4 times as rapid in the hip One could assume from these data that as in the knee. Also, in the weight bearing RNA and protein synthesis are significantly area of osteoarthritic hips, it is sharply re- decreased in these tissues, and that the duced to less than 60 per cent of the nor- degree of depression is proportional to the mal mean value. In the non-weight bearing severity of the disease. Before accepting area, there is a much wider scatter but these observations, it is necessary to be certhere appears to be little or no decline in tain that the results expressed above are rate. valid and that they indeed reflect the metFig. 4 demonstrates the results of cyti- abolic activity of the tissue. dine-H3 incorporation. In normal animals There is ample evidence that glycine-H3, RESULTS 447 PROTEIN AND RIBONUCLEIC SYNTHESIS -I in 4 2.00 5 1.80 ZP f \ J 4 I WT. BEARINO 2.00 1.60 z a v, 1.40 1.20 1.00 1.00 0.80 0.60 0.80 o o 0 0.40 0.60 0.20 0.40 Fig. 3.-Glycine-H3 incorporation into the cartilage of femoral heads of normal and osteoarthritic dogs. Data is expressed as multiples of values for the knee of the same animal (recorded as counts/pgm. DNA/min.). It is apparent that the cartilage from normal hips incorporates glycine-H3 about 1.4 times as rapidly as the knees. The cartilage from the weight bearing areas of arthritic hips shows marked reduction in glycine-H3 incorporation, and that from the non-weight bearing areas a slight reduction (or at least no increase.) incorporated into the cartilage cell, is utilized in protein-polysaccharide synthesis. Prior studies in this laboratory have indicated the shunt into nucleic acid metabolism23 is insignificant in articular cartilage,20*24 and that the majority of the labeled substrate is entering the proteinp o l y ~ a c c h a r i d e . 2Degradation ~~~~ rates for the protein-polysaccharides are much longer than the experimental time in this ~ t u d y , 2 ~negating ~~7 the possibility that the counts obtained are indicative of decay rates as well as the rate of incorporation. Cytidine, although a precursor of both RNA and DNA,28,2gmay be assumed to be entering RNA synthesis in a tissue where DNA synthesis is minimal.29 Although there is some increase in DNA synthesis in osteoarthritic ~artilage,2*~O the amount is so small as to be insignificant, and it is therefore assumed that the rate of cytidine-H3 incorporation is indicative of RNA synthesis. 0.20 If:. AV = 0.38 AV = 0.76 I AV = 1.051 Fig. 4.-Cytidine-H3 incorporation into the cartilage of femoral heads of normal and osteoarthritic dogs. Data is expressed as multiples of values for the knee of the same animal (see text). Cartilage from normal hips incorporates cytidine-H3 at approximately the same rate as the knees. There is a reduction in the rate in the weight bearing area of the arthritic hips and a wide scatter but probably no change in the non-weight bearing area. (Each point on the graph represents the mean of 10 determinations on cartilage from only the femoral head.) Before accepting the validity of these counts, it is important to point out that there are 2 sources of potential error which cannot be controlled. The quantity of labeled substrate which is present in a tissue at the time of counting will depend not only on the synthesis of t h e product and the rate of degradation but also on the pool of unlabeled substrate present in and around the ce11.3I It is therefore possible that the intracellular pools for glycine or cytidine (or even sulfate) vary with the degree of osteoarthritis and possibly independently of each other, thus affecting the rates of incorporation of the labeled substrates to a lesser or greater extent. A second problem exists in attempting to define the metabolism of a damaged cell by comparing it with a "healthy" one. It is possible that glycine or cytidine utilization by the 448 MANKIN AND LAINC Table 1.-Average DNA Concentrations in Normal and Osterarthritir of DNA per mgm. ( d r y weight). Cartilag-mgm. Krires ( 3 0 ) 17.4 & 5.0 Normal Hips (6) Osteoarthritic Hills (9) U'eiglit Ilearing Area Non-weight Bearing Area Weight Ilearing Area Nori-weight Bearing Area 16.7 r+. 6.2 (96%) 15.9 ? 4.5 (91%) 21.2 ? 6.1 (117%) 13.8 _t 5.4 (80%) osteoarthritic cartilage cell is abnormal in terms of either product or pathway, thus to some extent invalidating these results. If, however, one assumes that there is little variation in the pools or utilization, it is possible to conclude that the rates of incorporation of glycine-HI and cytidineH I are indicative of the rates of protein and RNA synthesis respectively in both normal and abnormal cartilage. The DNA determinations, as performed by the method of Honting and Jones,22 are indicators of the number of cells present per milligram of tissue and therefore reflect, not only cell replication, but any change in ratio of cells to quantity of matrix. Table 1 illustrates the variations in quantity of DNA in the weight bearing and non-weight bearing areas of the osteoarthritic hips as cornpared with the normal hips and knees. It is quite evident that there is an increase in the amount of DNA per milligram of tissue in the more severly damaged (weight bearing) areas of the femoral heads, probably indicating a decrease in matrix rather than an absolute increase in the number of cells. Such an observation is certainly consistent with the histological appearance (Fig. 2 ) , in which there is obvious degradation of matrix and fibrillization of the articular cartilage with relatively good preservation of the cells. If the data are combined, it is possible to define a rate of synthesis of RNA and pro- tein per microgram of DNA, and thus establish an index of cellular metabolism for the osteoarthritic cartilage as compared to the hips of the normal animals. It is quite evident that, barring the possible sources of error described above, there is a decrease in metabolic activity in the severely affected areas in the weight bearing portion of the femoral head. At first glance, these results vary considerably from those reported by Collins and his co-workers who have shown, by both qualitative autoradiography and quantitative assay, an increased S'"O,= fixation in articular cartilage from osteoarthritic human jointsHIn critical analysis, however, it is apparent that species variation or technical difference in the procedures, or variation in the pathology, make a positive statement of this sort impossible. If one wishes to speculate that such a dichotomy exists, the findings may imply that there is some variation in the synthesis or degradation of the protein-polysaccharides in osteoarthritic cartilage which is associated with a decline in RNA and protein synthesis and an increase in sulfate incorporation. In conclusion, the authors have found that in cartilage from the femora of osteoarthritic hips of dogs, there is a substantial decrease in protein and RNA synthesis as compared with normals, and this decrease seems proportional to the severity of the process. SUMMARY The in vitro rates of incorporation in glycine-H:<and cytidine-H3 were determined for the articular cartilage from the femoral heads of 9 German Shepherd dogs with osteoarthritis secondary to congenital dysplasia, and 3 normal animals of approximately 449 PROTEIN AND RIBONUCLEIC SYNTHESIS the same size and weight. These data were expressed per pgm of DNA and compared, using the cartilage from the knees as internal control. On the basis of prior studies, these rate values can be equated to the rates of synthesis of protein and RNA respectively. From the data obtained, it is apparent that protein and RNA synthesis are markedly decreased in the most severely involved areas and, if not decreased, at least riot increased in the less damaged areas. SUhlMAFUO I N INTERLINGUA Le rapiditate del incorporation de tritiate glycina e de tritiate cytidina esseva determinate in vitro in specimens de cartilagine articular ab le capites femoral de 9 canes de pastor german con osteoarthritis secundari a congenite dysplasia e de 3 tal canes normal de approximativemente le mesme grandor e peso. Le valores esseva exprimite como fig de acido desoxyribonucleic e comparate con le us0 de cartilagine ab le genu como control0 interne. A base de studios effectuate in le passato, il pare justificate equar le valores obtenite con le rapiditates de synthese de proteina e de acido ribonucleic, respectivemente. Ab le datos obtenite le conclusion pote esser derivate que le synthese de proteina e de acido ribonucleic es marcatemente relentate in le areas le plus severmente afficite. In areas minus servmente afficite, ille synthese-si non relentatees certo non accelerate. REFERENCES 1. Bennett, G. A., Bauer, W., and Waine, H.: Changes in the Knee Joint at Various Ages. New York, Commonwealth Fund, 1942. 2. Collins, D. H.: The Pathology of Articular and Spinal Diseases. London, E. Arnold, 1949. 3. Sokoloff, L.: The Pathology and pathogenesis of osteoarthritis. In Hollander, W. L., ed.: Arthritis and Allied Conditions: 7th Ed. Philadelphia, Lea and Febiger, 1966, p. 849. 4. IIirsch, C.: The Pathogenesis of chondromalacia of the patella. A physical, histologic and chemical study. Acta. Chir. Scand. 1944, 90, Suppl. 83. 5. Matthews, B. F.: Composition of articular cartilage in osteoarthritis: changes in collagen to chondroitin sulfate ratio. Brit. Med. J. 2:660, 1953. 6. Shetlar, M. 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