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PEMBERTON LECTURE

DEGRADATIVE ENZYMES IN OSTEOARTHRITIC HUMAN ARTICULAR CARTILAGE DAVID S. HOWELL On this occasion of the 23rd Pemberton Memorial Lecture, I am provided an irresistible excuse to review some thoughts and data on the nature of osteoarthritis with special emphasis upon degradation of cartilage. A 1962 Public Health survey, reporting on xrays of hands, projected about 40.5 million Americans to have some form of osteoarthritis (l), and 85% of a sampled population (age 75-79 years) were afflicted. Knowledge of this enormous prevalence has evoked a relatively minor increase, if any, in research on osteoarthritis, perhaps due to lack of dramatic aspects of osteoarthritis as opposed to some aspects of the lifethreatening diseases. Also, there seems to be a prevalent feeling on the part of the laiety and professionals alike that osteoarthritis is a hopeless, irreversible, age-related tissue deterioration. Grounds for a more optimistic view are found From the Arthritis Division of Veterans Administration Hospital and of the Department of Medicine, University of Miami School of Medicine. Supported by Career Development and Research support funds, United States Veterans Administration Hospital, Miami, Florida; and grants AM-08662 and AM-05038 from the National Institutes of Health and The Arthritis Foundation Center Grant. David S. Howell, M.D.: Professor of Medicine, University of Miami School of Medicine. and Medical Investigator, Veterans Administration Hospital. Address reprint requests to David S. Howell M.D., University of Miami School of Medicine, P.O. Box 875, Biscayne Annex, Miami, Florida 33152. Submitted for publication July 15, 1974; accepted September 16, 1974. Arthritis and Rheumatism, Vol. IS, No. 2 (March-April 1975)

in the fact that cartilage provides some degree of active reparative machinery throughout life. Crude evidence that this restorative process successfully combats wear-and-tear in articular cartilage is provided in persons coming to autopsy at age 80-90 years. Most of the articular cartilages in such persons are predominantly glistening pale yellow and resilient. Also, fundamental chemical and physical properties of these articular cartilages so far give little evidence of ugerelated deterioration (Linn, Sokoloff, Bollet, et al (2,3)). This remarkable preservation of cartilage follows a period of the first 3 5 4 0 years of life during which several maturational chemical alterations occur (3). Also, such preservation of cartilage quality with aging is in sharp contrast to the universal age-related attrition of bone mass in humans, which begins at age 35-40 years and leads steadily toward overt diffuse bone biomaterial changes, ie, osteoporosis (4). Despite absence of detectable diffuse changes in articular cartilage with aging, except for a diffuse light yellowish pigment (3), localized tan, brown, or colorless “softened” spots appear after age 10 with increasing frequency in certain joints (such as the knee and hip) studied extensively ( 5 ) . Such spots observed at a u t o p sies in different age groups are surrounded by apparently normal cartilage (6). Only a small proportion of such lesions appear to proceed to cartilage ulceration and appearance of symptomatic osteoarthritis (6). Some workers have felt that osteoarthritis results, in part, either from nutritional failure of carti-

HOWELL

168

ALTERED PHYSICAL FORCES I N JOINT OR PART OF JOINT

J

MATRIX EXPOSURE TO LOCAL OR EXTRA-CARTILAGINOUS ENZYMES

EROSION OF CARTILAGE

J

INCREASED DEGRADATION OF PROTEOGLYCANS

f

DECREASED CONCENTRAT ION OF PROTEOGLYCANS

G

I

PROL IFERAT ION OF CHONDROCMES LOCALLY INCREASED SYNTHESIS B, OF PROTEOGLYCA NS PROLIFERATION OF BONE CELLS

LOSS OF ELASTICITY AND SELF-LUBRICATION PROPERTIES

Fig 1. Working hypothesis on series of events leading to the pathologic picture of osteoarthritis. Reprodured by permission of f h e authors and Transactions of the Association of American Physicians.

lage in nonweight-bearing sites due to increased joint congruence with aging (7) or from conversely abnormal joint development with unfavorable biomechanical effects during the aging process (8). Controversy has arisen as to whether a n osteoarthritic lesion in weight-bearing sites is merely an extension of the same kind of degenerative lesions with eventual ulceration and cartilage denudation (as observed in the aforementioned region), or whether such osteoarthritic lesions represent somewhat different pathologic processes (3,6). New interest focuses on whether subchondral bone-stiffening is a pathogenetically important factor that develops in the earliest stages of osteoarthritis (9), and whether in females at menopause the osteoarthritis of fingers with an apparent inheritable pattern (lo), as well as a postmenopausal polyarticular syndrome described by Kellgren (1 l), should be considered separate as to etiology. To what extent is bony remodeling at joint margins a “normal” function of aging (12)? Is this remodeling principally stimulated secondary to the action of cartilage degradation products in the course of osteoarthritis, or does remodeling occur without intervention of cartilage degradation (3, 12,13)? Are immunologic phenomena involved in osteoarthritis? It is difficult to choose from the various hypotheses on the origin of osteoarthritis any single scientific arena within which to focus research for a medical prophylactic or treatment regimen. At present, no medical measures, except possibly in the realm

of physical therapy, home exercise programs, and measures for prevention of microtrauma, can be hoped to improve cartilage nutrition, indirectly influence joint congruity, and retard subchondral bone stiffening. Advances in surgical management have been remarkable, particularly in the area of total joint replacement, but these will not be reviewed here. Where, then, to direct new efforts in pharmacotherapy? Bollet postulated that, regardless of specific plausible multiple etiologies of primary osteoarthritis, there is a final common pathway of cartilage degradation (Figure 1) (14). By this view, the final common pathway leads to cartilage cellular injury whether from physical, nutritional, or toxicologic sources. There ensues surface abrasion or entry of synovial fluid enzymes and release of cell enzymes with destruction of cartilage matrix. T h e documented loss or alteration of proteoglycans in several studies has produced some general support for the above theory (15-20). T h e finding of accelerated cartilage repair in osteoarthritic cartilage (21) of early to moderate severity, as opposed to advanced severity, and the concurrent maintenance of total proteoglycan content in early disease are strongly suggestive of a response to accelerated breakdown of such cartilage. Among the unsolved problems have been: a) the nature of enzymes involved in normal human articular cartilage catabolism, and b) the combination of enzymes that starts and continues the degradation in osteoarthritis. Could potent irreversible inhibi-

169

DEGRADATIVE ENZYMES

TIDE MARK

Fig 2. Schematized section of an osteoarthritic ulceration indicating site of tissue collection. Reproduced by permission of the authors and the Journal of Clinical Investigation.

tors of degradative enzymes be utilized successfully to prevent or to retard the disease? The answers to such questions are being studied further in several laboratories, and my emphasis tonight is placed on the efforts from our laboratory and from other workers collaborating with us; literature review will be abbreviated.

TISSUE COLLECTION Our recent approach has been to use an arthroscope to obtain fresh cartilage at the time of a diagnostic study, and to save very minute slivers of fresh tissue for histologic and biochemical evaluation. As employed here, arthroscopy displayed other advantages because it could be performed on an outpatient basis using a local anesthetic. The direct viewing of apparently normal versus fibrillated cartilage at biopsy allowed clear-cut sampling of certain regions. The osteoarthritic cartilage was sampled just beyond the border of an ulcerative lesion that seemed the most plausible site at which to detect early biochemical events (Figure 2). For control cartilage we routinely sampled nonweight-bearing* regions of the *Differences in glycosaminoglycan composition between weightbearing and nonbearing sites of a joint were recently described (22).

intercondylar fossa of the knee. All arthroscopic sampling was incidental to diagnostic studies. However, the number of such studies in which this sampling could be taken ethically has been limited. The second source of samples was open surgery on knees in which disease was often more advanced and total knee replacement contemplated. Collection during surgery was necessary to obtain an adequate number of samples; effects of tourniquets, premedications, and general anesthetics, as well as tissue trauma of the operation, admittedly constituted uncontrolled variables that we believe were not serious in view of the currently studied aspects of cartilage metabolism. After tissue collection the dissected cartilage was quick-frozen in alcohol-acetone mixture and saved for study of the state of proteoglycans and of enzymatic factors that might be degrading tissues, and for other studies described below.

THE PROTEOGLYCANS OF OSTEOARTHRITIC CARTILAGES Proteoglycans comprise about one-half the d r y weight of cartilage and, because of their biologic importance in understanding osteoarthritis, I must digress briefly to describe proteoglycan structure (Figure 3). This subject has been studied intensively and in

170

several laboratories has led to considerable recent advances, which are discussed in detail elsewhere (23-34). Electron micrographs of the proteoglycan molecules, spread out in a monolayer on a cytochrome C solution by Drs. Rosenberg, Hellman, and KleinSchmidt (33), have the startling appearance of a cluster of Christmas trees (Figure 3). T h e “trunk” of each “tree” is thought to comprise a core protein of a proteoglycan subunit (Figure 4). T h e symmetrically arranged “branches” of the tree correspond to multiple linear glycosaminoglycans. The cluster comprises a proteoglycan aggregate first discovered and defined by Hascall and Sajdera (24,25). By one hypothesis the aggregate appears to be comprised of subunits, attached at intervals along a linear cartilage hyaluronate molecule (28), probably stabilized at each linkage site by a link glycoprotein (32). Each subunit molecule of proteoglycan consists of core proteins of molecular weight (about 200,000 Daltons).’ Linear glycosaminoglycans (GAG) of molecular weight 1 1,00036,000 are attached to each subunit core protein (35, 36). The chemical nature of the GAG-core protein link has been characterized (30), and the protein core-hyaluronate link is under intensive investigation (28). Aggregates consisting of proteoglycan subunits, collagen, and 2.7 S protein are found in bovine articular cartilage (34). Among the exciting features of the proteoglycan molecules are their enormous size, and their physical properties, which make them ideal “stuffing material” for the interstices between cartilage collagen fibers. Proteoglycans are largely responsible for reduced diffusion of all but select small molecules through cartilage matrix, as shown by Maroudas (37). Thus, their size drastically reduces diffusion of large molecules through cartilage matrix (37). Plasma proteins, except perhaps small amounts of albumin, are totally excluded.? The elasticity of the cartilage was shown by Harris et al to be dissipated by selective degradation of proteoglycans, whereas collagen provided for the integrity of cartilage shape (39). Also, breakdown of such proteoglycans might interfere, speculatively, with the reparative response of collagen in osteoarthritic lesions, a process thought to be in*Because of their polydisperse nature, no exact figure for molecular size of subunits or aggregates can be given. tInfhmmatory cartilage degradation permits entry of synovial fluid and plasma immunoglobulins -I- complexes into surface cartilage layers with important implications in respect to sequestered antigens (88); however, in osteoarthritis, cartilage has not been shown to sequester such deposits.

HOWELL

Fig 3. Top. Electron micrograph of a proteoglycan subunit molecule. A 3-dimensional structure is converted to a flat form by spreading the molecule in a surface film in a solution of cytochrome C . Arrows point to chondroitin sulfate side chains attached to a protein core whose appearance suggests a helical conformation. Bottom. (A) Similar electron micrograph showing a star-shaped aggregate proteoglycan form, and (B), larger aggregate form. Reproduced by permission of the authors and the Journal of Biological Chemistry.

appropriate in osteoarthritis. Certainly, hallmark p e p tides (35) of cartilage collagen [a1(1I)l3 are replaced by repair tissue of osteoarthritic cartilage [al(I)],(as)l bone collagen peptides (40). In these cartilages the half life of proteoglycan “5s has been estimated to be 20-500 days (41,42). It seems highly desirable to measure proteoglycan molecular size distributions in the early nonulcerative lesions. Such measurements would give a means for measuring disaggregation and degradation of proteoglycans by any factors that could be isolated

DEGRADATIVE ENZYMES

171

PROTEOGLYCAN COMPLEX (MW 26-40x106D) (PGC) I

. 1

KERATAN SO^ 7I CHONDROITIN 6-504 4-SO4

I

SUBUNIT

€

MW 2 ~ 1 D0 ~

-

GLYC(Ir:TEIN

HYALURONATE

I

-