Variations in site and levels of expression of chondrocyte nucleotide pyrophosphohydrolase with aging.

The aim of this study was to identify changes in cartilage intermediate layer protein/nucleotide pyrophosphohydrolase (CILP/NTPPH) expression in articular cartilage during aging. Adult (3-4 years old) and young (7-10 days old) porcine articular hyaline cartilage and fibrocartilage were studied by Northern blot analysis, in situ hybridization, and immunohistochemistry using a complementary DNA (cDNA) probe encoding porcine CILP/NTPPH and antibody to a synthetic peptide corresponding to a CILP/NTPPH sequence. Northern blot analysis of chondrocytes showed lower expression of CILP/NTPPH messenger RNA (mRNA) in young cartilage than in adult cartilage. In adult cartilage, extracellular matrix from the surface to the middeep zone was immunoreactive for CILP/NTPPH, especially in the pericellular matrix surrounding the middeep zone chondrocytes. In young cartilage, chondrocytes were moderately immunoreactive for CILP/NTPPH throughout all zones except the calcified zone. The matrix of young cartilage was negative except in the superficial zone. In young cartilage, CILP/NTPPH mRNA expression was undetectable. In adult cartilage, chondrocytes showed strong mRNA expression for CILP/NTPPH throughout middeep zones. Protein and mRNA signals were not detectable below the tidemark. CILP/NTPPH secretion into matrix around chondrocytes increases with aging. In this extracellular site it may generate inorganic pyrophosphate and contribute to age-related calcium pyrophosphate dihydrate crystal deposition disease.

Molecular cloning and expression of a porcine chondrocyte nucleotide pyrophosphohydrolase.

The porcine 127-kDa nucleotide pyrophosphohydrolase (NTPPHase) had been previously purified from the conditioned culture media of porcine articular cartilage. Protein sequencing of an internal 61-kDa proteolytic fragment of NTPPHase (61-kDa NTPPHase) determined the 26 N-terminal amino acids. This sequence was used to amplify a DNA fragment, which was used as a probe to clone the gene encoding the 61-kDa NTPPHase from a porcine chondrocyte cDNA library. DNA sequence analysis showed the cDNA insert to be 2509 bp, corresponding to a predicted open reading frame (ORF) encoding 599 amino acids. The 26 N-terminal amino acids of the 61-kDa NTPPHase were located within the ORF immediately downstream of a putative protease recognition region, RRKRR. This is consistent with this cDNA insert representing an internal proteolytic fragment of the full length 127-kDa NTPPHase. BLAST and FASTA analysis confirmed that the deduced amino acid sequence of 61-kDa NTPPHase was unique and did not possess a high degree of homology to sequence in the non-redundant protein and nucleotide databases. Proteins that possess limited homology (< 17%) with the 61-kDa NTTPPHase include several prokaryotic and eukaryotic ATP pyrophosphate-lyases (adenylate cyclase). Northern blot analysis of porcine chondrocyte RNA showed that the DNA encoding the 61-kDa NTPPHase hybridized to a single 4.0-kb RNA transcript. This DNA probe also hybridized to a single species of human chondrocyte RNA. Expression of a 61-kDa protein was detected by coupled in-vitro transcription/translation. Western blot analysis of this in-vitro transcription/translation reaction detected a 61-kDa protein, using an antibody raised against the peptide sequence that was originally used to clone the 61-kDa NTPPHase. These data indicate the successful in-vitro cloning and expression of the porcine chondrocyte 61-kDa NTPPHase. Future studies that utilize the gene encoding the 61-kDa NTPPHase may allow the characterization of the role of NTPPHase in calcium pyrophosphate dihydrate (CPPD) crystal deposition disease.

Crystals and arthritis.

Monosodium urate, calcium pyrophosphate dihydrate, and basic calcium phosphate (carbonate-substituted hydroxyapatite and octacalcium phosphate) crystal aggregates are associated with gout, pseudogout, and cartilage degeneration (osteoarthritis, Milwaukee Shoulder/Knee Syndrome), respectively. Hyperuricemia is a frequent but nonspecific and inconstant feature of gout just as an elevated synovial fluid inorganic pyrophosphate level is an inconstant feature of pseudogout. Monosodium urate, calcium pyrophosphate dihydrate, or basic calcium phosphate crystals can cause acute inflammation associated with phagocytosis by neutrophilic leukocytes. Each induces neutral protease synthesis and secretion and arachidonic acid metabolism by synoviocytes and macrophages in a dose-dependent fashion, postulated to produce the damage to bone, cartilage, and other joint tissues that is perceived clinically as tophaceous destruction or degenerative joint disease. Crystals containing calcium are potent mitogens. All three types of crystals are more common in older persons and will attract additional attention as the mean age of our population increases. Gout is perhaps the most treatable disease in medicine, although mistakes in diagnosis and in choice of appropriate therapy are very common. Acute pseudogout and acute calcific periarthritis are readily treated medically, but the chronic effects of crystals containing calcium are not. New approaches using drugs derived from scientific study of the biologic effects of these crystals may become useful therapeutically.

Effect of topical capsaicin in the therapy of painful osteoarthritis of the hands.

Topical capsaicin 0.075% was evaluated for the treatment of the painful joints of rheumatoid arthritis (RA) and osteoarthritis (OA) in a 4 week double blind, placebo controlled randomized trial. Twenty-one patients were selected, all of whom had either RA (n = 7) or OA (n = 14) with painful involvement of the hands. Assessments of pain (visual analog scale), functional capacity, morning stiffness, grip strength, joint swelling and tenderness (dolorimeter) were performed before randomization. Treatment was applied to each painful hand joint 4 times daily with reassessment at 1, 2 and 4 weeks after entry. One subject did not complete the study. Capsaicin reduced tenderness (p less than 0.02) and pain (p less than 0.02) associated with OA, but not RA as compared with placebo. A local burning sensation was the only adverse effect noted. These findings suggest that topical capsaicin is a safe and potentially useful drug for the treatment of painful OA of the hands.

Arthritis associated with calcium oxalate crystals in an anephric patient treated with peritoneal dialysis.

We report a case of calcium oxalate arthropathy in a woman undergoing intermittent peritoneal dialysis who was not receiving pharmacologic doses of ascorbic acid. She developed acute arthritis, with calcium oxalate crystals in Heberden's and Bouchard's nodes, a phenomenon previously described in gout. Intermittent peritoneal dialysis may be less efficient than hemodialysis in clearing oxalate, and physicians should now consider calcium oxalate-associated arthritis in patients undergoing peritoneal dialysis who are not receiving large doses of ascorbic acid.

Crystal identification in human synovial fluids. Methods and interpretation.

Gout is largely solved, both from diagnostic and therapeutic standpoints. Acute gout is easily suppressed and joint destruction can be prevented and at least reversed by lowering the serum uric acid level with relatively safe and very effective drugs. But the arthritides associated with the calcium-containing crystals remain untreatable by other than symptomatic or surgical means. If we had a method or a drug to remove CPPD or BCP crystal deposits from joints, would it make any difference in the severity of the arthritis? Which of the paradigms shown in Figure 5 holds for these crystals? If joint damage directly follows crystal deposition as in gout, then crystal removal should prove prophylactic. The unusual pattern of joint degeneration associated with polyarticular CPPD crystal deposition and the initial appearance of CPPD crystals in radiographically normal cartilage favors this idea. But radiologic chondrocalcinosis appearing in knees subjected years before to meniscectomy but not in the contralateral knees suggests that crystal deposition, in these cases at least, is secondary to trauma or surgery. If degeneration of cartilage precedes crystal deposition, as it probably does in the case of BCP crystals, then crystal removal may not be particularly helpful. Dieppe and his colleagues proposed that the calcium crystals provide a positive feedback (amplification) loop. This represents the minimalistic view of their importance. The biologic consequences of the calcium crystal deposition diseases are now being explored at the molecular level. Much more data are needed before more than speculative answers to the questions posed here can be formulated. Calcium crystal deposition is more common in older persons. The degenerative and destructive arthropathies associated with them will predictably become increasingly common as our population ages.

Clinical aspects of basic calcium phosphate crystal deposition.

Our present understanding of the mechanisms of pathologic calcification is quite limited. Therefore, no reliable method exists to prevent calcium crystal deposition. Low doses of warfarin have been employed in some cases of soft tissue calcification because it depresses the synthesis of the vitamin K-dependent GLA protein (gamma carboxyglutamic acid), which has been implicated in the process of calcification. Reports of success must be tempered by the lack of a controlled study. Probenecid has also been utilized in the treatment of calcinosis.

Arthritis associated with crystals containing calcium.

Varying combinations of acute inflammatory and/or chronic degenerative arthritis have been found to be associated with crystals of calcium pyrophosphate dihydrate (CPPD) and/or basic calcium phosphates (BCPs). Since the arthropathies associated with CPPDs and/or BCPs occur in older individuals, while diagnosis and treatment for monosodium urate monohydrate crystal deposition disease (gout) have become extremely precise and effective, joint problems associated with calcium crystals have become more common than those associated with monosodium urate monohydrate crystals. The classification, pathogenesis, clinical manifestations, and treatment of CPPD and BCP crystal deposition are discussed.

Identification of collagen subtypes in synovial fluid sediments from arthritic patients.

Collagen fibers in synovial fluid sediment were described a decade ago. Since then, tissue-specific collagen molecules (types) have been characterized. Techniques were devised to identify the collagen types in joint fluid sediment. Collagens were found in 12 of 17 pellets prepared from fluid aspirates from 17 knee joints of patients with various forms of arthritis. Collagen types I and III and polypeptide chains A and B (basement membrane collagen) were specifically identified in four of seven fluids from patients with active systemic lupus erythematosus (SLE) and in a single fluid from a patient with severe septic arthritis. This "collagen profile" was identical to that of rheumatoid synovium. Type II collagen, characteristic of hyaline articular cartilage, was found in two of six fluids from osteoarthritic joints. The presence of sufficient collagen (about 5 micrograms) to permit typing was correlated with roentgenographic evidence of joint space narrowing; the presence of the "synovial" collagen profile was correlated with decreased joint fluid pH.

Clearance of calcium pyrophosphate dihydrate crystals in vivo. I. Studies using 169Yb labeled triclinic crystals.

Synthetic triclinic calcium pyrophosphate dihydrate crystals were uniformly trace-labeled with Ytterbium-169 (169Yb), a pure gamma-emitting isotope with a halflife of 31 days. The solubility of the labeled crystals was similar to that of cold synthetic crystals. The clearance rate of labeled sterile crystals, sieved to obtain the desired size, was determined after injection of microgram quantities into 4 arthritic huuman and 3 normal adult rabbit joints and corrected by the observed rate of clearance of free 169Yb. The derived rate constants were then used to calculate the time required for half of the injected dose of CPPD to be cleared from the joint. Crystal clearance was found in all instances. Crystal removal from normal rabbit joints was much more rapid than from the much larger human arthritic joints and was inversely proportional to the size of the crystals injected.

Crystal shedding and acute pseudogout. An hypothesis based on a therapeutic failure.

Four patients with chondrocalcinosis of the knees volunteered for joint lavage. Preliminary experiments indicated that disodium EDTA and magnesium ions were potent solubilizers of CPPD crystals. The procedure was a therapeutic failure in that insignificant amounts of CPPD were removed and all 4 subjects developed postlavage attacks of pseudogout. It is hypothesized that the acute attack of pseudogout is a result of crystal shedding and may be triggered by any factor that enhances CPPD solubility.

Inorganic pyrophosphate pool size and turnover rate in arthritic joints.

Recent studies have shown elevated inorganic pyrophosphate (PPi) levels in most knee joint fluid supernates from patients with pseudogout (PG) or osteoarthritis (OA) and more modestly elevated levels in some supernates from patients with gout or rheumatoid arthritis (RA) relative to PPi levels found in the venous blood plasma of normal or arthritic subjects. We measured the intraarticular PPi pool and its rate of turnover to better understand the significance of the joint fluid-plasma PPi gradient. Preliminary studies in rabbits showed that (32-P)PPi passed from joint space to blood and vice versa without detectable hydrolysis. Incubation of natural or synthetic calcium pyrophosphate dihydrate (CPPD) microcrystals with synovial fluid in vitro in the presence of (32P)PPi tracer showed no change in PPi specific activity in the supernate over a 19-h period so that exchange of PPi in solution with that in CPPD microcrystals could be ignored. Clearance rates of (32P)PPi and of (33P)Pi, as determined by serially sampling the catheterized knee joints of volunteers with various types of arthritis over a 3-h period, were nearly identical. The (32P)PPi/(32P)Pi was determined in each sample. A mixture of a large excess of cold PPi did not influence the clearance rate of either nuclide. The quantity of PPi turned over per hous was calculated from the pool size as determined by isotope dilution and the turnover rate. The residual joint fluid nuclide was shown to be (32P)PPi. The PPi pool was generally smaller and the rate of turnover was greater in clinically inflamed joints. The mean plus or minus SEM pool size (mu-moles) and turnover rate (percent/hour) in PG knees was 0.23 plus or minus 0.07 and 117 plus or minus 11.9, hydrolysis rate (%/h) to Pi was 27.7 plus or minus 13.2; in OA knees: 0.45 plus or minus 0.26 and 72 plus or minus 9.2, hydrolysis 6.9 plus or minus 0.9; in gouty knees: 0.8 plus or minus 0.41 and 50 plus or minus 11.6, hydrolysis 9.8 plus or minus 2.8; and in RA knees: 0.14 plus or minus 0.14 and 114 plus or minus 35.8, hydrolysis 236 plus or minus 116. PPi turnover (mumoles/hour) correlated with the degree of OA change present in the joint as graded by radiologic criteria irrespective of the clinical diagnosis. Mean PPi turnover in joints with advanced OA was greater than in those with mild or moderate changes (P smaller than 0.001), but the mild and moderate groups showed no significant difference. We conclude that synovial PPi turnover and elevated PPi fluid concentrations are not specific for PG patients, and that these factors alone cannot be the only determinants of CPPD crystal deposition.