Increased expression of carbonic anhydrase I in the synovium of patients with ankylosing spondylitis.

BACKGROUND: One of the most distinctive features of ankylosing spondylitis (AS) is new bone formation and bone resorption at sites of chronic inflammation. Previous studies have indicated that the hyperplasia and inflammation of synovial tissues are significantly related to the pathogenic process of AS. The present study used a proteomic approach to identify novel AS-specific proteins by simultaneously comparing the expression profiles of synovial membranes from patients with AS, rheumatoid arthritis (RA) and osteoarthritis (OA). METHODS: Synovial tissues were collected from the hip joints of patients with AS and knee joints of patients with RA or OA (n = 10 for each disease) during joint replacement surgery. Proteins extracted from the synovial tissues were separated by 2-D electrophoresis (2-DE), and the proteins with significantly increased expression in the AS samples were subjected to MALDI-TOF/TOF-MS analysis. The results were verified using western blotting and immunohistochemistry. Levels of the candidate proteins in synovial fluids from knee joints (n = 40 for each disease) were measured using ELISA. RESULTS: The proteomic approach revealed significantly increased expression of carbonic anhydrase I (CA1) in the synovial membrane of patients with AS as compared with the RA and OA tissue samples. Immunohistochemistry and western blotting analysis confirmed the findings described above. The ELISA detected a higher level of CA1 in synovial fluids from patients with AS than those with OA. The mean value of the CA1 level was also higher in AS patients as compared with RA patients. This study also detected increased expression of alpha-1-antitrypsin in the synovial tissues from AS patients, which is in agreement with other reports. CONCLUSION: In vitro experiments by other groups indicated that CA1 catalyzes the generation of HCO3- through the hydration of CO2, which then combines with Ca2+ to form a CaCO3 precipitate. Calcification is an essential step of bone formation. Substantial evidence indicates that carbonic anhydrase also stimulates bone resorption. Hence, overexpression of CA1 in the synovial tissues of AS patients may promote improper calcification and bone resorption in AS.

Expression and activity of COX-1 and 2 and 5-LOX in joint tissues from dogs with naturally occurring coxofemoral joint osteoarthritis.

Understanding the neurobiology of pain in naturally occurring models of osteoarthritis (OA) may improve the understanding of human OA pain. Both COX and LOX have been associated with joint pain. This study evaluated COX-1, COX-2, and 5-LOX expression and activity in a naturally occurring canine model of secondary OA. Hip joint capsule with synovial tissue (HJC) and femoral head subchondral bone (FH) was collected from normal dogs and dogs undergoing total hip replacement for coxofemoral joint OA. Tissues were analyzed for COX-1, COX-2, and LOX protein, and PGE(2) and LTB(4). Significantly more COX-2 protein was present in OA HJC than normal joints (p = 0.0009). There was no significant difference in COX-1 or LOX protein, although LOX protein was increased (p = 0.069). PGE(2) concentration in normal and OA HJC was similar (p = 1.0). LTB(4) concentration in OA HJC was significantly greater than normal HJC (p = 0.028). Significantly more COX-1 (p = 0.0098), COX-2 (p = 0.0028), and LOX (p = 0.0095) protein was present in OA FH tissue compared to normal FH tissue. There were no differences in PGE(2) or LTB(4) concentration in normal and OA FH tissue (p = 0.77 and p = 0.11). Together, these data suggest both COX-2 and 5-LOX are appropriate targets for the management of pain associated with naturally occurring OA.

Double-knockout of ADAMTS-4 and ADAMTS-5 in mice results in physiologically normal animals and prevents the progression of osteoarthritis.

OBJECTIVE: To phenotypically characterize ADAMTS-4- and ADAMTS-5-double-knockout mice, and to determine the effect of deletion of ADAMTS-4 and ADAMTS-5 on the progression of osteoarthritis (OA) in mice. METHODS: Mice lacking the catalytic domain of ADAMTS-4 and ADAMTS-5 were crossed to generate ADAMTS-4/5-double-knockout animals. Twelve-week-old and 1-year-old male and female ADAMTS-4/5-double-knockout mice were compared with age- and sex-matched wild-type (WT) mice by evaluating terminal body weights, organ weights, clinical pathology parameters, PIXImus mouse densitometry findings, and macroscopic and microscopic observations. ADAMTS-4/5-double-knockout mice were challenged by surgical induction of joint instability to determine the importance of these genes in the progression of OA. Articular and nonarticular cartilage explants from WT and ADAMTS-4/5-double-knockout mice were treated with interleukin-1 (IL-1) plus retinoic acid ex vivo, to examine proteoglycan degradation. RESULTS: There were no genotype-related phenotype differences between ADAMTS-4/5-double-knockout and WT mice through 1 year of age, with the exception that female ADAMTS-4/5-double-knockout mice had a lower mean terminal body weight at the 12-week time point. Eight weeks after surgical induction of joint instability, OA was significantly less severe in ADAMTS-4/5-double-knockout mice compared with WT mice. Following stimulation of cartilage explants with IL-1 plus retinoic acid, aggrecanase-mediated degradation in ADAMTS-4/5-double-knockout mice was ablated, to a level comparable with that in ADAMTS-5-knockout mice. CONCLUSION: Dual deletion of ADAMTS-4 and ADAMTS-5 generated mice that were phenotypically indistinguishable from WT mice. Deletion of ADAMTS-4/5 provided significant protection against proteoglycan degradation ex vivo and decreased the severity of murine OA. These effects in the ADAMTS-4/5-double-knockout mice were comparable with those observed with deletion of ADAMTS-5 alone.

Changes in immunolocalisation of beta-dystroglycan and specific degradative enzymes in the osteoarthritic synovium.

OBJECTIVE: To investigate the immunolocalisation of beta-dystroglycan (beta-DG) and specific matrix metalloproteinases (MMPs)-3, -9, -13 and a disintegrin like and metalloproteinase thrombospondin type 1 motif 4 (ADAMTS-4) within the joint tissues of patients with osteoarthritis (OA) and unaffected controls. DESIGN: Cartilage, synovium and synovial fluid were obtained from the hip joints of five osteoarthritic (patients undergoing total hip replacement) and five control hip joints (patients undergoing hemiarthroplasty for femoral neck fracture). The samples were analysed for beta-DG protein using Western blot technique and by immunohistochemistry for tissue distribution of beta-DG, MMP-3, -9, -13, and ADAMTS-4. RESULTS: beta-DG was detected in the smooth muscle of both normal and osteoarthritic synovial blood vessels. Importantly, beta-DG was detected in endothelium of blood vessels of OA synovium, but not in the control endothelium. In the endothelium of osteoarthritic synovial blood vessels, beta-DG co-localised with MMP-3 and -9. MMP-13 and ADAMTS-4 showed no endothelial staining, and only weak staining of the vascular smooth muscle was found. In contrast, we did not detect beta-DG protein in cartilage or synovial fluid. CONCLUSIONS: beta-DG has been shown to have a role in angiogenesis, and our results demonstrate for the first time that there are clear differences in beta-DG staining between OA and control synovial blood vessels. The specific immunolocalisation of beta-DG within endothelium of inflamed OA blood vessels and its co-localisation with MMP-3 and -9, reported to have pro-angiogenic roles and believed to be involved in beta-DG cleavage, may also suggest that beta-DG plays a role in angiogenesis accompanying OA.

Mithramycin downregulates proinflammatory cytokine-induced matrix metalloproteinase gene expression in articular chondrocytes.

Interleukin-1 (IL-1), IL-17 and tumor necrosis factor alpha (TNF-alpha) are the main proinflammatory cytokines implicated in cartilage breakdown by matrix metalloproteinase (MMPs) in arthritic joints. We studied the impact of an anti-neoplastic antibiotic, mithramycin, on the induction of MMPs in chondrocytes. MMP-3 and MMP-13 gene expression induced by IL-1beta, TNF-alpha and IL-17 was downregulated by mithramycin in human chondrosarcoma SW1353 cells and in primary human and bovine femoral head chondrocytes. Constitutive and IL-1-stimulated MMP-13 levels in bovine and human cartilage explants were also suppressed. Mithramycin did not significantly affect the phosphorylation of the mitogen-activated protein kinases, extracellular signal-regulated kinase, p38 and c-Jun N-terminal kinase. Despite effective inhibition of MMP expression by mithramycin and its potential to reduce cartilage degeneration, the agent might work through multiple unidentified mechanisms.

Investigation of alcohol metabolizing enzyme genes in Chinese alcoholics with avascular necrosis of hip joint, pancreatitis and cirrhosis of the liver.

AIMS AND METHODS: Alcoholism may cause a range of diseases including avascular necrosis of the hip joint (AVN), cirrhosis of the liver, pancreatitis and oesophageal carcinoma. Chinese alcoholic patients diagnosed with AVN have a higher incidence of cirrhosis than of acute pancreatitis or oesophageal cancer. Thus, the aim of this study was to investigate genetic differences in polymorphisms of the alcohol-metabolizing enzymes ADH2, ADH3, ALDH2 and P4502E1 for subgroups of Chinese alcoholic patients, defined by diagnoses of AVN (n = 51), acute pancreatitis (n = 92) and liver cirrhosis (n = 159), and for 280 non-alcoholic patients. RESULTS: Analysis revealed that ADH2*1 allele frequency was significantly lower for the alcoholic AVN than for the cirrhosis subgroup. However, no significant difference was found between the alcoholic AVN and pancreatitis subgroups. Furthermore, ALDH2*2 prevalence was not found to differ significantly between the alcoholic subgroups. When compared with our previously published data for alcoholic patients with oesophageal carcinoma, ADH2*1 carriage was significantly less frequent for the alcoholic AVN patients in the current study. Further, ALDH2*2 carriage was significantly less frequent for the alcoholic AVN subgroup than for the oesophageal carcinoma patients. CONCLUSIONS: The allele frequencies for ADH2*1 and ALDH2*2 are different when comparing subpopulations of alcoholics defined by presence of specific alcohol-induced diseases, suggesting that genetic variation in alcohol-metabolizing enzyme genes accounts for, at least in part, the specific types of organ damage observed. We also found the combination of AVN and cirrhosis to be more prevalent than that of AVN and acute pancreatitis. In contrast, the ADH2 and ALDH2 allele frequencies for the AVN subgroup were more similar to those of the acute-pancreatitis than to the cirrhosis subgroup. These data indicate the possibility that other genetic variations may also influence the type of organ-specific complications in Chinese alcoholics.

Nitric oxide synthase in tissues around failed hip prostheses.

Nineteen patients who had undergone hip revision surgery for aseptic loosening of joint prostheses were studied. Tissue samples were harvested at the interface between bone and implant, either at the stem or at the cotyle level. Immunohistochemistry was performed on tissue sections to detect nitric oxide synthase (NOS), the enzyme which enables the synthesis of nitric oxide (NO), a molecule which can activate bone resorption. Quantitative analysis of the positive cells and correlation with the presence of particulate wear debris and radiological data were performed. The authors observed a trend towards a moderate increase in positive cells due to inducible NOS in tissues containing particulate wear debris, especially of a plastic material. This increase, however, did not achieve statistical significance. On the contrary, there was a statistical correlation between iNOS (inducible NOS) and the severity of osteolysis around the prosthetic implant. Pharmacological control of the biosynthesis of NO may be considered in the prevention or treatment of loosening.

Patterns of osteocytic endothelial nitric oxide synthase expression in the femoral neck cortex: differences between cases of intracapsular hip fracture and controls.

Evidence indicates that extensive amalgamation of adjacent resorbing osteons is responsible for destroying the microstructural integrity of the femoral neck's inferior cortex in osteoporotic hip fracture. Such osteonal amalgamation is likely to involve a failure to limit excessive resorption, but its mechanistic basis remains enigmatic. Nitric oxide (NO) inhibits osteoclastic bone destruction, and in normal bone cells its generation by endothelial nitric oxide synthase (eNOS, the predominant bone isoform) is enhanced by mechanical stimuli and estrogen, which both protect against fracture. To determine whether eNOS expression in osteocytes reflects their proposed role in regulating remodeling, we have examined patterns of osteocyte eNOS immunolabeling in the femoral neck cortex of seven cases of hip fracture and seven controls (females aged 68-96 years). The density of eNOS+ cells (mm(-2)) was 53% lower in the inferior cortex of the fracture cases (p < 0.0004), but was similar in the superior cortex. eNOS+ osteocytes were, on average, 22% further from their nearest blood supply, than osteocytes in general (p < 0.0001) and the nearest eNOS+ osteocyte was 57% further from its nearest canal surface (p < 0.0001). This differential distribution of eNOS+ osteocytes was significantly more pronounced in the cortices of fracture cases (p < 0.0001). We conclude that the normal regional and osteonal pattern of eNOS expression by osteocytes is disrupted in hip fracture, particularly at sites that are loaded most by physical activity. These results suggest that eNOS+ osteocytes may normally act as sentinels confining resorption within single osteons. A reduction in their number, coupled to an increase in their remoteness from canal surfaces, may thus permit the irreversible merging of resorbing osteons, and thus contribute to the marked increase in the fragility of osteoporotic bone.

High levels of expression of collagenase-3 (MMP-13) in pathological conditions associated with a foreign-body reaction.

A foreign-body-type host response can contribute to the induction and release of collagenolytic tissue-destructive enzymes of pathogenetic significance. Our aim was to analyse collagenase-3 in two conditions with putative involvement of foreign-body reactions. Synovial membrane-like tissue samples were obtained from cases of aseptic loosening of a total hip replacement (THR) and osteoarthritis (OA). The reverse transcription polymerase chain reaction (RT-PCR) disclosed that all the samples from patients contained collagenase-3 mRNA compared with only three out of ten control samples. The identity of the RT-PCR amplification product was confirmed by nucleotide sequencing. Immunohistochemical staining showed that collagenase-3 was present in endothelial cells, macrophages and fibroblasts, including those found in the synovial lining. This finding was confirmed by avidin-biotin-peroxidase complex-alkaline phosphatase-anti-alkaline phosphatase double staining and the specificity of the staining by antigen preabsorption using recombinant human collagenase-3. Collagenase-3 was released into the extracellular space and thus found in the synovial fluid in all patient samples as shown by Western blotting. The similar extent of collagenase-3 expression in aseptic loosening and OA compared with the low expression in control synovial membrane suggests involvement of a similar, foreign-body-based pathogenetic component in both. Comparative analysis of collagenase-3 and of foreign particles indicates that paracrine factors rather than phagocytosis per se are responsible for the induction of collagenase-3. We suggest that due to its localisation and substrate specificity, collagenase-3 may play a significant pathogenetic role in accelerating tissue destruction in OA and in aseptic loosening of a THR.

Immunolocalization of inducible nitric oxide synthase in synovium and cartilage in rheumatoid arthritis and osteoarthritis.

Nitric oxide has been implicated as a mediator of inflammatory arthritis, and recent work has shown that pro-inflammatory cytokines stimulate NO production in vitro by activation of the inducible nitric oxide synthase (iNOS) pathway. In order to identify the cellular sources of NO production within the joint, we have used immunohistochemical techniques to study the distribution of iNOS in synovium and cartilage from normal and diseased joints. iNOS was most strongly expressed in the synovial lining layer, subsynovium, vascular smooth muscle and chondrocytes from patients with rheumatoid arthritis (RA). Analysis of serial sections, coupled with double immunofluorescent staining, showed that the CD68+ macrophages in the synovial lining layer and, to a lesser extent, fibroblasts were the predominant source of iNOS within synovium, whereas T cells, B cells and neutrophils were negative. A similar pattern of iNOS staining was seen in osteoarthritis, but fewer cells were iNOS positive and the intensity of staining, particularly in cartilage, was much weaker than in RA. In contrast, no evidence of iNOS was detected in non-inflammatory synovium or in cartilage derived from normal joints (fractured neck of femur). In conclusion, these data support the hypothesis that synovium and cartilage are important sources of increased NO production in patients with inflammatory arthritis. Localization of iNOS at these sites within the inflamed joint raises the possibility that increased local production of NO may contribute to the pathogenesis of inflammatory arthritis by increasing synovial blood flow and by modulating cellular function within synovium and articular cartilage.

Role of mesenchymal collagenase in the loosening of total hip prosthesis.

Fibroblast-type interstitial collagenase (E.C. 3.4.24.7) was associated with loosening of total hip prostheses in eight patients: there were four cemented stems and one cementless stem with the common type of loosening and two cemented stems and one cementless acetabular component with aggressive granulomatous lesions. The authors used a specific, well-characterized, heterologous, affinity-purified, polyclonal rabbit anti-human fibroblast collagenase antiserum applied in avidin-biotin-peroxidase-complex (ABC) staining. In the aggressive granulomatous type of loosening, collagenase was found in most of the fibroblast- and macrophagelike cells, including multinuclear giant cells and epithelioid cells in periprosthetic tissue. Collagenase-positive cells also were found in the periprosthetic tissue associated with common loosening. Collagenase was also found in capillary and postcapillary venule endothelial cells in the richly vascularized aggressive granulomatous tissue. Collagenase was extracted directly from the tissue samples and incubated with soluble Type I collagen. Collagen degradation products then were analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, and the three-fourths length degradation product quantitated by gel scanning densitometry. In both aggressive granulomatosis and the common type of loosening, extractable collagenase was found in tissue. No significant differences between the sample groups were detected in respect to total measurable collagenase, however. The extractable collagenase was present in a latent form that could be activated by the organomercurial procollagenase activator, phenylmercuric chloride (PMC). It is likely that interstitial collagenase contributes to rapid growth of reactive infiltrative tissue, loosening of the prosthesis associated with aggressive granulomatosis, and the periprosthetic lytic process associated with the common type of hip prosthesis loosening.

Cellular enzymatic activities in the articular cartilage of osteoarthritic and osteoporotic hip joints of humans: a quantitative cytochemical study.

Quantitative cytochemical methods were applied to measure the activity of several oxidative enzymes in human articular cartilage of the femoral head obtained from osteoarthritic patients (OA) and from patients with fractured femoral neck (OP) due to primary osteoporosis. In both conditions, lactate dehydrogenase (LDH) was found to be the most active one followed by two additional cytosolic enzymes: glyceraldehyde 3-phosphate dehydrogenase (GAPD) and glucose 6-phosphate dehydrogenase (G6PD). On the other hand, the activity of mitochondrial enzymes such as succinate dehydrogenase (SDH) and beta-hydroxyacyl dehydrogenase (HOAD), appeared much lower in degree. Except for HOAD, all the other enzymes exhibited a high degree of activity along the inner zone in the cartilage, i.e., zone 3b, indicative of an apparently more active metabolism in the osteochondral junction. G6PD activity was significantly higher (p less than 0.01) in OP than in OA patients. By contrast, SDH appeared more active in specimens obtained from OA patients. The remaining enzymes showed no appreciable activity differences between cartilages of OP and OA patients. These findings suggest that oxidative enzyme activity in chondrocytes involved in osteoarthritis does not differ substantially from that in cartilage of OP patients.

A histochemical study of alkaline and acid phosphatase activity in subchondral bone from osteoarthrotic human hips.

In order to analyze the presence of alkaline and acid phosphatase activity in osteoarthrotic subchondral bone frozen sections from 24 femoral heads were prepared for semiquantitative analysis, using the enzyme-histochemical methods described by Burstone and by Barka and Anderson. Different areas of the subchondral bone, viz. weight-bearing, nonweight-bearing and osteophytes as well as central regions were analyzed. Furthermore, the cartilagenous changes were determined by histological-histochemical grading. There were wide variations within the same femoral head with significantly greater activity of both alkaline and acid phosphatase in weight-bearing than in nonweight-bearing areas and in subchondral bone than in central regions. Osteophytes excluded, the enzyme activities correlated directly with the severity of the cartilage lesions. These enzyme activities presumably reflect the degree of bone regeneration and bone resorption respectively.

A histochemical study of alkaline and acid phosphatase activity in osteoarthritic synovial membrane.

In order to determine the localization and activity of alkaline and acid phosphatase in the synovial membrane of osteoarthritic hip joints, enzymo-histochemical analyses were performed using Burstone's and Barka & Anderson's methods. Frozen sections of synovial biopsy material from 12 osteoarthritic and 6 control hip joints were studied. Alkaline phosphatase was found located in fibroblasts below the lining cells and in capillaries and precapillary arterioles. Acid phosphatase was seen in the lysosomes in the lining cells. Semiquantitative evaluation by means of initial time determination showed significantly greater activity in osteoarthritic synovia than in the control group. Whilst the increased activity of lysosomal enzymes is presumably implicated in the joint cartilage damage seen in osteoarthritis, the significance of elevated alkaline phosphatase levels is not yet clear.