The effect of neonatal gene therapy on skeletal manifestations in mucopolysaccharidosis VII dogs after a decade.

Mucopolysaccharidosis (MPS) VII is a lysosomal storage disease due to deficient activity of ß-glucuronidase (GUSB), and results in glycosaminoglycan accumulation. Skeletal manifestations include bone dysplasia, degenerative joint disease, and growth retardation. One gene therapy approach for MPS VII involves neonatal intravenous injection of a gamma retroviral vector expressing GUSB, which results in stable expression in liver and secretion of enzyme into blood at levels predicted to be similar or higher to enzyme replacement therapy. The goal of this study was to evaluate the long-term effect of neonatal gene therapy on skeletal manifestations in MPS VII dogs. Treated MPS VII dogs could walk throughout their lives, while untreated MPS VII dogs could not stand beyond 6 months and were dead by 2 years. Luxation of the coxofemoral joint and the patella, dysplasia of the acetabulum and supracondylar ridge, deep erosions of the distal femur, and synovial hyperplasia were reduced, and the quality of articular bone was improved in treated dogs at 6 to 11 years of age compared with untreated MPS VII dogs at 2 years or less. However, treated dogs continued to have osteophyte formation, cartilage abnormalities, and an abnormal gait. Enzyme activity was found near synovial blood vessels, and there was 2% as much GUSB activity in synovial fluid as in serum. We conclude that neonatal gene therapy reduces skeletal abnormalities in MPS VII dogs, but clinically-relevant abnormalities remain. Enzyme replacement therapy will probably have similar limitations long-term.

Paeoniflorin inhibits Rho kinase activation in joint synovial tissues of rats with collagen-induced rheumatoid arthritis.

Paeoniflorin (PF) has many effects, such as anti-inflammation, immune-regulation, abirritation, and so on. However, the protective mechanisms of PF on rheumatoid arthritis (RA) was not completely known. Thus, we explored deeply the protective mechanisms in a collagen-induced RA (CIA) rat model. CIA was induced in rats by intradermal injection of bovine type II collagen in complete Freund's adjuvant. Later, the CIA rats received oral administration of PF (50 and 100 mg/kg) once a day from the day 21, with the treatment lasting for 14 days. A variety of indicators were measured for evaluation of anti-rheumatism effect, including paw swelling, arthritis scores, and histopathological changes. And the contents of pro-inflammatory cytokines, including tumor necrosis factor alpha (TNF-α), interleukin-1 beta (IL-1ß), and interleukin-6 (IL-6) in the serum, as well as p-NF-κB p65 and p-MYPT1 in the joint synovial tissues were detected to explore the possible mechanisms. The results demonstrated that PF treatment significantly ameliorated the symptoms in CIA rats, reduced the levels of pro-inflammatory cytokines and paw swelling, down-regulated the expressions of p-NF-κB p65 and p-MYPT1. The present results revealed that PF could effectively improve collagen-induced RA in rats by inhibiting Rho kinase activation in the joint synovial tissues, in turn down-regulating expression of p-NF-κB p65 and reducing contents of pro-inflammatory cytokines. Moreover, PF may be an effective agent for RA.

Bovine joint capsule and fibroblasts derived from joint capsule express aggrecanase activity.

Bovine joint capsule was maintained in explant culture in the presence of bovine aggrecan monomer and it was shown that the aggrecan monomer was degraded. Amino-terminal sequence analysis of the resulting aggrecan core protein fragments revealed that the core protein was cleaved at five specific sites attributed to glutamyl endopeptidases referred to as aggrecanase activity. Fibroblast cultures were established from explant cultures of joint capsule and when these cells were exposed to aggrecan, cleavage of the core protein of aggrecan at the aggrecanase sites was observed. Inclusion of either retinoic acid or interleukin-1alpha in medium of either joint capsule explant cultures or fibroblast cultures did not increase the rate of cleavage of exogenous aggrecan present in the culture medium. When aggrecan monomer was incubated with conditioned medium from explant cultures of joint capsule maintained in medium, degradation could be detected after 10 min. After a 6-h incubation period the same fragments of aggrecan core protein were observed as those for tissue or cells incubated directly with aggrecan monomer. RT-PCR analysis of mRNA extracted from joint capsule fibroblasts showed that these cells express both aggrecanase-1 and -2 [ADAMTS-2 (Tang) and ADAMTS-5].

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.

Degradative enzymes in osteoarthritis.

A central feature of the osteoarthritic disease process involves erosive destruction of the articular cartilage extracellular matrix (ECM) on the surfaces of diarthrotic joints. The resultant loss of joint function makes studies on mechanisms underlying ECM degradation critical for treatment of the disease and prevention of disability. Candidate pathways to account for the loss of cartilage involve expression of a combination of proteases that degrade the major cartilage matrix macromolecules, aggrecan and type II collagen. The specific types of enzymatic activities associated with the progressive removal of ECM and severity of joint disease include the matrix metalloproteinases, collagenase, gelatinase and aggrecanase(s). The degradative enzymes originate in synovial cells, cartilage cells, the chondrocytes, distributed within the ECM and leukocytes that actively invade the joint space. Specific enzymes arising from each of these tissues exhibit selective ECM degrading properties; the different categories of these tissue-derived enzymes will be discussed in this chapter. A perspective on the efficacy of existing agents and the potential for development of novel therapeutic agents is also included. While the degradative enzymes serve as a focal point for therapeutic intervention, a fundamental understanding of the mechanisms underlying degradative enzyme expression in osteoarthritis remains an important goal for prevention of disease.

Effect of psychological stress on the structure of the temporomandibular joint and the expression of MMP-3 and TIMP-3 in the cartilage in rats.

Our aim was to observe the effects of psychological stress on the structure of the temporomandibular joint (TMJ), and to evaluate the expression of matrix metallopeptidase-3 (MMP-3) and tissue inhibitor of metalloproteinase-3 (TIMP-3) in condylar chondrocytes in rats. The rats were divided into 3 groups of 12 according to the duration of psychological stress: 3 weeks or 6 weeks, and 6 weeks of recovery. A fourth group of 12 rats was used as controls. Each rat was evaluated by the open-field test and the weight measured. The results confirmed psychological stress in 24 of the 36 rats (67%). The tissues of the TMJ were stained with haematoxylin and eosin and pathological changes were studied under a light microscope. MMP-3 and TIMP-3 expression was investigated using the SP kit. The experimental groups showed thinning of articular cartilage, shedding of collagen fibres, cracks in the articular discs, and other structural changes that were aggravated with time, from three weeks to six weeks. The 6-week recovery group showed an improvement in these changes, which indicated the initiation of joint repair. The MMP-3 expression rate correlated with the degree of joint lesion, while the TIMP-3 rate showed an opposite trend and was highest in the 6-week recovery group. Our findings clearly indicate that psychological stress may play an important part in the development of TMJ diseases in rats; further studies should be made to extrapolate the results to other models before clinical use.

Phospholipase D1 has a pivotal role in interleukin-1ß-driven chronic autoimmune arthritis through regulation of NF-κB, hypoxia-inducible factor 1α, and FoxO3a.

Interleukin-1ß (IL-1ß) is a potent proinflammatory and immunoregulatory cytokine playing an important role in the progression of rheumatoid arthritis (RA). However, the signaling network of IL-1ß in synoviocytes from RA patients is still poorly understood. Here, we show for the first time that phospholipase D1 (PLD1), but not PLD2, is selectively upregulated in IL-1ß-stimulated synoviocytes, as well as synovium, from RA patients. IL-1ß enhanced the binding of NF-κB and ATF-2 to the PLD1 promoter, thereby enhancing PLD1 expression. PLD1 inhibition abolished the IL-1ß-induced expression of proinflammatory mediators and angiogenic factors by suppressing the binding of NF-κB or hypoxia-inducible factor 1α to the promoter of its target genes, as well as IL-1ß-induced proliferation or migration. However, suppression of PLD1 activity promoted cell cycle arrest via transactivation of FoxO3a. Furthermore, PLD1 inhibitor significantly suppressed joint inflammation and destruction in IL-1 receptor antagonist-deficient (IL-1Ra(-/-)) mice, a model of spontaneous arthritis. Taken together, these results suggest that the abnormal upregulation of PLD1 may contribute to the pathogenesis of IL-1ß-induced chronic arthritis and that a selective PLD1 inhibitor might provide a potential therapeutic molecule for the treatment of chronic inflammatory autoimmune disorders.

Proteolytic cleavage of versican during limb joint development.

Versican is highly expressed in developing joint interzones during limb morphogenesis. This study was undertaken to examine whether proteolytic cleavage of versican occurs that could potentially impact its function during the process of embryonic synovial joint formation. Using an antibody to the DPEAAE neoepitope generated by ADAMTS proteolysis, versican amino terminal cleavage fragments were detected in joint interzones at 12-16 days post coitum (dpc). ADAMTS-1 localization overlapped that of DPEAAE-reactive versican fragments suggesting it as one possible protease activity involved in processing of versican in the interzone. Results show that increased cleavage of versican in the interzone accompanies cavitation and suggests that proteolytic modification of versican may be important during the process of synovial joint maturation.

Differential stimulation of three forms of hyaluronan synthase by TGF-beta, IL-1beta, and TNF-alpha.

This study compares the regulation of three isoforms of hyaluronan synthase (HAS1, HAS2, and HAS3) transcripts and hyaluronan (HA) production by cytokines in human synovial fibroblastic cells derived from tissue from patients with rheumatoid arthritis (RA) and osteoarthritis (OA). Levels of HAS mRNA of the cells with or without stimulation were detected using a real-time fluorescence polymerase chain reaction detection system. Concentrations of HA in the culture supernatants of the cells were measured by a sandwich binding protein assay. Molecular weight of HA was evaluated by agarose gel electrophoresis. The relative proportions of the expression pattern of HAS isoforms was similar between RA and OA tissue-derived cells. HAS1 mRNA was upregulated by transforming growth factor-beta and HAS3 mRNA was upregulated by interleukin-1beta and somewhat by tumor necrosis factor-alpha in the RA cells. HAS2 remained unchanged. Differences in the expression pattern of HAS1, HAS2, and HAS3 mRNA by cytokines suggest that these three isoforms are independently and differentially regulated, and each isoform of HAS may have a different role in arthritic joint disease.