Enzyme replacement therapy is currently available for three of the mucopolysaccharidoses (MPSs) but has limited effects on the skeletal lesions. We investigated the involvement of the Toll-like receptor 4 (TLR4) signaling pathway in the pathogenesis of MPS bone and joint disease, and the use of the anti-TNF-α drug, Remicade (Centocor, Inc.), for treatment. TLR4 KO (TLR4 (lps−/−) ) mice were interbred with MPS VII mice to produce double-KO (DKO) animals. The DKO mice had longer and thinner faces and longer femora as revealed by micro-computed tomography analysis compared with MPS VII mice. Histological analyses also revealed more organized and thinner growth plates. The serum levels of TNF-α were normalized in the DKO animals, and the levels of phosphorylated STAT1 and STAT3 in articular chondrocytes were corrected. These findings led us to evaluate the effects of Remicade in MPS VI rats. When initiated at 1 month of age, i.v. treatment prevented the elevation of TNF-α, receptor activator of NF-κB, and other inflammatory molecules not only in the blood but in articular chondrocytes and fibroblast-like synoviocytes (FLSs). Treatment of 6-monthold animals also reduced the levels of these molecules to normal. The number of apoptotic articular chondrocytes in MPS VI rats was similarly reduced, with less infiltration of synovial tissue into the underlying bone. These studies revealed the important role of TLR4 signaling in MPS bone and joint disease and suggest that targeting TNF-α may have positive therapeutic effects.bone and joint disease | inflammation | glycosaminoglycans | growth plate

The mucopolysaccharidoses (MPS) are inherited metabolic disorders resulting from the defective catabolism of glycosaminoglycans. In this report, we find that the stimulation of MPS connective tissue cells by the inflammatory cytokines causes enhanced secretion of several matrix-degrading metalloproteinases (MMPs). In addition, expression of tissue inhibitor of metalloproteinase-1 was elevated, consistent with the enhanced MMP activity. These findings were not restricted to one particular MPS disorder or species, and are consistent with previous observations in animal models with chemically induced arthritis. Bromodeoxyuridine incorporation studies also revealed that MPS chondrocytes proliferated up to 5-fold faster than normal chondrocytes, and released elevated levels of transforming growth factor-beta, presumably to counteract the marked chondrocyte apoptosis and matrix degradation associated with MMP expression. Despite this compensatory mechanism, studies of endochondral ossification revealed a reduction in chondrodifferentiation in the growth plates. Thus, although MPS chondrocytes grew faster, most of the newly formed cells were immature and could not mineralize into bone. Our studies suggest that altered MMP expression, most likely stimulated by inflammatory cytokines and nitric oxide, is an important feature of the MPS disorders. These data also identify several proinflammatory cytokines, nitric oxide, and MMPs as novel therapeutic targets and/or biomarkers of MPS joint and bone disease. This information should aid in the evaluation of existing therapies for these disorders, such as enzyme replacement therapy and bone marrow transplantation, and may lead to the development of new therapeutic approaches. The accumulation of partially degraded glycosaminoglycans (GAGs) in late endosomes and lysosomes of connective tissue cells is characteristic of a family of heritable lysosomal storage diseases known as the mucopolysaccharidoses (MPS) (1). The MPS family consists of 12 chronic and progressive syndromes, each with a specific enzyme deficiency leading to a characteristic pattern of accumulating GAGs within lysosomes. Among the different MPS disorders, there are several clinical characteristics present in most affected individuals. These include anterior hypoplasia of lumbar vertebrae, enlarged diaphyses of the long bones, underdeveloped epiphyseal centers, marked dwarfism, degenerative joint disease, dysostosis multiplex, facial dysmorphia, organomegaly, corneal clouding, and in most syndromes mental retardation (1). Cartilage is a major site of pathology in these diseases, leading to painful joints, poor joint mobility, poor bone growth, and an abnormal larynx and trachea producing breathing abnormalities often requiring tracheotomies.

Farber disease (FD) is a severe inherited disorder of lipid metabolism characterized by deficient lysosomal acid ceramidase (ACDase) activity, resulting in ceramide accumulation. Ceramide and metabolites have roles in cell apoptosis and proliferation. We introduced a single-nucleotide mutation identified in human FD patients into the murine Asah1 gene to generate the first model of systemic ACDase deficiency. Homozygous Asah1P361R/P361R animals showed ACDase defects, accumulated ceramide, demonstrated FD manifestations and died within 7–13 weeks. Mechanistically, MCP-1 levels were increased and tissues were replete with lipid-laden macrophages. Treatment of neonates with a single injection of human ACDase-encoding lentivector diminished the severity of the disease as highlighted by enhanced growth, decreased ceramide, lessened cellular infiltrations and increased lifespans. This model of ACDase deficiency offers insights into the pathophysiology of FD and the roles of ACDase, ceramide and related sphingolipids in cell signaling and growth, as well as facilitates the development of therapy.

SUMMARY: Mucopolysaccharidosis (MPS) Type VI (Maroteaux-Lamy Disease) is the lysosomal storage disease characterizedby deficient arylsulfatase B activity and the resultant accumulation of dermatan sulfate-containing glycosaminoglycans (GAGs). A major feature of this and other MPS disorders is abnormal cartilage and bone development leading to short stature, dysostosis multiplex, and degenerative joint disease. To investigate the underlying cause(s) of degenerative joint disease in the MPS disorders, articular cartilage and cultured articular chondrocytes were examined from rats and cats with MPS VI. An age-progressive increase in the number of apoptotic chondrocytes was identified in the MPS animals by terminal transferase nick-end translation (TUNEL) staining and by immunohistochemical staining with anti-poly (ADP-ribose) polymerase (PARP) antibodies. Articular chondrocytes grown from these animals also released more nitric oxide (NO) and tumor necrosis factor alpha (TNF-␣) into the culture media than did control chondrocytes. Notably, dermatan sulfate, the GAG that accumulates in MPS VI cells, induced NO release from normal chondrocytes, suggesting that GAG accumulation was responsible, in part, for the enhanced cell death in the MPS cells. Coculture of normal chondrocytes with MPS VI cells reduced the amount of NO release, presumably because of the release of arylsulfatase B by the normal cells and reuptake by the mutant cells. As a result of the enhanced chondrocyte death, marked proteoglycan and collagen depletion was observed in the MPS articular cartilage matrix. These results demonstrate that MPS VI articular chondrocytes undergo cell death at a higher rate than normal cells, because of either increased levels of dermatan sulfate and/or the presence of inflammatory cytokines in the MPS joints. In turn, this leads to abnormal cartilage matrix homeostasis in the MPS individuals, which further exacerbates the joint deformities characteristic of these disorders. (Lab Invest 2001, 81:1319 -1328.

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