Joint and Bone Disease in Mucopolysaccharidoses VI and VII: Identification of New Therapeutic Targets and BioMarkers Using Animal Models

Abstract

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.

Involvement of the Toll-like receptor 4 pathway and use of TNF-α antagonists for treatment of the mucopolysaccharidoses

“…Normal and MPS VI rat FLSs were cultured on coverslips, fixed with 4% (v/v) paraformaldehyde/PBS, incubated overnight with primary TLR4 antibody (Santa Cruz Biotechnology), and then incubated with the appropriate secondary antibody (4,5). LysoTracker Green (Invitrogen) was used to visualize lysosomes and bis-benzamide Hoechst dye (SigmaAldrich) was used to visualize nuclei (for details, see SI Materials and Methods).…”

“…The fixed bones were decalcified in 8% (v/v) formic acid (Sigma Chemical) for 5 days, paraffin-embedded, sectioned (5 μm), and stained with H&E or TUNEL. Primary FLS and articular chondrocyte cultures were established as previously described (4,5), and expression of molecules involved in TLR4 signaling and autophagy was assessed (see above).…”

“…Our laboratory has used MPS animal models to document the molecular, biochemical, and cellular changes occurring in these tissues. We reported that GAG storage in MPS induces a complex sequence of molecular abnormalities, leading to inflammation, apoptosis (cartilage), and hyperplasia (synovial membranes), which result in poorly organized and metabolically abnormal connective tissue matrices (3)(4)(5).…”

“…Treatment of group 2 rats for 24 weeks maintained their serum TNF-α at normal. Previously, we found that MPS VI rats also had elevated serum levels of receptor activator of NF-κB (RANKL) (4). This inflammatory molecule has many potential pathological effects, including a potent effect on osteoclastogenesis (34).…”

Upregulation of elastase proteins results in aortic dilatation in mucopolysaccharidosis I mice

“…In addition, these proteins may contribute to manifestations of disease in other organs, such as skin, ligaments, and tendons. Indeed, MMPs contributed to joint disease in other MPS models [44] while cathepsin S was upregulated in the brain of MPS I mice [45]. If the genetic cross that is in progress shows that deficiency of one or both of these elastase proteins can reduce aortic disease in MPS I, it might be possible to treat MPS I patients with inhibitors of MMP-12 [46] and/or cathepsin S [47].…”