Cole-Carpenter syndrome is caused by a heterozygous missense mutation in P4HB.

Cole-Carpenter syndrome is a severe bone fragility disorder that is characterized by frequent fractures, craniosynostosis, ocular proptosis, hydrocephalus, and distinctive facial features. To identify the cause of Cole-Carpenter syndrome in the two individuals whose clinical results were presented in the original description of this disorder, we performed whole-exome sequencing of genomic DNA samples from both individuals. The two unrelated individuals had the same heterozygous missense mutation in exon 9 of P4HB (NM_000918.3: c.1178A>G [p.Tyr393Cys]), the gene that encodes protein disulfide isomerase (PDI). In one individual, the P4HB mutation had arisen de novo, whereas in the other the mutation was transmitted from the clinically unaffected father who was a mosaic carrier of the variant. The mutation was located in the C-terminal disulfide isomerase domain of PDI, sterically close to the enzymatic center, and affected disulfide isomerase activity in vitro. Skin fibroblasts showed signs of increased endoplasmic reticulum stress, but despite the reported importance of PDI for collagen type I production, the rate of collagen type I secretion appeared normal. In conclusion, Cole-Carpenter syndrome is caused by a specific de novo mutation in P4HB that impairs the disulfide isomerase activity of PDI.

Recessive osteogenesis imperfecta caused by missense mutations in SPARC.

Secreted protein, acidic, cysteine-rich (SPARC) is a glycoprotein that binds to collagen type I and other proteins in the extracellular matrix. Using whole-exome sequencing to identify the molecular defect in two unrelated girls with severe bone fragility and a clinical diagnosis of osteogenesis imperfecta type IV, we identified two homozygous variants in SPARC (GenBank: NM_003118.3; c.497G>A [p.Arg166His] in individual 1; c.787G>A [p.Glu263Lys] in individual 2). Published modeling and site-directed mutagenesis studies had previously shown that the residues substituted by these mutations form an intramolecular salt bridge in SPARC and are essential for the binding of SPARC to collagen type I. The amount of SPARC secreted by skin fibroblasts was reduced in individual 1 but appeared normal in individual 2. The migration of collagen type I alpha chains produced by these fibroblasts was mildly delayed on SDS-PAGE gel, suggesting some overmodification of collagen during triple helical formation. Pulse-chase experiments showed that collagen type I secretion was mildly delayed in skin fibroblasts from both individuals. Analysis of an iliac bone sample from individual 2 showed that trabecular bone was hypermineralized on the material level. In conclusion, these observations show that homozygous mutations in SPARC can give rise to severe bone fragility in humans.

Aggrecan heterogeneity in articular cartilage from patients with osteoarthritis.

BACKGROUND: Aggrecan degradation is the hallmark of cartilage degeneration in osteoarthritis (OA), though it is unclear whether a common proteolytic process occurs in all individuals. METHODS: Aggrecan degradation in articular cartilage from the knees of 33 individuals with OA, who were undergoing joint replacement surgery, was studied by immunoblotting of tissue extracts. RESULTS: Matrix metalloproteinases (MMPs) and aggrecanases are the major proteases involved in aggrecan degradation within the cartilage, though the proportion of aggrecan cleavage attributable to MMPs or aggrecanases was variable between individuals. However, aggrecanases were more associated with the increase in aggrecan loss associated with OA than MMPs. While the extent of aggrecan cleavage was highly variable between individuals, it was greatest in areas of cartilage adjacent to sites of cartilage erosion compared to sites more remote within the same joint. Analysis of link protein shows that in some individuals additional proteolytic mechanisms must also be involved to some extent. CONCLUSIONS: The present studies indicate that there is no one protease, or a fixed combination of proteases, responsible for cartilage degradation in OA. Thus, rather than targeting the individual proteases for OA therapy, directing research to techniques that control global protease generation may be more productive.

Efficient processing of procathepsin K to the mature form.

The proteolysis of collagen fibrils by cathepsin K is a hallmark of bone catabolism and tissue degeneration. The production of active recombinant cathepsin K is central for our ability to study the mechanisms by which these processes occur. Here we report an efficient processing method for the preparation of recombinant cathepsin K expressed in Pichia pastoris. Methanol precipitation of crude media and autoactivation in the absence of a reducing agent allows for the reversible inhibition of the enzyme prior to subsequent purification steps. The resultant purified enzyme is both resistant to autolysis and effective at cleaving collagen.

Alarmins S100A8 and S100A9 elicit a catabolic effect in human osteoarthritic chondrocytes that is dependent on Toll-like receptor 4.

OBJECTIVE: S100A8 and S100A9 are two Ca(2+) binding proteins classified as damage-associated molecular patterns or alarmins that are found in high amounts in the synovial fluid of osteoarthritis (OA) patients. The purpose of this study was to investigate whether S100A8 and/or S100A9 can interact with chondrocytes from OA patients to increase catabolic mediators. METHODS: Using immunohistochemistry, we stained for S100A8 and S100A9 protein, matrix metalloproteinases (MMPs), and a cartilage-breakdown epitope specific for MMPs (VDIPEN) in cartilage from OA donors. Isolated chondrocytes or explants from OA and non-OA donors were stimulated with S100A8 and/or S100A9. Messenger RNA and protein levels of MMPs, cytokines, and cartilage matrix molecules were determined with quantitative reverse transcription-polymerase chain reaction and Luminex techniques, respectively. For receptor blocking studies, specific inhibitors for Toll-like receptor 4 (TLR-4), receptor for advanced glycation end products (RAGE), and carboxylated glycans were used. RESULTS: In cartilage from OA patients, the expression of S100A8 and S100A9 protein close to chondrocytes was associated with proteoglycan depletion and expression of MMP-1, MMP-3, and VDIPEN. Stimulation of chondrocytes with S100A8 and S100A9 caused a strong up-regulation of catabolic markers (MMPs 1, 3, 9, and 13, interleukin-6 [IL-6], IL-8, and monocyte chemotactic protein 1) and down-regulation of anabolic markers (aggrecan and type II collagen), thereby favoring cartilage breakdown. Blocking TLR-4, but not carboxylated glycans or RAGE, inhibited the S100 effect. The catabolic S100 effect was significantly more pronounced in chondrocytes from OA patients as compared to those from non-OA patients, possibly due to higher TLR-4 expression. CONCLUSION: S100A8 and S100A9 have a catabolic effect on human chondrocytes that is TLR-4 dependent. OA chondrocytes are more sensitive than normal chondrocytes to S100 stimulation.

Partially redundant functions of Adamts1 and Adamts4 in the perinatal development of the renal medulla.

Adamts4 encodes a widely-expressed proteinase thought to be involved in processes ranging from cartilage metabolism to ovarian follicle development. To study its physiological roles, Adamts4-null mice were created by gene targeting. Unexpectedly, these were found to be phenotypically normal, suggesting that other gene(s) may compensate for its loss. Adamts4(-/-) mice were, therefore, crossed with a strain lacking Adamts1, whose pattern of expression and substrate specificity overlap that of Adamts4. Most (>95%) Adamts1(-/-) ;Adamts4(-/-) mice died within 72 hr after birth with a marked thinning of the renal medulla. The renal defect was not observed in embryonic Adamts1(-/-) ;Adamts4(-/-) kidneys, but became apparent around birth. The few (<5%) Adamts1(-/-) ;Adamts4(-/-) animals to reach adulthood had the same renal phenotype seen in newborns. This study is thus the first to report Adamts4 expression and function in the mammalian kidney, and to demonstrate that Adamts1 and Adamts4 play redundant and essential roles in perinatal kidney development.

The pattern recognition receptor CD36 is a chondrocyte hypertrophy marker associated with suppression of catabolic responses and promotion of repair responses to inflammatory stimuli.

Multiple inflammatory mediators in osteoarthritis (OA) cartilage, including S100/calgranulin ligands of receptor for advanced glycation end products (RAGE), promote chondrocyte hypertrophy, a differentiation state associated with matrix catabolism. In this study, we observed that RAGE knockout was not chondroprotective in instability-induced knee OA in 8-wk-old mice. Hence, we tested the hypothesis that expression of the alternative S100/calgranulin and patterning receptor CD36, identified here as a marker of growth plate chondrocyte hypertrophy, mediates chondrocyte inflammatory and differentiation responses that promote OA. In rat knee joint destabilization-induced OA, RAGE expression was initially sparse throughout cartilage but increased diffusely by 4 wk after surgery. In contrast, CD36 expression focally increased at sites of cartilage injury and colocalized with developing chondrocyte hypertrophy and aggrecan cleavage NITEGE neoepitope formation. However, CD36 transfection in normal human knee-immortalized chondrocytes (CH-8 cells) was associated with decreased capacity of S100A11 and TNF-alpha to induce chondrocyte hypertrophy and ADAMTS-4 and matrix metalloproteinase 13 expression. S100A11 lost the capacity to inhibit proteoglycans synthesis and gained the capacity to induce proteoglycan synthesis in CD36-transfected CH-8 cells. Moreover, S100A11 required the p38 MAPK pathway kinase MKK3 to induce NITEGE development in mouse articular cartilage explants. However, CH-8 cells transfected with CD36 demonstrated decreased S100A11-induced MKK3 and p38 phosphorylation. Therefore, RAGE and CD36 patterning receptor expression were linked with opposing effects on inflammatory, procatabolic responses to S100A11 and TNF-alpha in chondrocytes.

Rho/ROCK and MEK/ERK activation by transforming growth factor-alpha induces articular cartilage degradation.

Identification and characterization of therapeutic targets for joint conditions, such as osteoarthritis (OA), is exceedingly important for addressing the increasing burden of disease. Transforming growth factor-alpha (TGFalpha) is upregulated by articular chondrocytes in experimentally induced and human OA. To test the potential involvement of TGFalpha, which is an activator of epidermal growth factor receptor (EGFR) signaling, in joint degeneration and to identify signaling mechanisms mediating articular chondrocyte responses to TGFalpha, rat chondrocytes and osteochondral explants were treated with TGFalpha and various inhibitors of intracellular signaling pathways. Stimulation of EGFR signaling in articular chondrocytes by TGFalpha resulted in the activation of RhoA/ROCK (Rho kinase), MEK (MAPK/ERK kinase)/ERK (extracellular-signal-regulated kinase), PI3K (phosphoinositide 3-kinase) and p38 MAPK (mitogen-activated protein kinase) pathways. Modification of the chondrocyte actin cytoskeleton was stimulated by TGFalpha, but inhibition of only Rho or ROCK activation prevented morphological changes. TGFalpha suppressed expression of anabolic genes including Sox9, type II collagen and aggrecan, which were rescued only by inhibiting MEK/ERK activation. Furthermore, catabolic factor upregulation by TGFalpha was prevented by ROCK and p38 MAPK inhibition, including matrix metalloproteinase-13 and tumor necrosis factor-alpha, which are well known to contribute to cartilage digestion in OA. To assess the ability of TGFalpha to stimulate degradation of mature articular cartilage, type II collagen and aggrecan cleavage fragments were analyzed in rat osteochondral explants exposed to exogenous TGFalpha. Normal articular cartilage contained low levels of both cleavage fragments, but high levels were observed in the cartilage treated with TGFalpha. Selective inhibition of MEK/ERK and Rho/ROCK activation greatly reduced or completely prevented excess type II collagen and aggrecan degradation in response to TGFalpha. These data suggest that TGFalpha is a strong stimulator of cartilage degradation and that Rho/ROCK and MEK/ERK signaling have critical roles in mediating these effects.

A mouse model of human mucopolysaccharidosis IX exhibits osteoarthritis.

Hyaluronidases are endoglycosidases that hydrolyze hyaluronan (HA), an abundant component of the extracellular matrix of vertebrate connective tissues. Six human hyaluronidase-related genes have been identified to date. Mutations in one of these genes cause a deficiency of hyaluronidase 1 (HYAL1) resulting in a lysosomal storage disorder, mucopolysaccharidosis (MPS) IX. We have characterized a mouse model of MPS IX and compared its phenotype with the human disease. The targeted Hyal1 allele in this model had a neomycin resistance cassette in exon 2 that replaced 753 bp of the coding region containing the predicted enzyme active site. As a result, Hyal1(-/-) animals had no detectable wild-type Hyal1 transcript, protein or serum activity. Hyal1 null animals were viable, fertile and showed no gross abnormalities at 1 year and 8 months of age. Histological studies of the knee joint showed a loss of proteoglycans occurring as early as 3 months that progressed with age. An increased number of chondrocytes displaying intense pericellular and/or cytoplasmic HA staining were detected in the epiphyseal and articular cartilage of null mice, demonstrating an accumulation of HA. Elevations of HA were not detected in the serum or non-skeletal tissues, indicating that osteoarthritis is the key disease feature in a Hyal1 deficiency. Hyal3 expression was elevated in Hyal1 null mice, suggesting that Hyal3 may compensate in HA degradation in non-skeletal tissues. Overall, the murine MPS IX model displays the key features of the human disease.

Cleavage of type II collagen by cathepsin K in human osteoarthritic cartilage.

Cathepsin K is a cysteine protease of the papain family that cleaves triple-helical type II collagen, the major structural component of the extracellular matrix of articular cartilage. In osteoarthritis (OA), the anabolic/catabolic balance of articular cartilage is disrupted with the excessive cleavage of collagen II by collagenases or matrix metalloproteinases. A polyclonal antibody against a C-terminal neoepitope (C2K) generated in triple-helical type II collagen by the proteolytic action of cathepsin K was prepared and used to develop an enzyme-linked immunosorbent assay to study the generation of this epitope and the effects of its presence in normal adult and osteoarthritic femoral condylar articular cartilage. The generation of the C2K epitope in explant culture and the effect of a specific cathepsin K inhibitor were studied. The neoepitope, which is not generated by the collagenase matrix metalloproteinase-13, increased with age in articular cartilage and was significantly elevated in osteoarthritic cartilage compared with adult nonarthritic cartilage. Moreover, in explants from three of eight OA patients, the generation of the neoepitope in culture was significantly reduced by a specific, nontoxic inhibitor of cathepsin K. These data suggest that cathepsin K is involved in the cleavage of type II collagen in human articular cartilage in certain OA patients and that it may play a role in both OA pathophysiology and the aging process.

Hyaluronidase 3 (HYAL3) knockout mice do not display evidence of hyaluronan accumulation.

Hyaluronidases are endoglycosidases that initiate the breakdown of hyaluronan (HA), an abundant component of the vertebrate extracellular matrix. In humans, six paralogous genes encoding hyaluronidase-like sequences have been identified on human chromosomes 3p21.3 (HYAL2-HYAL1-HYAL3) and 7q31.3 (SPAM1-HYAL4-HYALP1). Mutations in one of these genes, HYAL1, were reported in a patient with mucopolysaccharidosis (MPS) IX. Despite the broad distribution of HA, the HYAL1-deficient patient exhibited a mild phenotype, suggesting other hyaluronidase family members contribute to constitutive HA degradation. Hyal3 knockout (Hyal3-/-) mice were generated to determine if HYAL3 had a role in constitutive HA degradation. Hyal3-/- mice were viable, fertile, and exhibited no gross phenotypic changes. X-ray analysis, histological studies of joints, whole-body weights, organ weights and the serum HA levels of Hyal3-/- mice were normal. No evidence of glycosaminoglycan accumulation, including vacuolization, was identified in the Hyal3-/- tissues analyzed. Remarkably, the only difference identified in Hyal3-/- mice was a subtle change in the alveolar structure and extracellular matrix thickness in lung-tissue sections at 12-14 months-of-age. We conclude that HYAL3 does not play a major role in constitutive HA degradation.

Crystal structure of the parasite protease inhibitor chagasin in complex with a host target cysteine protease.

Chagasin is a protein produced by Trypanosoma cruzi, the parasite that causes Chagas' disease. This small protein belongs to a recently defined family of cysteine protease inhibitors. Although resembling well-known inhibitors like the cystatins in size (110 amino acid residues) and function (they all inhibit papain-like (C1 family) proteases), it has a unique amino acid sequence and structure. We have crystallized and solved the structure of chagasin in complex with the host cysteine protease, cathepsin L, at 1.75 A resolution. An inhibitory wedge composed of three loops (L2, L4, and L6) forms a number of contacts responsible for high-affinity binding (K(i), 39 pM) to the enzyme. All three loops interact with the catalytic groove, with the central loop L2 inserted directly into the catalytic center. Loops L4 and L6 embrace the enzyme molecule from both sides and exhibit distinctly different patterns of protein-protein recognition. Comparison with a 1.7 A structure of uncomplexed chagasin, also determined in this study, demonstrates that a conformational change of the first binding loop (L4) allows extended binding to the non-primed substrate pockets of the enzyme active site cleft, thereby providing a substantial part of the inhibitory surface. The mode of chagasin binding is generally similar, albeit distinctly different in detail, when compared to those displayed by cystatins and the cysteine protease inhibitory p41 fragment of the invariant chain. The chagasin-cathepsin L complex structure provides details of how the parasite protein inhibits a host enzyme of possible importance in host defense. The high level of structural and functional similarity between cathepsin L and the T. cruzi enzyme cruzipain gives clues to how the cysteine protease activity of the parasite can be targeted. This information will aid in the development of synthetic inhibitors for use as potential drugs for the treatment of Chagas disease.

The cysteine-rich domain of the secreted proprotein convertases PC5A and PACE4 functions as a cell surface anchor and interacts with tissue inhibitors of metalloproteinases.

The proprotein convertases PC5, PACE4 and furin contain a C-terminal cysteine-rich domain (CRD) of unknown function. We demonstrate that the CRD confers to PC5A and PACE4 properties to bind tissue inhibitors of metalloproteinases (TIMPs) and the cell surface. Confocal microscopy and biochemical analyses revealed that the CRD is essential for cell surface tethering of PC5A and PACE4 and that it colocalizes and coimmunoprecipitates with the full-length and C-terminal domain of TIMP-2. Surface-bound PC5A in TIMP-2 null fibroblasts was only observed upon coexpression with TIMP-2. In COS-1 cells, plasma membrane-associated PC5A can be displaced by heparin, suramin, or heparinases I and III and by competition with excess exogenous TIMP-2. Furthermore, PC5A and TIMP-2 are shown to be colocalized over the surface of enterocytes in the mouse duodenum and jejunum, as well as in liver sinusoids. In conclusion, the CRD of PC5A and PACE4 functions as a cell surface anchor favoring the processing of their cognate surface-anchored substrates, including endothelial lipase.

Collagen type I degradation fragments act through the collagen receptor LAIR-1 to provide a negative feedback for osteoclast formation.

The major organic component of bone is collagen type I. Osteoclasts are terminally differentiated multinucleated cells of hematopoietic origin that are essential for physiological development of bone and teeth. We examined if osteoclast differentiation from murine bone marrow precursors is affected by collagen type I, or by its degradation products produced by human recombinant cathepsin K. Osteoclasts formation was dose-dependently inhibited in the presence of full length collagen type I or its 30-75 kDa degradation products added to the osteoclast differentiation media for the duration of an experiment. Collagen degradation fragments signaled through SH-2 phosphatases, inhibiting calcium signaling and NFATc1 translocation in osteoclast precursors. Osteoclasts and their precursors expressed a collagen receptor of leukocyte receptor complex family, LAIR-1. Importantly, collagen fragments failed to inhibit osteoclast formation from LAIR-1 deficient murine osteoclast precursors. This study demonstrates that collagen degradation fragments inhibit osteoclast formation acting through LAIR-1, providing a novel mechanism for the physiologically-relevant negative control of osteoclastogenesis.

Measurement of glycosaminoglycan release from cartilage explants.

Quantitation of glycosaminoglycans (GAGs) in the form of aggrecan fragments released from cartilage in culture is a simple way to determine the efficacy of different cytokines alone or in combination in simulating cartilage catabolism. Two approaches for GAG assay are described, with special attention being paid to the advantages and limitations of each method.

Protease analysis by neoepitope approach reveals the activation of MMP-9 is achieved proteolytically in a test tissue cartilage model involved in bone formation.

A principle of regulation of matrix metalloproteinase (MMP) activity has been introduced as the cysteine-switch mechanism of activation (Springman et al. 1990). According to this mechanism, a critical Cys residue found in the auto-inhibitory propeptide domain of latent proenzyme is important to determine whether or not activation is turned on or off. The mechanism further allows for multiple modes of activation. To determine whether or not activation is accomplished proteolytically within a rat test cartilage model, protease analysis by the neoepitope approach, which relies upon a set of antibodies, was applied. One is used to identify the MMP-9 proenzyme bearing the critical cysteine residue, the other to identify any enzyme present bearing a new NH2-terminus 89FQTFD. This is indicative of MMP-9 lacking the cysteine switch. The antibody set has been applied to frozen tissue sections and analyzed by light and electron microscopic methods. Results reveal that activation of the MMP-9 protease involves limited proteolysis resulting in propeptide domain release. Here we report the observed changes of protease form to indigenous cells and extracellular matrix, thereby making it possible to uncover the features of MMP-9 activation within a specified set of tissue circumstances where a cartilage model is transformed into definitive bone. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials.

Insect cell conditioned medium contains an endoglycosidase able to liberate chondroitin sulfate chains from aggrecan.

Culture medium obtained from baculovirus-infected High Five insect cells contains an endoglycosidase activity capable of releasing chondroitin sulfate chains from aggrecan, decorin and biglycan. Release appears to occur by cleavage within the linkage region of the chondroitin sulfate chain, but not all chains are amenable to release. Culture medium from Sf9 insect cells does not contain this activity. The endoglycosidase may become a useful reagent for biochemical research for releasing intact chondroitin sulfate chains from proteoglycans. It may also represent an impediment to such research when baculovirus systems are used to generate recombinant proteoglycans.

Variations in aggrecan structure modulate its susceptibility to aggrecanases.

Proteoglycan aggregates and purified aggrecan from adult and fetal bovine cartilage and adult and neonatal human cartilage were subjected to in vitro degradation by recombinant aggrecanase-1 and aggrecanase-2. The ability of the aggrecanases to cleave within the aggrecan IGD (interglobular domain) and CS2 domain (chondroitin sulphate-rich domain 2) was monitored by SDS/PAGE and immunoblotting. Aggrecanase-2 showed a similar ability to cleave within the IGD of adult and immature aggrecan, whereas aggrecanase-1 was less efficient in cleavage in the IGD of immature aggrecan, for both the bovine and the human substrates. Both aggrecanases showed a similar ability to cleave within the CS2 domain of bovine aggrecan irrespective of age, but showed a much lower ability to cleave within the CS2 domain of human aggrecan. Equivalent results were obtained whether aggrecan was present in isolation or as part of proteoglycan aggregates. When proteoglycan aggregates were used, neither aggrecanase was able to cleave link protein. Thus, for aggrecan cleavage by aggrecanases, variations in cleavage efficiency exist with respect to the species and age of the animal from which the aggrecan is derived and the type of aggrecanase being used.

Reduction in disease progression by inhibition of transforming growth factor α-CCL2 signaling in experimental posttraumatic osteoarthritis.

OBJECTIVE: Transforming growth factor α (TGFα) is increased in osteoarthritic (OA) cartilage in rats and humans and modifies chondrocyte phenotype. CCL2 is increased in OA cartilage and stimulates proteoglycan loss. This study was undertaken to test whether TGFα and CCL2 cooperate to promote cartilage degradation and whether inhibiting either reduces disease progression in a rat model of posttraumatic OA. METHODS: Microarray analysis was used to profile expression of messenger RNA (mRNA) for Tgfa, Ccl2, and related genes in a rat model of posttraumatic OA. Rat primary chondrocytes and articular cartilage explants were treated with TGFα in the presence or absence of MEK-1/2, p38, phosphatidylinositol 3-kinase, Rho-associated protein kinase, or CCR2 inhibitors and immunostained for markers of cartilage degradation. The rat model was used to administer pharmacologic inhibitors of TGFα (AG1478) and CCL2 (RS504393) signaling for up to 10 weeks and assess histopathology and serum biomarkers of cartilage synthesis (C-propeptide of type II collagen [CPII]) and breakdown (C2C). RESULTS: Tgfa and Ccl2 mRNA were simultaneously up-regulated in articular cartilage in the rat model of posttraumatic OA. TGFα induced expression of CCL2, Mmp3, and Tnf in primary chondrocytes. Cleavage of type II collagen and aggrecan (by matrix metalloproteinases and ADAMTS-4/5, respectively) induced by TGFα was blocked by pharmacologic inhibition of CCL2 in cartilage explants. In vivo pharmacologic inhibition of TGFα or CCL2 signaling reduced Osteoarthritis Research Society International cartilage histopathology scores and increased serum CPII levels, but only TGFα inhibition reduced C2C levels intreated versus untreated rat OA cartilage. CONCLUSION: TGFα signaling stimulates cartilage degradation via a CCL2-dependent mechanism, but pharmacologic inhibition of the TGFα-CCL2 axis reduces experimental posttraumatic OA progression in vivo.

Repeated exposure to high-frequency low-amplitude vibration induces degeneration of murine intervertebral discs and knee joints.

OBJECTIVE: High-frequency, low-amplitude whole-body vibration (WBV) is being used to treat a range of musculoskeletal disorders; however, there is surprisingly limited knowledge regarding its effect(s) on joint tissues. This study was undertaken to examine the effects of repeated exposure to WBV on bone and joint tissues in an in vivo mouse model. METHODS: Ten-week-old male mice were exposed to vertical sinusoidal vibration under conditions that mimic those used clinically in humans (30 minutes per day, 5 days per week, at 45 Hz with peak acceleration at 0.3g). Following WBV, skeletal tissues were examined by micro-computed tomography, histologic analysis, and immunohistochemistry, and gene expression was quantified using real-time polymerase chain reaction. RESULTS: Following 4 weeks of WBV, intervertebral discs showed histologic hallmarks of degeneration in the annulus fibrosus, disruption of collagen organization, and increased cell death. Greater Mmp3 expression in the intervertebral disc, accompanied by enhanced collagen and aggrecan degradation, was found in mice exposed to WBV as compared to controls. Examination of the knee joints after 4 weeks of WBV revealed meniscal tears and focal damage to the articular cartilage, changes resembling osteoarthritis. Moreover, mice exposed to WBV also demonstrated greater Mmp13 gene expression and enhanced matrix metalloproteinase-mediated collagen and aggrecan degradation in articular cartilage as compared to controls. No changes in trabecular bone microarchitecture or density were detected in the proximal tibia. CONCLUSION: Our experiments reveal significant negative effects of WBV on joint tissues in a mouse model. These findings suggest the need for future studies of the effects of WBV on joint health in humans.