Mechanical loading rescues mechanoresponsiveness in a human osteoarthritis explant model despite Wnt activation.

OBJECTIVES: Optimizing rehabilitation strategies for osteoarthritis necessitates a comprehensive understanding of chondrocytes' mechanoresponse in both health and disease, especially in the context of the interplay between loading and key pathways involved in osteoarthritis (OA) development, like canonical Wnt signaling. This study aims to elucidate the role of Wnt signaling in the mechanoresponsiveness of healthy and osteoarthritic human cartilage. METHODS: We used an ex-vivo model involving short-term physiological mechanical loading of human cartilage explants. First, the loading protocol for subsequent experiments was determined. Next, loading was applied to non-OA-explants with or without Wnt activation with CHIR99021. Molecular read-outs of anabolic, pericellular matrix and matrix remodeling markers were used to assess the effect of Wnt on cartilage mechanoresponse. Finally, the same set-up was used to study the effect of loading in cartilage from patients with established OA. RESULTS: Our results confirm that physiological loading maintains expression of anabolic genes in non-OA cartilage, and indicate a deleterious effect of Wnt activation in the chondrocyte mechanoresponsiveness. This suggests that loading-induced regulation of chondrocyte markers occurs downstream of canonical Wnt signaling. Interestingly, our study highlighted contrasting mechanoresponsiveness in the model of Wnt activation and the established OA samples, with established OA cartilage maintaining its mechanoresponsiveness, and mechanical loading rescuing the chondrogenic phenotype. CONCLUSION: This study provides insights into the mechanoresponsiveness of human cartilage in both non-OA and OA conditions. These findings hold the potential to contribute to the development of strategies that optimize the effect of dynamic compression by correcting OA pathological cell signaling.

Hypoxia and Wnt signaling inversely regulate expression of chondroprotective molecule ANP32A in articular cartilage.

OBJECTIVES: ANP32A is a key protector of cartilage health, via preventing oxidative stress and Wnt hyper-activation. We aimed to unravel how ANP32A is regulated in cartilage. METHODS: A bioinformatics pipeline was applied to identify regulators of ANP32A. Pathways of interest were targeted to study their impact on ANP32A in in vitro cultures of the human chondrocyte C28/I2 cell-line and primary human articular chondrocytes (hACs) from up to five different donors, using Wnt-activator CHIR99021, hypoxia-mimetic IOX2 and a hypoxia chamber. ANP32A was evaluated using real-time quantitative polymerase chain reaction (RT-qPCR) and Western blot. In vivo, the effect of hypoxia was examined by immunohistochemistry in mice injected intra-articularly with IOX2 after destabilization of the medial meniscus. Effects of Wnt hyper-activation were investigated using Frzb-knockout mice and wild-type mice treated intra-articularly with CHIR99021. Wnt inhibition effects were assessed upon intra-articular injection of XAV939. RESULTS: The hypoxia and Wnt signaling pathways were identified as networks controlling ANP32A expression. In vitro and in vivo experiments demonstrated increases in ANP32A upon hypoxic conditions (1.3-fold in hypoxia in C28/I2 cells with 95% confidence interval (CI) [1.11-1.54] and 1.90-fold in hACs [95% CI: 1.56-2] and 1.67-fold in ANP32A protein levels after DMM surgery with IOX2 injections [95% CI: 1.33-2.08]). Wnt hyper-activation decreased ANP32A in chondrocytes in vitro (1.23-fold decrease [95% CI: 1.02-1.49]) and in mice (1.45-fold decrease after CHIR99021 injection [95% CI: 1.22-1.72] and 1.41-fold decrease in Frzb-knockout mice [95% CI: 1.00-1.96]). Hypoxia and Wnt modulated ataxia-telangiectasia mutated serine/threonine kinase (ATM), an ANP32A target gene, in hACs (1.89-fold increase [95% CI: 1.38-2.60] and 1.41-fold decrease [95% CI: 1.02-1.96]). CONCLUSIONS: Maintaining hypoxia and limiting Wnt activation sustain ANP32A and protect against osteoarthritis.

ANP32A represses Wnt signaling across tissues thereby protecting against osteoarthritis and heart disease.

OBJECTIVES: To investigate how ANP32A, previously linked to the antioxidant response, regulates Wnt signaling as unraveled by transcriptome analysis of Anp32a-deficient mouse articular cartilage, and its implications for osteoarthritis (OA) and diseases beyond the joint. METHODS: Anp32a knockdown chondrogenic ATDC5 cells were cultured in micromasses. Wnt target genes, differentiation markers and matrix deposition were quantified. Wnt target genes were determined in articular cartilage from Anp32a-deficient mice and primary human articular chondrocytes upon ANP32A silencing, using qPCR, luciferase assays and immunohistochemistry. Co-immunoprecipitation, immunofluorescence and chromatin-immunoprecipitation quantitative PCR probed the molecular mechanism via which ANP32A regulates Wnt signaling. Anp32a-deficient mice were subjected to the destabilization of the medial meniscus (DMM) OA model and treated with a Wnt inhibitor and an antioxidant. Severity of OA was assessed by cartilage damage and osteophyte formation. Human Protein Atlas data analysis identified additional organs where ANP32A may regulate Wnt signaling. Wnt target genes were determined in heart and hippocampus from Anp32a-deficient mice, and cardiac hypertrophy and fibrosis quantified. RESULTS: Anp32a loss triggered Wnt signaling hyper-activation in articular cartilage. Mechanistically, ANP32A inhibited target gene expression via histone acetylation masking. Wnt antagonist treatment reduced OA severity in Anp32a-deficient mice by preventing osteophyte formation but not cartilage degradation, contrasting with antioxidant treatment. Dual therapy ameliorated more OA features than individual treatments. Anp32a-deficient mice also showed Wnt hyper-activation in the heart, potentially explaining the cardiac hypertrophy phenotype found. CONCLUSIONS: ANP32A is a novel translationally relevant repressor of Wnt signaling impacting osteoarthritis and cardiac disease.

Complement component C1q is produced by isolated articular chondrocytes.

OBJECTIVE: Inflammation and innate immune responses may contribute to development and progression of Osteoarthritis (OA). Chondrocytes are the sole cell type of the articular cartilage and produce extracellular-matrix molecules. How inflammatory mediators reach chondrocytes is incompletely understood. Previous studies have shown that chondrocytes express mRNA encoding complement proteins such as C1q, suggesting local protein production, which has not been demonstrated conclusively. The aim of this study is to explore C1q production at the protein level by chondrocytes. DESIGN: We analysed protein expression of C1q in freshly isolated and cultured human articular chondrocytes using Western blot, ELISA and flow cytometry. We examined changes in mRNA expression of collagen, MMP-1 and various complement genes upon stimulation with pro-inflammatory cytokines or C1q. mRNA expression of C1 genes was determined in articular mouse chondrocytes. RESULTS: Primary human articular chondrocytes express genes encoding C1q, C1QA, C1QB, C1QC, and secrete C1q to the extracellular medium. Stimulation of chondrocytes with pro-inflammatory cytokines upregulated C1QA, C1QB, C1QC mRNA expression, although this was not confirmed at the protein level. Extracellular C1q bound to the chondrocyte surface dose dependently. In a pilot study, binding of C1q to chondrocytes resulted in changes in the expression of collagens with a decrease in collagen type 2 and an increase in type 10. Mouse articular chondrocytes also expressed C1QA, C1QB, C1QC, C1R and C1S at the mRNA level. CONCLUSIONS: C1q protein can be expressed and secreted by human articular chondrocytes and is able to bind to chondrocytes influencing the relative collagen expression.

Exostosin-1 enhances canonical Wnt signaling activity during chondrogenic differentiation.

OBJECTIVE: Exostosin-1 (Ext1) encodes a glycosyltransferase required for heparan sulfate (HS) chain elongation in HS-proteoglycan biosynthesis. HS chains serve as binding partners for signaling proteins, affecting their distribution and activity. The Wnt/ß-catenin pathway emerged as critical regulator of chondrogenesis. Yet, how EXT1 and HS affect Wnt/ß-catenin signaling during chondrogenesis remains unexplored. METHOD: Ext1 was stably knocked-down or overexpressed in ATDC5 chondrogenic cells cultured as micromasses. HS content was determined using ELISA. Chondrogenic markers Sox9, Col2a1, Aggrecan, and Wnt direct target gene Axin2 were measured by RT-qPCR. Proteoglycan content was evaluated by Alcian blue and DMMB assay, canonical Wnt signaling activation by ß-catenin Western blot and TOP/FOP assay. ATDC5 cells and human articular chondrocytes were treated with Wnt activators CHIR99021 and recombinant WNT3A. RESULTS: Ext1 knock-down reduced HS, and increased chondrogenic markers and proteoglycan accumulation. Ext1 knock-down reduced active Wnt/ß-catenin signaling. Conversely, Ext1 overexpressing cells, with higher HS content, showed decreased chondrogenic differentiation and enhanced Wnt/ß-catenin signaling. Wnt/ß-catenin signaling activation led to a down-regulation of Ext1 expression in ATDC5 cells and in human articular chondrocytes. CONCLUSIONS: EXT1 affects chondrogenic differentiation of precursor cells, in part via changes in the activity of Wnt/ß-catenin signaling. Wnt/ß-catenin signaling controls Ext1 expression, suggesting a regulatory loop between EXT1 and Wnt/ß-catenin signaling during chondrogenesis.

Increased susceptibility to develop spontaneous and post-traumatic osteoarthritis in Dot1l-deficient mice.

OBJECTIVE: We earlier identified that the histone methyltransferase Disruptor of telomeric silencing 1-like (DOT1L) is as a master protector of cartilage health via limiting excessive activation of the Wnt pathway. However, cartilage-specific homozygous Dot1l knockout mice exhibited a severe growth phenotype and perinatal death, which hampered their use in induced or ageing models of osteoarthritis (OA). The aim of this study was to generate and examine haploinsufficient and inducible conditional Dot1l-deficient mouse models to evaluate the importance of DOT1L during post-traumatic or ageing-associated OA onset and progression. METHOD: We used cartilage-specific heterozygous and postnatal tamoxifen-inducible Dot1l knockout mice and performed destabilization of the medial meniscus (DMM) and ageing as OA models. Mice were examined histologically using X-rays and micro-computed tomography (µCT), and cartilage damage and osteophyte formation were assessed based on OARSI guidelines. Immunohistochemistry of DOT1L, H3K79me2, TCF1 and COLX was performed. RESULTS: Both Dot1l-deficient strains exhibit a phenotype characterized by joint remodeling with extensive osteophyte formation and ectopic ossification upon ageing, indicating accelerated development of spontaneous osteoarthritis. In the DMM-induced OA mouse model, absence of Dot1l resulted in increased cartilage damage. Wnt signalling hyper-activation and ectopic chondrocyte hypertrophy were observed in the articular cartilage of both Dot1l-deficient mice. CONCLUSIONS: This study demonstrated the functional relevance of DOT1L in vivo during the development of OA using genetically modified mice. Thus, maintaining or enhancing DOT1L activity during ageing or after trauma might prevent OA onset and progression.

Loss of Frzb and Sfrp1 differentially affects joint homeostasis in instability-induced osteoarthritis.

OBJECTIVE: To investigate the specific role of Frizzled-related protein (FRZB) and Secreted frizzled-related protein 1 (SFRP1) in the onset and progression of Osteoarthritis (OA) using Frzb(-/-) and Sfrp1(-/-) mice in the destabilization of medial meniscus model (DMM), a slowly progressing model of OA. Secreted frizzled-related proteins (SFRPs) were identified as secreted Wingless-type (Wnt) antagonists. The Wnt signaling cascade is a major regulator in cartilage development, homeostasis and degeneration. METHODS: The DMM model was surgically induced in eight-week-old male C57/Bl6 Frzb(-/-), Sfrp1(-/-) or wild-type mice by transection of the medial meniscotibial ligament. Cartilage damage in the femoral and tibial articular surfaces was calculated following the Osteoarthritis Research Society International (OARSI) histopathology initiative guidelines. Histomorphometry was used to evaluate the subchondral bone plate thickness. RESULTS: OA severity scores were significantly higher in the tibia of Frzb(-/-) mice as compared to littermates, whereas no interaction was seen between genotype and intervention in Sfrp1(-/-) mice. Moreover, the DMM model resulted in significantly greater subchondral bone changes compared to sham but was not different between Frzb(-/-) mice and littermates. In contrast, the subchondral bone properties in Sfrp1(-/-) mice were significantly different from littermates. CONCLUSION: Using the DMM model, we demonstrated that FRZB and SFRP1 differentially modulate joint homeostasis in two distinct compartments of the joint. These data highlight the fine-tuning of Wnt signaling in joint homeostasis and disease, show differential regulation of the cascade in cartilage and subchondral bone, and provide further evidence for a role of endogenous Wnt modulators as key players in OA.

The coupling of bone and cartilage turnover in osteoarthritis: opportunities for bone antiresorptives and anabolics as potential treatments?

Osteoarthritis (OA) is the most common form of arthritic disease, and a major cause of disability and impaired quality of life in the elderly. OA is a complex disease of the entire joint, affecting bone, cartilage and synovium that thereby presents multiple targets for treatment. This manuscript will summarise emerging observations from cell biology, preclinical and preliminary clinical trials that elucidate interactions between the bone and cartilage components in particular. Bone and cartilage health are tightly associated. Ample evidence has been found for bone changes during progression of OA including, but not limited to, increased turnover in the subchondral bone, undermineralisation of the trabecular structure, osteophyte formation, bone marrow lesions and sclerosis of the subchondral plate. Meanwhile, a range of investigations has shown positive effects on cartilage health when bone resorption is suppressed, or deterioration of the cartilage when resorption is increased. Known bone therapies, namely oestrogens, selective oestrogen receptor modifiers (SERMs), bisphosphonates, strontium ranelate, calcitonin and parathyroid hormone, might prove useful for treating two critical tissue components of the OA joint, the bone and the cartilage. An optimal treatment for OA likely targets at least these two tissue components. The patient subgroups for whom these therapies are most appropriate have yet to be fully defined but would likely include, at a minimum, those with high bone turnover.

Rituximab treatment induces the expression of genes involved in healing processes in the rheumatoid arthritis synovium.

OBJECTIVE: Rituximab displays therapeutic benefits in the treatment of patients with rheumatoid arthritis (RA) resistant to tumor necrosis factor (TNF) blockade. However, the precise role of B cells in the pathogenesis of RA is still unknown. We undertook this study to investigate the global molecular effects of rituximab in synovial biopsy samples obtained from anti-TNF-resistant RA patients before and after administration of the drug. METHODS: Paired synovial biopsy samples were obtained from the affected knee of anti-TNF-resistant RA patients before (time 0) and 12 weeks after (time 12) initiation of rituximab therapy. Total RNA was extracted, labeled according to standard Affymetrix procedures, and hybridized on GeneChip HGU133 Plus 2.0 slides. Immunohistochemistry and quantitative real-time reverse transcriptase-polymerase chain reaction experiments were performed to confirm the differential expression of selected transcripts. RESULTS: According to Student's paired t-tests, 549 of 54,675 investigated probe sets were differentially expressed between time 0 and time 12. Pathway analysis revealed that genes down-regulated between time 0 and time 12 were significantly enriched in immunoglobulin genes and genes involved in chemotaxis, leukocyte activation, and immune responses (Gene Ontology annotations). In contrast, genes up-regulated between time 0 and time 12 were significantly enriched in transcripts involved in cell development (Gene Ontology annotation) and wound healing (Gene Set Enrichment Analysis). At baseline, higher synovial expression of immunoglobulin genes was associated with response to therapy. CONCLUSION: Rituximab displays unique effects on global gene expression profiles in the synovial tissue of RA patients. These observations open new perspectives in the understanding of the biologic effects of the drug and in the selection of patients likely to benefit from this therapy.

Detection, identification and in vivo treatment responsiveness of bone morphogenetic protein (BMP)-activated cell populations in the synovium of patients with rheumatoid arthritis.

OBJECTIVE: To characterise the bone morphogenetic protein (BMP) target cells positive for phosphorylated (P)-SMAD1/5, in rheumatoid arthritis (RA) synovium. METHODS: Synovial biopsies were obtained by needle arthroscopy. Anti-P-SMAD1/5 antibodies were used for Western blot (WB) on protein extracts from RA and normal synovium and for immunostaining of synovial biopsy sections. Positive cells were further identified by double staining for CD3, CD20, CD68, CD138, CD90, alpha smooth muscle actin (SMA), endoglin (CD105) and von Willebrand factor (VWF). In sections from early patients with RA taken before and under antirheumatic treatment, the degree of inflammation and activation of the BMP pathway were quantified. RESULTS: P-SMAD1/5 protein was detected by WB in RA and to a lesser extent in normal synovium. Different P-SMAD1/5 positive cell populations were identified in RA synovium, mainly in perivascular and sublining cells. P-SMAD1/5 positive perivascular cells were alphaSMA positive and located around VWF positive endothelial cells. Some CD90 positive synovial fibroblasts were P-SMAD1/5 positive, as was part of the CD68 positive synovial cells but other cells of the haematopoietic lineage showed no SMAD1/5 phosphorylation. Treatment resulted in an absolute but not relative decrease in BMP activation in the synovium. CONCLUSION: BMP-activated cells belong to distinct stromal compartments in RA synovium and some of them express markers associated with the mesenchymal progenitor cell lineage. Antirheumatic treatment effectively downregulates synovial inflammation, but BMP activation in the synovium does persist albeit reduced.

Deletion of frizzled-related protein reduces voluntary running exercise performance in mice.

OBJECTIVE: To study the effect of frizzled-related protein (Frzb) deletion in mice on voluntary running wheel exercise performance and osteoarthritis. METHODS: At the age of 7 weeks, Frzb(-/-) and wild-type mice were grouped and a running wheel was introduced into the cage. At week 8, all mice were caged solitarily with a running wheel available. Mice were allowed free exercise for 6-12 months and distances run were recorded daily. Non-running mice were used as additional control group. X-rays of knees and hips were taken at different time points. At the end of the experiment, mice were sacrificed and joints were processed for histological evaluation. Cartilage damage, synovitis and osteophyte formation were scored. Muscle fiber composition of the soleus and extensor digitorum longus was studied by immunofluorescence. RESULTS: At the age of 6 months, both female and male wild-type mice showed a significantly greater exercise performance than the Frzb(-/-) mice (P<0.05). At 1 year, the difference was still significant for male mice, but not for females. Running exercise did not significantly affect severity of osteoarthritis. No statistical differences in osteoarthritis severity were seen between Frzb(-/-) mice and wild-type mice. No differences were seen in muscle composition between Frzb(-/-) mice and wild-type mice. CONCLUSION: Absence of Frzb in mice reduced voluntary exercise performance in running wheels. These experiments demonstrate that the effects of genes in mice can also be evaluated using functional outcomes such as running wheel exercise performance, similar to evolving practice in human clinical trials.

Differences in pathophysiology between rheumatoid arthritis and ankylosing spondylitis.

Rheumatoid arthritis and ankylosing spondylitis are common and severe chronic inflammatory skeletal diseases. Recognizing the differences rather than emphasizing similarities is important for a better understanding of the disease processes, the identification of specific therapeutic targets and in the long-term better treatment options for the individual patients. We discuss a number of pathophysiological differences between rheumatoid arthritis and ankylosing spondylitis by looking at the anatomical characteristics, differences and similarities in the autoimmune and autoinflammatory reactions, association with other immune mediated inflammatory diseases, structural outcome, and their potential significance for further therapeutic developments. Further research into the differences between these diseases should focus on the specific nature of the immune/inflammatory components, the role of resident cells in the joint and joint-associated tissues, the types and mechanisms of tissue remodeling and the characteristics of the articular cartilage. Better insights into their individual characteristics may lead to better therapeutic strategies, specific targets and useful biomarkers.

Absence of GDF5 does not interfere with LPS Toll-like receptor signaling.

OBJECTIVES: Growth and differentiation factor 5 (GDF5), member of TGFBeta superfamily, has been implicated in limb development, and is known to play an important role in joint formation. Its absence leads to brachypodism in mice and a number of skeletal malformation syndromes in humans. Recently, an association was shown between osteo-arthritis and a 5' UTR polymorphism in GDF5 gene. In addition, the role of GDF5 may reach beyond the musculoskeletal system. GDF5 appears present in a lipopolysaccharide (LPS) receptor cluster. Absence of GDF5 may limit the response to LPS. This may have consequences for immune responses and macrophage function in general, and for arthritis in particular. Here we compared the sensitivity of Gdf5(Bp-J/Bp-J) mice and wild type (WT) mice to LPS. METHODS: Peritoneal macrophages from Gdf5(Bp-J/Bp-J) mice and WT mice were stimulated for 18h with LPS (0, 10 or 100 ng/ml). The supernatant was collected and TNF release was measured by ELISA and by an indirect luciferase assay using LNF-luc C3 cells. Gdf5(Bp-J/Bp-J) mice and WT mice were injected with LPS i.p. (30 mg/kg) and LPS induced lethality was checked every 3 hours for 36 hours. RESULTS: Gdf5(Bp-J/Bp-J) macrophages showed no difference in TNF expression upon LPS stimulation measured by ELISA and by indirect luciferase assay. Gdf5(Bp-J/Bp-J) mice died upon a lethal dose of LPS, as is seen in WT controls. CONCLUSION: Absence of Gdf5 appears not to affect the LPS response. Mice with a reduced expression of Gdf5 can be used in disease models which are dependent on LPS boost.

Inhibition of osteoclasts does not prevent joint ankylosis in a mouse model of spondyloarthritis.

OBJECTIVES: The relationship between inflammation, destruction and new tissue formation leading to ankylosis, determines the severity and prognosis of patients with SpA. Recent data in mice and men suggest that new cartilage and bone formation and subsequent ankylosis are uncoupled from chronic inflammation. These data challenge the hypothesis that inflammation and tissue damage trigger an excessive repair response in SpA. We tested whether inhibition of bone erosion by targeting osteoclasts, would prevent or influence joint ankylosis in a mouse model. METHODS: Male DBA/1 mice from different litters were caged together at the age of 8 weeks. Treatment with zoledronic acid (ZA) (100 ng/g) or placebo was started at the age of 10 weeks and administered every 2 weeks. Clinical incidence and severity of arthritis were evaluated twice a week until the age of 26 weeks. At this point, bone density measurements were performed, mice were sacrificed and severity of arthritis was evaluated by histology. RESULTS: Treatment with ZA did not affect incidence or clinical severity of arthritis in male DBA/1 mice. ZA treatment significantly increased bone mineral density and content as demonstrated by dual X-ray densitometry and peripheral quantitative CT. However, the treatment did not affect histomorphological appearance of arthritis or ankylosis. CONCLUSIONS: These data suggest that bone erosion at the enthesis does not necessarily precede entheseal ankylosis. Therefore, these observations further support the concept that inflammation and new tissue formation in SpA are at least partially uncoupled events and may be different therapeutic targets.

Activation of nuclear factor kappa B and mitogen activated protein kinases in psoriatic arthritis before and after etanercept treatment.

OBJECTIVE: To study activation of intracellular pathways depending on nuclear factor kappa B (NFkappaB) and mitogen activated kinases (MAPK) in the synovium of patients with psoriatic arthritis before and after treatment with etanercept. METHODS: Synovial biopsies were obtained by needle arthroscopy of the knee in 9 patients with active psoriatic arthritis before the initiation of etanercept. Follow-up biopsies were taken in the same knee after 6 months. Synovitis was studied by histology. Pathway activation was studied by immunofluorescense for phosphorylated ERK, phosphorylated p38, phosphorylated JNK or phosphorylated inhibitor of kappa B (IkappaBalpha) using digital image analysis. RESULTS: Histological severity scores were significantly reduced after etanercept treatment. Activation of NFkappaB signaling was found in the lining layer, and in infiltrating and peri-vascular cells in the sublining zone. Activated p38 was present in both lining and sublining layer. In the sublining layer, positive cells were found in inflammatory infiltrates, in perivascular zones and in the endothelium. Activated ERK was mainly present in the sublining layer, both in mononuclear cell infiltrates and perivascularly. Occasional positive cells were found in the lining layer. Activation of JNK was recognized in cells of the lining layer, in some of the sublining cell infiltrates and the perivascular compartment. CONCLUSIONS: Etanercept therapy resulted in a significant decrease in NFkappaB, JNK and ERK, but not in p38 activation. Persistent activation of these pathways, albeit reduced, may trigger positive feedback loops and flares of arthritis after cessation of etanercept.

Animal models in systemic sclerosis.

Systemic sclerosis (SSc) is a complex systemic disease. It is characterised by fibrosis, of the skin and internal organs, as lungs, kidney, heart and gastrointestinal tract. As the aetiology of SSc is unknown, numerous animal models have been developed to better understand the pathogenesis of the disease and to test potentially useful therapeutic interventions. Although several animal models have been described, predominantly in mice, none reproduces precisely all manifestations of SSc. However, all animal models display tissue fibrotic changes similar to those present in SSc. This review is focused on the principal animal models and the molecular pathways involved. Animal models can be divided into two groups. In one group the pathologic phenotype is the result of a genetic mutation ( tight skin 1 and tight skin 2,UCD 200). In the second group, the pathologic alterations are induced in normal animals by manipulation of their immune system (sclerodermatous graft-vs-host disease (Scl GVHD) induced by transplantation, or by administration of exogenous substances). In the future, the development of additional animal models may become important in the further understanding of the alteration of the molecular pathways that regulate a physiologic processes to induce tissue fibrosis, the hallmark of this disease, and to identify and test targeted therapeutic agents.

Niemann-Pick disease type B: an unusual clinical presentation with multiple vertebral fractures.

We report here a unique case of a 55-year-old woman presenting with a clinical picture of Parkinson disease, severe back pain, splenomegaly, and pronounced dyspnea. Radiographic examination of the spine showed multiple vertebral fractures. Niemann-Pick disease type B was diagnosed by findings of lipid-loaded histiocytes and a strongly reduced sphingomyelinase enzyme activity. She was homozygous for the deletion of codon 608 (delR608), which encodes an arginine residue in the Acid Sphingomyelinase gene. To investigate the cause of the unusual vertebral fractures, we screened for polymorphisms previously described as possibly associated with increased risk for osteoporosis and fractures. Our patient was heterozygous for the polymorphisms of the vitamin D receptor gene, the estrogen receptor gene, and the collagen 1A1gene. Increased physical activity after Parkinson treatment, a genetic predisposition, together with worsening disease due to interfering medications could explain the dramatic presentation of this patient. She was treated with cholesterol lowering drugs such as statins to decrease sphingomyelin synthesis, avoidance of drugs that inhibit sphingomyelinase, and bisphosphonates. No new fractures have occurred, but the interstitial lung disease has progressed.

Osteoprotegerin and osteoprotegerin-ligand balance: a new paradigm in bone metabolism providing new therapeutic targets.

Osteoprotegerin-ligand, also called Osteoclast Differentiation Factor or RANK-ligand, its receptor RANK and its decoy receptor Osteoprotegerin are key molecules regulating osteoclast differentiation and activation. In this view we discuss structure and expression of these molecules, the genetic models addressing their function and their role in in vivo models of osteoclast differentiation and activation. The new paradigm that has evolved from these studies, is not only important in normal bone homeostasis but also appears to play a role in different diseases that affect the skeleton, such as osteoporosis, inflammatory joint disease and cancer. It has opened a new era in bone research by increasing our molecular knowledge and providing new therapeutic targets in bone disease.