Pathogenesis of ligaments ossification in spondyloarthritis: insights and doubts.

Despite intensive research in spondyloarthritis pathogenesis, some important questions still remain unanswered, particularly concerning enthesis new bone formation. Several evidences suggest that it prevalently occurs by endochondral ossification, however it remains to identify factors that can induce and influence its initiation and progression. Recent progress, achieved in animal models and in vitro and genetic association studies, has led us to hypothesize that several systemic factors (adipokines and gut hormones) and local factors (BMP and Wnt signaling) as well as angiogenesis and mechanical stress are involved. We critically review and summarize the available data and delineate the possible mechanisms involved in enthesis ossification, particularly at spinal ligament level. KEY MESSAGES Complete understanding of spondyloarthritis pathophysiology requires insights into inflammation, bone destruction and bone formation, which are all located in entheses and lead all together to ankylosis and functional disability. Several factors probably play a role in the pathogenesis of bone formation in entheses including not only cytokines but also several systemic factors such as adipokines and gut hormones, and local factors, such as BMP and Wnt signaling, as well as angiogenesis and mechanical stress. Data available about pathophysiology of new bone formation in spondyloarthritis are limited and often conflicting and future studies are needed to better delineate it and to develop new therapeutic approaches.

Relationship between Body Mass Composition, Bone Mineral Density, Skin Fibrosis and 25(OH) Vitamin D Serum Levels in Systemic Sclerosis.

A reduced bone mineral density (BMD) is observed in several rheumatic autoimmune diseases, including Systemic Sclerosis (SSc); nevertheless, data concerning the possible determinants of bone loss in this disease are not fully investigated. The aim of this study is to evaluate the relationship between BMD, body mass composition, skin sclerosis and serum Vitamin D levels in two subsets of SSc patients. 64 post-menopausal SSc patients, classified as limited cutaneous (lcSSc) or diffuse cutaneous (dcSSc) SSc, were studied. As control, 35 healthy post-menopausal women were recruited. Clinical parameters were evaluated, including the extent of skin involvement. BMD at lumbar spine, hip, femoral neck and body mass composition were determined by dual-energy X-ray absorptiometry. Serum calcium, phosphorus, alkaline phosphatase, urine pyridinium cross-links, intact parathyroid hormone and 25-hydroxyvitamin D (25OHD) were measured. BMD at spine, femoral neck and total hip was significantly lower in SSc patients compared to controls. In dcSSc subset, BMD at spine, femoral neck and total hip was significantly lower compared to lcSSc. No differences in both fat and lean mass were found in the three study groups even if patients with dcSSc showed a slightly lower total body mass compared to healthy controls. Total mineral content was significantly reduced in dSSc compared to both healthy subjects and lcSSc group. Hypovitaminosis D was observed both in healthy post-menopausal women and in SSc patients, but 25OHD levels were significantly lower in dcSSc compared to lcSSc and inversely correlated with the extent of skin thickness. These results support the hypothesis that the extent of skin involvement in SSc patients could be an important factor in determining low circulating levels of 25OHD, which in turn could play a significant role in the reduction of BMD and total mineral content.

Yes-associated protein (YAP) is a negative regulator of chondrogenesis in mesenchymal stem cells.

INTRODUCTION: The control of differentiation of mesenchymal stromal/stem cells (MSCs) is crucial for tissue engineering strategies employing MSCs. The purpose of this study was to investigate whether the transcriptional co-factor Yes-associated protein (YAP) regulates chondrogenic differentiation of MSCs. METHODS: Expression of total YAP, its paralogue transcriptional co-activator with PDZ-binding motif (TAZ), and individual YAP transcript variants during in vitro chondrogenesis of human MSCs was determined by quantitative reverse transcription polymerase chain reaction (RT-PCR). YAP expression was confirmed by western blotting. To determine the effect of high YAP activity on chondrogenesis, C3H10T1/2 MSC-like cells were transduced with human (h)YAP and treated in micromass with bone morphogenetic protein-2 (BMP-2). Chondrogenic differentiation was assessed by alcian blue staining and expression of chondrocyte-lineage genes. BMP signalling was determined by detection of pSmad1,5,8 by western blotting and expression of BMP target genes by quantitative RT-PCR. Finally, YAP and pYAP were detected in mouse embryo hindlimbs by immunohistochemistry. RESULTS: YAP, but not TAZ, was downregulated during in vitro chondrogenesis of human MSCs. One of the YAP transcript variants, however, was upregulated in high-density micromass culture. Overexpression of hYAP in murine C3H10T1/2 MSCs inhibited chondrogenic differentiation. High YAP activity in these cells decreased Smad1,5,8 phosphorylation and expression of the BMP target genes Inhibitor of DNA binding/differentiation (Id)1, Id2 and Id3 in response to BMP-2. In developing mouse limbs, Yap was nuclear in the perichondrium while mostly phosphorylated and cytosolic in cells of the cartilage anlage, suggesting downregulation of Yap co-transcriptional activity during physiological chondrogenesis in vivo. CONCLUSIONS: Our findings indicate that YAP is a negative regulator of chondrogenic differentiation of MSCs. Downregulation of YAP is required for chondrogenesis through derepression of chondrogenic signalling. Therapeutic targeting of YAP to promote cartilage repair and prevent secondary osteoarthritis is an exciting prospect in rheumatology.

Combined effects of infliximab and methotrexate on rheumatoid arthritis osteoblastic cell metabolism.

The goal of this study is to investigate the in vitro effects of two disease-modifying anti-rheumatic drugs, largely used in the treatment of rheumatoid arthritis (RA), [infliximab (IFX) and methotrexate (MTX)], in RA primary osteoblast cell cultures. MTX inhibited proliferation and metabolic activity in RA osteoblasts was able to increase apoptosis. Conversely, IFX increased the proliferation, osteocalcin production and the alkaline phosphatase activity. Interestingly, IFX appeared to antagonise the negative effect exerted by MTX. Both drugs significantly reduced the IL-6 production in osteoblasts when used alone, and the combination of the two agents resulted in a significant additional reduction of IL-6 synthesis, with an apparent additive effect. The present study suggests that MTX exerts negative direct effects on bone metabolism in RA patients, but the combined treatment with anti-TNF-α can be beneficial for the interaction of MTX with bone cells.

Extracellular matrix modulates angiogenesis in physiological and pathological conditions.

Angiogenesis is a multistep process driven by a wide range of positive and negative regulatory factors. Extracellular matrix (ECM) plays a crucial role in the regulation of this process. The degradation of ECM, occurring in response to an angiogenic stimulus, leads to degradation or partial modification of matrix molecules, release of soluble factors, and exposure of cryptic sites with pro- and/or antiangiogenic activity. ECM molecules and fragments, resulting from proteolysis, can also act directly as inflammatory stimuli, and this can explain the exacerbated angiogenesis that drives and maintains several inflammatory diseases. In this review we have summarized some of the more recent literature data concerning the molecular control of ECM in angiogenesis in both physiological and pathological conditions.

TNF-related apoptosis-inducing ligand (TRAIL) in rheumatoid arthritis: what's new?

TNF-related apoptosis-inducing ligand (TRAIL) is a type II transmembrane protein of the TNF superfamily that serves as an extracellular signal that triggers programmed cell death in tumor cells, without affecting normal cells. Recently, scientists have turned their attention to the emerging role of TRAIL in immune and autoimmune responses. TRAIL has been shown to down-regulate the self-antigens in autoimmune diseases, such as rheumatoid arthritis (RA) by exerting its apoptotic effect on activated T cells and synoviocytes and by its local anti-inflammatory effect. The impact of TRAIL molecular variants and agonistic monoclonal antibodies in the regulation of TRAIL activity in arthritis animal models strongly supports the idea of testing the role of TRAIL in humans, with the aim of developing new effective therapies that promote apoptosis of synoviocytes and/or infiltrating lymphocytes, by targeting TRAIL. The aim of this review is to summarize recent progress and current knowledge of TRAIL functions in RA.

Immunomodulatory effects of vitamin D in peripheral blood monocyte-derived macrophages from patients with rheumatoid arthritis.

1,25-Dihydroxyvitamin D (1,25(OH)2D3), the active form of vitamin D, modulates both innate and adaptive immune responses. Emerging epidemiological data has also demonstrated disease-modifying and immunomodulatory effects of vitamin D in a wide range of human autoimmune diseases, including rheumatoid arthritis (RA). To evaluate in vitro effects of 1,25(OH) 2D3 in primary cultures of peripheral blood monocyte-derived macrophages of RA patients, monocyte/macrophages, isolated from peripheral blood mononuclear cells of RA patients and healthy subjects by exploiting their ability to adhere to plastic, were treated with increasing concentrations of 1,25(OH)2D3 for 48 h. TNF-α, IL-1 α, IL-1ß, IL-6 and RANKL production was determined by ELISA and nitric oxide (NO) release using the Griess method. Immunocytochemistry analysis was also performed to evaluate alterations in transmembrane TNF-α expression after 1,25(OH) 2D3 treatment. A significant dose-dependent decrease in TNF-α and RANKL production by cultured RA macrophages after 1,25(OH)2D3 treatment was found, whereas a significant reduction in normal cells was observed only at higher concentrations. IL-1 α, IL-1ß and IL-6 levels were reduced by 1,25(OH) 2D3 at higher concentrations in all cell populations. TNF-α immunostaining was less intense in treated cells compared with untreated. 1,25(OH) 2D3 significantly reduced NO levels regardless of the concentration used. Vitamin D downregulated proinflammatory mediators in monocyte-derived macrophages, and RA cells appeared more sensitive than normal cells. These effects further provide a rationale for the therapeutic value of vitamin D supplementation in the treatment for RA.

Bone effects of biologic drugs in rheumatoid arthritis.

Biologic agents used in the treatment of rheumatoid arthritis (RA) are able to reduce both disease activity and radiographic progression of joint disease. These drugs are directed against several proinflammatory cytokines (TNF α , IL-6, and IL-1) which are involved both in the pathogenesis of chronic inflammation and progression of joint structural damage and in systemic and local bone loss typically observed in RA. However, the role of biologic drugs in preventing bone loss in clinical practice has not yet clearly assessed. Many clinical studies showed a trend to a positive effect of biologic agents in preventing systemic bone loss observed in RA. Although the suppression of inflammation is the main goal in the treatment of RA and the anti-inflammatory effects of biologic drugs exert a positive effect on bone metabolism, the exact relationship between the prevention of bone loss and control of inflammation has not been clearly established, and if the available biologic drugs against TNF α , IL-1, and IL-6 can exert their effect on systemic and local bone loss also through a direct mechanism on bone cell metabolism is still to be clearly defined.

RANKL/OPG ratio and DKK-1 expression in primary osteoblastic cultures from osteoarthritic and osteoporotic subjects.

OBJECTIVE: To evaluate the expression of Dickkopf-1 protein factor (DKK-1), DKK-2, and ß-catenin, components of the Wnt pathway, in human osteoarthritic (OA) and osteoporotic (OP) osteoblasts and to correlate it to cell metabolic activity, proliferation, and receptor activator of nuclear factor-κB ligand/osteoprotegerin (RANKL/OPG) expression. METHODS: Primary human osteoblast cultures were obtained from healthy, OA, and OP donors. In each cell population we evaluated DKK-1, DKK-2, nonphosphorylated ß-catenin and RANKL/OPG expression, osteocalcin and alkaline phosphatase (ALP) synthesis, and cell proliferation, both in basal condition and after vitamin D3 stimulation. RESULTS: DKK-1 and DKK-2 showed opposite patterns of expression in OA and OP osteoblasts. The RANKL/OPG ratio was significantly higher in the OP group because of a greater expression of RANKL, whereas it was significantly lower in the OA group because of a higher expression of OPG. Treatment with vitamin D3 increased the RANKL/OPG ratio and DKK-2 expression and reduced DKK-1 expression in each cell population, but did not affect ß-catenin levels. Both osteocalcin and ALP production and cell proliferation were enhanced in OA cells and reduced in the OP ones. CONCLUSION: These data confirm that OA and OP are characterized by opposite bone changes, consisting of reduced bone remodeling processes with increased osteoblast activity in OA, and enhanced bone resorptive activity with reduction of osteoblast metabolism in OP, and suggest that the Wnt pathway is involved in the pathogenesis of both diseases.

In vitro and in vivo angiogenic activity of osteoarthritic and osteoporotic osteoblasts is modulated by VEGF and vitamin D3 treatment.

Vascular Endothelial Growth Factor (VEGF) is a potent angiogenic factor, which also regulates bone remodeling. Osteoblasts not only respond to VEGF stimulation, but also express and synthesize this factor. The present study was aimed to evaluate in vitro differences in VEGF production and expression of cultured human osteoblastic cells derived from healthy donors and from subjects affected by osteoarthritis and osteoporosis, under basal conditions than after vitamin D3, and to investigate the angiogenic activity of culture media obtained by these cells in chick embryo chorioallantoic membrane (CAM) assay. The results showed that normal and pathological osteoblasts produce and express VEGF and 1,25 dihydroxy-vitamin D3 treatment increases protein and m-RNA VEGF levels. In addition culture media of pathological osteoblasts induce a strong angiogenic response, greater than observed with culture medium of normal cells, suggesting the involvement of osteoblast-derived VEGF in the pathogenesis of bone diseases.

Systemic effects of Wnt signaling.

Wnt signaling plays a key role in several physiological and pathological aspects. Even if Wnt signal was first described more than 20 years ago, its role in systemic effects, such as angiogenesis and vascular disorders, bone biology, autoimmune diseases, neurological diseases, and neoplastic disorders, was only recently emerged through the use of animal and in vitro models. Moreover, Wnt signaling inhibitors, such as DKK-1, may be advantageously considered targets for the treatment of several diseases, including osteoporosis, vascular diseases, inflammatory diseases, neurological diseases, and cancer. Nevertheless, further studies are required to provide a complete understanding of this complex signaling pathway, and especially of its role in human diseases, considering the possible advantageous effects of Wnt signaling inhibitors on the progression of disease conditions.

Osteocalcin: skeletal and extra-skeletal effects.

Osteocalcin (OC) is a non-collagenous, vitamin K-dependent protein secreted in the late stage of osteoblasts differentiation. The presence of the three residues of γ-carbossiglutamatic acid, specific of the active form of OC protein, allows the protein to bind calcium and consequently hydroxyapatite. The osteoblastic OC protein is encoded by the bone γ-carbossiglutamate gene whose transcription is principally regulated by the Runx2/Cbfa1 regulatory element and stimulated by vitamin D(3) through a steroid-responsive enhancer sequence. Even if data obtained in literature are controversial, the dual role of OC in bone can be presumed as follows: firstly, OC acts as a regulator of bone mineralization; secondly, OC regulates osteoblast and osteoclast activity. Recently the metabolic activity of OC, restricted to the un-carboxylated form has been demonstrated in osteoblast-specific knockout mice. This effect is mediated by the regulation of pancreatic ß-cell proliferation and insulin secretion and adiponectin production by adipose tissue and leads to the regulation of glucose metabolism and fat mass. Nevertheless, clinical human studies only demonstrated the correlation between OC levels and factors related to energy metabolism. Thus further investigations in humans are required to demonstrate the role of OC in the regulation of human energy metabolism. Moreover, it is presumable that OC also acts on blood vessels by inducing angiogenesis and pathological mineralization. This review highlights the recent studies concerning skeletal and extra-skeletal effects of OC.

Expression of vascular endothelial growth factor in normal, osteoarthritic and osteoporotic osteoblasts.

To evaluate vascular endothelial growth factor (VEGF) mRNA expression and protein synthesis in primary human osteoblast cultures from healthy, osteoporotic and osteoarthritic subjects. Normal primary human osteoblast cultures were obtained from healthy subjects undergoing surgery for the reduction in traumatic fractures. Pathological osteoblasts were obtained from patients undergoing to total hip replacement for osteoporotic hip fracture or advanced osteoarthritis. VEGF mRNA expression and protein synthesis were evaluated in cultured cells, by semiquantitative real-time PCR and ELISA, respectively, both under basal conditions than after vitamin D3 stimulation. Osteoarthritic osteoblasts showed a significantly higher VEGF expression compared to the normal and OP osteoblasts, both under basal conditions than in the presence of vitamin D3, whereas no difference was found between osteoporotic and normal osteoblast. Vitamin D3 significantly enhanced VEGF expression in normal and pathological osteoblasts. This preliminary study supports the hypothesis that VEGF is involved in the pathogenic mechanisms underlying the bone alterations typical of osteoarthritis and confirms the crucial role of vitamin D3 supplementation in metabolic bone diseases.

Bisphosphonates: effects on osteoblast.

PURPOSE: Bisphosphonates are synthetic analogues of pyrophosphate usually used in treating bone disorders such as osteoporosis, Paget's disease, fibrous dysplasia, hypercalcemia of malignancy, and inflammation-related bone loss. Though therapeutic effects of bisphosphonates depend primarily on their inhibitory effect on osteoclasts, increasing attention is being given to other effector cells, such as osteoblasts. This review focuses on the presumed effect of bisphosphonates on osteoblasts. METHODS: A review of the literature was conducted to evaluate the pharmacodynamic effects of bisphosphonates including inhibition of osteoclasts and apoptosis of osteocytes and osteoblasts as well as their potential stimulatory effects on the proliferation of osteoblasts. RESULTS: Studies have demonstrated that bisphosphonates may stimulate proliferation of osteoblasts and inhibit apoptosis of osteocytes and osteoblasts. CONCLUSION: Considering that osteoblasts may be involved in bone disorders, such as osteoporosis, osteopetrosis, osteogenesis imperfecta, and Paget's disease, and that bisphosphonates may stimulate proliferation of osteoblasts and inhibit apoptosis of osteocytes and osteoblasts, it is conceivable that a role for bisphosphonates exists in these diseases beyond merely the osteoclast influence.

Osteoblast physiology in normal and pathological conditions.

Osteoblasts are mononucleated cells that are derived from mesenchymal stem cells and that are responsible for the synthesis and mineralization of bone during initial bone formation and later bone remodelling. Osteoblasts also have a role in the regulation of osteoclast activity through the receptor activator of nuclear factor κ-B ligand and osteoprotegerin. Abnormalities in osteoblast differentiation and activity occur in some common human diseases such as osteoporosis and osteoarthritis. Recent studies also suggest that osteoblast functions are compromised at sites of focal bone erosion in rheumatoid arthritis.

Botulinum toxin type A in the treatment of focal hand dystonia after surgical treatment for thumb duplication.

Dystonia is a set of disorders characterized by abnormal postures and unwanted muscle spasms that interfere with motor performance. Focal dystonias, the most common, affect just 1 body part. A 25-year-old woman had a focal hand dystonia with pain and muscle spasms in the right hand after surgical treatment for thumb duplication. According to Wassel's classification, she was type II, with duplication of the distal phalanx. She had undergone Bilhaut-Cloquet surgery to remove a radial supernumerary thumb. During repetitive movements, she reported pain and muscle cramping in the right thumb and wrist. Force was rated, according to the Medical Research Council scale, from grades 0 to 5, and the patient had a score of 4. No sign of joint instability was found in her hand, and normal active and passive range of motion were found for interphalangeal or metacarpophalangeal joints of right thumb. However, right thenar muscle hypoplasia was found. Repetitive activity acted as an environmental trigger for the reconstructed thumb on thenar muscle hypoplasia as residual anatomical modification. Surface electromyography showed movements suggestive of dystonia. After 5 days of treatment with botulinum toxin type A, with both the flexor pollicis longus and the abductor pollicis brevis of right hand injected with a dose of 20 microM each, the patient reported a regression of most signs and symptoms. Two-month follow-up revealed that clinical effects of botulinum toxin type A were still present.