Christopher A. Holding

Osteoprotegerin (OPG) is localized to the Weibel-Palade bodies of human vascular endothelial cells and is physically associated with von Willebrand factor†

Recent studies demonstrate roles for osteoprotegerin (OPG) in both skeletal and extra‐skeletal tissues. Although its role in preventing osteoclast (OC) formation and activity is well documented, emerging evidence suggests a role of OPG in endothelial cell survival and the prevention of arterial calcification. In this communication, we show that vascular endothelial cells in situ, and human umbilical vein endothelial cells (HUVEC) in vitro, express abundant OPG. In HUVEC, OPG co‐localizes with P‐selectin and von Willebrand factor (vWF), within the Weibel‐Palade bodies (WPB). Treatment of HUVEC with the pro‐inflammatory cytokines, tumor necrosis factor (TNF)‐α and IL‐1β, resulted in mobilization from the WPBs and subsequent secretion of OPG protein into the culture supernatant. Furthermore, TNF‐α treatment of HUVEC resulted in a sustained increase in OPG mRNA levels and protein secretion over the 24‐h treatment period. Reciprocal immunoprecipitation experiments revealed that while not associated with P‐Selectin, OPG is physically complexed with vWF both within the WPB and following secretion from endothelial cells. Interestingly, this association was also identified in human peripheral blood plasma. In addition to its interaction with vWF, we show that OPG also binds with high avidity to the vWF reductase, thrombospondin (TSP‐1), raising the intriguing possibility that OPG may provide a link between TSP‐1 and vWF. In summary, the intracellular localization of OPG in HUVEC, in association with vWF, together with its rapid and sustained secretory response to inflammatory stimuli, strongly support a modulatory role in vascular injury, inflammation and hemostasis.

Factors regulating osteoclast formation in human tissues adjacent to peri-implant bone loss: expression of receptor activator NFκB, RANK ligand and osteoprotegerin

Aseptic bone loss adjacent to orthopedic joint implants is a common cause of joint implant failure in humans. This study investigates the expression of key regulators of osteoclast formation, receptor activator NFkappaB (RANK), Receptor activator of NFkappaB ligand (RANKL) and osteoprotegerin (OPG), in the peri-implant tissues of patients with osteolysis compared with levels in synovial tissues from osteoarthritic and healthy subjects. Immunohistochemical studies demonstrated that significantly higher levels of RANKL protein (p<0.05) were found in the peri-implant tissues of patients with implant failure than in similar tissues from osteoarthritic and healthy subjects. In contrast, OPG protein levels were similar in all tissues. RANKL, expressed as mRNA and protein, was predominantly associated with cells containing wear particles. Dual labeling studies showed that the cells expressing RANKL protein were macrophages. In situ hybridization studies confirmed that mRNA encoding for these proteins is also expressed by cells in the peri-implant tissues. In addition, RANK mRNA was expressed in cells that contained wear particles. These findings show that abnormally high levels of RANKL are expressed in peri-implant tissues of patients with prosthetic loosening and that these abnormal levels of RANKL may significantly contribute to aseptic implant loosening.

The induction of a catabolic phenotype in human primary osteoblasts and osteocytes by polyethylene particles

Polyethylene (PE) wear particles are associated with the osteolysis seen in aseptic loosening that leads to orthopaedic implant failure. While cells of the monocyte/macrophage lineage are implicated, evidence is now emerging that osteoblastic cells may also be affected by PE. In this study we investigated the effect of PE particles on osteoblasts, using a novel in vitro cell culture system that was developed to juxtapose cells and PE particles, replicating the 3-dimensional (3D) environment near implants. This system allowed normal human bone-derived cells (NHBC) to undergo differentiation into a mature osteocyte-like phenotype over a 21-28-day culture period. PE particles induced an increase in mRNA expression of the osteocyte markers E11, DMP-1 and SOST/sclerostin. NHBC responded to PE particles by increasing the mRNA expression of several genes associated with osteoclast formation and activity (RANKL, IL-8 and M-CSF) and decreased the expression of the osteoclast antagonist, OPG. PE also appeared to induce a switch in the RUNX2 control of gene expression from that of promoting matrix production (type I collagen) to inducing the expression of pro-osteoclastogenic genes. These results suggest that PE particles switch mature osteoblastic cells from an anabolic to a more catabolic phenotype. This concept was further supported by the finding that PE-induced expression of RANKL mRNA in the mouse osteocyte cell line, MLO-Y4. Overall, our results suggest that PE particles directly induce a change in the phenotype of mature osteoblasts and osteocytes, consistent with the net loss of bone near orthopaedic implants.

Elevated expression of caspase-3 inhibitors, survivin and xIAP correlates with low levels of apoptosis in active rheumatoid synovium

IntroductionTumour necrosis factor-related apoptosis-inducing ligand (TRAIL) is a tumour necrosis factor (TNF) family member capable of inducing apoptosis in many cell types.MethodsUsing immunohistochemistry, terminal deoxynucleotidyl transferase biotin-dUTP nick end labelling (TUNEL) and real-time PCR we investigated the expression of TRAIL, TRAIL receptors and several key molecules of the intracellular apoptotic pathway in human synovial tissues from various types of arthritis and normal controls. Synovial tissues from patients with active rheumatoid arthritis (RA), inactive RA, osteoarthritis (OA) or spondyloarthritis (SpA) and normal individuals were studied.ResultsSignificantly higher levels of TRAIL, TRAIL R1, TRAIL R2 and TRAIL R4 were observed in synovial tissues from patients with active RA compared with normal controls (p < 0.05). TRAIL, TRAIL R1 and TRAIL R4 were expressed by many of the cells expressing CD68 (macrophages). Lower levels of TUNEL but higher levels of cleaved caspase-3 staining were detected in tissue from active RA compared with inactive RA patients (p < 0.05). Higher levels of survivin and x-linked inhibitor of apoptosis protein (xIAP) were expressed in active RA synovial tissues compared with inactive RA observed at both the protein and mRNA levels.ConclusionsThis study indicates that the induction of apoptosis in active RA synovial tissues is inhibited despite stimulation of the intracellular pathway(s) that lead to apoptosis. This inhibition of apoptosis was observed downstream of caspase-3 and may involve the caspase-3 inhibitors, survivin and xIAP.

Biological responses of human osteoblasts and osteoclasts to flame-sprayed coatings of hydroxyapatite and fluorapatite blends

The aim of this study was to determine how the activities of human osteoblastic cells and osteoclasts respond to substrates of thermal-sprayed mechanical blends of hydroxyapatite and fluorapatite with a view of determining an optimal blend ratio for osseointegration. Human osteoblastic cells and osteoclasts were grown on titanium alloy discs coated with blends of hydroxyapatite and fluorapatite, with concentrations ranging from 0 to 100% fluorapatite. Human osteoblastic cells attached in greater numbers and proliferated at a greater rate on blends containing 40% fluorapatite. Human osteoblastic cells grown on blends containing 40% fluorapatite for 7 days also expressed the highest levels of mRNA for several proteins involved with regulating bone metabolism (osteoprotegerin and receptor activator nuclear factor kappa B ligand), and bone formation (osteopontin, osteonectin and bone sialoprotein 1). Osteoclasts resorbed the dentine but poorly resorbed the hydroxyapatite-fluorapatite blends, particularly at high levels of fluorapatite. This in vitro study demonstrates that thermal-sprayed hydroxyapatitecoatings containing 40% fluorapatite may promote optimal bone growth and improve osseointegration of implants.

Inhibitors of histone deacetylases in class I and class II suppress human osteoclasts in vitro.

Histone deacetylase inhibitors (HDACi) suppress cancer cell growth, inflammation, and bone resorption. The aim of this study was to determine the effect of inhibitors of different HDAC classes on human osteoclast activity in vitro. Human osteoclasts generated from blood mononuclear cells stimulated with receptor activator of nuclear factor kappa B (RANK) ligand were treated with a novel compound targeting classes I and II HDACs (1179.4b), MS‐275 (targets class I HDACs), 2664.12 (targets class II HDACs), or suberoylanilide hydroxamic acid (SAHA; targets classes I and II HDACs). Osteoclast differentiation was assessed by expression of tartrate resistant acid phosphatase and resorption of dentine. Expression of mRNA encoding for osteoclast genes including RANK, calcitonin receptor (CTR), c‐Fos, tumur necrosis factor (TNF) receptor associated factor (TRAF)6, nuclear factor of activated T cells (NFATc1), interferon‐β, TNF‐like weak inducer of apoptosis (TWEAK), and osteoclast‐associated receptor (OSCAR) were assessed. Expression of HDACs 1–10 during osteoclast development was also assessed. 1179.4b significantly reduced osteoclast activity (IC50 < 0.16 nM). MS‐275 (IC50 54.4 nM) and 2664.12 (IC50 > 100 nM) were markedly less effective. A combination of MS‐275 and 2664.12 inhibited osteoclast activity similar to 1179.4b (IC50 0.35 nM). SAHA was shown to suppress osteoclast activity (IC50 12 nM). 1179.4b significantly (P < 0.05) reduced NFATc1, CTR, and OSCAR expression during the later stages of osteoclast development. Class I HDAC 8 and Class II HDAC5 were both elevated (P < 0.05) during osteoclast development. Results suggest that inhibition of both classes I and II HDACs may be required to suppress human osteoclastic bone resorption in vitro. J. Cell. Physiol. 226: 3233–3241, 2011.

TWEAK and Fn14 expression in the pathogenesis of joint inflammation and bone erosion in rheumatoid arthritis

IntroductionTNF-like weak inducer of apoptosis (TWEAK) has been proposed as a mediator of inflammation and bone erosion in rheumatoid arthritis (RA). This study aimed to investigate TWEAK and TWEAK receptor (Fn14) expression in synovial tissue from patients with active and inactive rheumatoid arthritis (RA), osteoarthritis (OA) and normal controls and assess soluble (s)TWEAK levels in the synovial fluids from patients with active RA and OA. Effects of sTWEAK on osteoclasts and osteoblasts were investigated in vitro.MethodsTWEAK and Fn14 expression were detected in synovial tissues by immunohistochemistry (IHC). Selected tissues were dual labelled with antibodies specific for TWEAK and lineage-selective cell surface markers CD68, Tryptase G, CD22 and CD38. TWEAK mRNA expression was examined in human peripheral blood mononuclear cells (PBMC) sorted on the basis of their expression of CD22. sTWEAK was detected in synovial fluid from OA and RA patients by ELISA. The effect of sTWEAK on PBMC and RAW 264.7 osteoclastogenesis was examined. The effect of sTWEAK on cell surface receptor activator of NF Kappa B Ligand (RANKL) expression by human osteoblasts was determined by flow cytometry.ResultsTWEAK and Fn14 expression were significantly higher in synovial tissue from all patient groups compared to the synovial tissue from control subjects (P < 0.05). TWEAK was significantly higher in active compared with inactive RA tissues (P < 0.05). TWEAK expression co-localised with a subset of CD38+ plasma cells and with CD22+ B-lymphocytes in RA tissues. Abundant TWEAK mRNA expression was detected in normal human CD22+ B cells. Higher levels of sTWEAK were observed in synovial fluids isolated from active RA compared with OA patients. sTWEAK did not stimulate osteoclast formation directly from PBMC, however, sTWEAK induced the surface expression of RANKL by human immature, STRO-1+ osteoblasts.ConclusionsThe expression of TWEAK by CD22+ B cells and CD38+ plasma cells in RA synovium represents a novel potential pathogenic pathway. High levels of sTWEAK in active RA synovial fluid and of TWEAK and Fn14 in active RA tissue, together with the effect of TWEAK to induce osteoblastic RANKL expression, is consistent with TWEAK/Fn14 signalling being important in the pathogenesis of inflammation and bone erosion in RA.

Inhibition of Apoptosis in Periodontitis

This study investigated whether the prolonged survival of inflammatory cells in periodontal disease could be due to the inhibition of apoptosis by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) decoy receptors and inhibition of the terminal stages of apoptosis signaling by inhibitor of apoptosis (IAP) family members. Gingival tissue samples were taken from healthy individuals and those with chronic periodontitis. The expression of TRAIL, TRAIL receptors, TUNEL, cleaved caspase-3, xIAP, and survivin was determined immunohistologically and at the level of mRNA expression. Higher levels of TRAIL and the TRAIL decoy receptor, TRAIL R4, were expressed in the diseased periodontal tissues (p < 0.005). Statistically (p < 0.05) higher levels of cleaved caspase-3 and the cleaved caspase-3 inhibitors, xIAP and survivin, were seen. Similar changes were seen at the level of mRNA. The results indicate that apoptosis in periodontitis may be inhibited by elevated expression of TRAIL decoy receptors and cleaved caspase-3 inhibitors.

Expression of tumor necrosis factor-like weak inducer of apoptosis (TWEAK) and its receptor, fibroblast growth factor-inducible 14 protein (Fn14), in healthy tissues and in tissues affected by periodontitis

BACKGROUND AND OBJECTIVE Host-derived enzymes, cytokines and other proinflammatory mediators play an integral role in periodontal destruction. The levels of tumor necrosis factor-like weak inducer of apoptosis (TWEAK) and its receptor, fibroblast growth factor-inducible 14 protein (Fn14), are elevated in tissues from a number of chronic inflammatory diseases. The aim of the present study was to investigate the expression of TWEAK and Fn14 at the protein and mRNA levels in gingival biopsies from periodontitis patients and from clinically healthy patients. MATERIALS AND METHODS Gingival biopsies were obtained from healthy sites (n = 7) and from sites affected by periodontitis (n = 27). The expression of TWEAK and Fn14 was investigated by immunohistochemistry in formalin-fixed, paraffin-embedded tissues. The levels of mRNA for TWEAK and Fn14 were also investigated by RT-PCR. RESULTS The expression of TWEAK and Fn14 proteins was significantly higher in periodontitis tissue than in healthy tissue. In periodontitis tissues, TWEAK and Fn14 proteins were mainly expressed by mononuclear leukocytes (morphologically resembling lymphocytes and plasma cells), by cells lining blood vessels, by spindle-shaped cells resembling fibroblasts and by multinucleated cells. The Fn14 mRNA level in periodontitis tissue was significantly higher than that in healthy tissue. A moderate correlation between TWEAK/Fn14 expression and inflammation and bone loss, but not pocket depth, was noted. CONCLUSION This study demonstrates higher expression of TWEAK protein and of Fn14 mRNA and protein in periodontitis tissues than in clinically healthy controls. Our data support the concept that TWEAK/Fn14 signaling is an additional player in the pathogenesis of periodontitis and adds to the increasing number of cytokine networks involved in periodontal inflammation.

Does Human Relaxin-2 Affect Peripheral Blood Mononuclear Cells to Increase Inflammatory Mediators in Pathologic Bone Loss?

Abstract: This study was designed to test the hypothesis that relaxin stimulates bone resorption by regulating the production of several mediators that stimulate osteoclast formation. The levels of mediators were measured in response to differing relaxin concentrations in supernatants from peripheral blood mononuclear cells (PBMCs), MCF‐7 breast cancer cells, and normal human osteoblasts. Although all cell types expressed mRNA for the relaxin receptor (LGR7), only PBMCs responded to relaxin at physiologic levels by increasing tumor necrosis factor‐α and interleukin‐1β secretion. The findings indicate that PBMCs should be studied in relation to the effect of relaxin on inflammation and bone destruction caused by osteoclasts.