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)-alpha and IL-1beta, resulted in mobilization from the WPBs and subsequent secretion of OPG protein into the culture supernatant. Furthermore, TNF-alpha 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.

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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 (IC(50) < 0.16 nM). MS-275 (IC(50) 54.4 nM) and 2664.12 (IC(50) > 100 nM) were markedly less effective. A combination of MS-275 and 2664.12 inhibited osteoclast activity similar to 1179.4b (IC(50) 0.35 nM). SAHA was shown to suppress osteoclast activity (IC(50) 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.

OPG expression on macrophage type synovial lining cells as well as endothelial cells is deficient in RA patients with active synovitis, in contrast to that seen in spondyloarthropathy patients with active synovitis. This deficiency in OPG expression in the inflamed joint of RA patients may be important in the development of radiologically defined joint erosions.

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The transforming growth factor betas are of major importance in the wound repair process; however, no studies to date have investigated the role of the transforming growth factor beta receptors in chronic venous leg ulcers or what effect healing has on these proteins. To determine whether the transforming growth factor beta peptides and their receptors are expressed in chronic venous wounds, we used immunofluorescent analysis and quantitative competitive reverse transcription polymerase chain reaction to identify the protein and mRNA expression, respectively. Biopsy samples from wounds and normal skin were collected from 12 patients with chronic venous leg ulcers and three patients undergoing reconstructive surgery, respectively. Additionally four of the chronic venous leg ulcer patients were re-biopsied between 2 and 8 wk after the first biopsy when the wounds had entered the healing phase. The tissue excised from the ulcers included the surrounding intact skin, the ulcer edge, and the ulcer base. Immunofluorescent staining for transforming growth factors beta1, beta2, and beta3 was observed within the epidermis of the skin surrounding the chronic venous ulcers and in fibroblasts and inflammatory cells of the dermis, although this staining was not as strong as that seen in normal unwounded skin. Very little staining could be seen within the ulcers for any of the ligands, however. In contrast the transforming growth factor beta type I receptor was observed throughout the ulcers and the normal unwounded skin biopsies, particularly in the basal epidermal cells. No immunofluorescence for the type II transforming growth factor beta receptor was observed in any of the ulcer biopsies investigated, although it was observed throughout the epidermis and in fibroblasts and inflammatory cells in the surrounding skin. Quantitative, competitive reverse transcription polymerase chain reaction was used to analyze mRNA expression for transforming growth factor beta1 and the type II receptor in the nonhealing ulcers and normal unwounded skin biopsies. These studies revealed that transforming growth factor beta1 and transforming growth factor beta receptor II mRNA was expressed in all the chronic nonhealing ulcers albeit at very low levels for the type II receptor. In marked contrast to the staining observed in nonhealing chronic ulcers, positive immunostaining was observed for the transforming growth factor betas and both the type I and type II receptors in healing ulcers. These results suggest that the absence of a viable receptor complex for the transforming growth factor betas in nonhealing chronic venous ulcers may contribute to wound chronicity.

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.