Abstract 46

Osteoprotegerin (OPG) is localised to the Weibel-Palade Bodies of Human Vascular Endothelial Cells and is reduced at sites if inflammatory bone loss.

Zannettino ACW¹, Holding CA², Diamond P¹, Atkins GJ³, Findlay DM³, Kostakis P¹, Farrugia A¹, Gamble J⁴ and Haynes DR²

¹Myeloma and Mesenchymal Research Laboratory, Division of Haematology Hanson Institute, Institute of Medical and Veterinary Science, Frome Road, Adelaide, Australia.
²Department of Pathology, Adelaide University, Adelaide, South Australia, 5005, Australia.
³Department of Orthopaedics and Trauma, Adelaide University, North Terrace, Adelaide, South Australia, 5000, Australia.
⁴Vascular Biology Laboratory, Division of Human Immunology, Hanson Institute, Institute of Medical and Veterinary Science, Frome Road, Adelaide, Australia.

Recent studies demonstrate roles for osteoprotegerin (OPG) in both skeletal and extra-skeletal tissues. Although its role in preventing osteoclast formation and activity is well documented, emerging evidence suggests a role of OPG in endothelial cell survival, and the prevention of arterial calcification. This study shows that vascular endothelial cells in situ, and human umbilical vein endothelial cells (HUVEC) in vitro, express abundant OPG. OPG expression by endothelial cells is markedly reduced in tissue near sites of bone loss in rheumatoid arthritis and periodontal disease. In HUVEC in vitro, OPG co-localises with P-selectin, within the Weibel-Palade bodies, where it is detectable using an antibody that recognises the dimeric form of human OPG. Treatment of HUVEC with the pro-inflammatory cytokines, TNF-_ and IL-1_ resulted in mobilization and secretion of OPG protein into the culture supernatant. Furthermore, using competitive RT-PCR and OPG-specific ELISA, TNF-_ treatment of HUVEC resulted in a sustained increase in OPG mRNA and protein over the 24 hour treatment period. Importantly, reciprocal immunoprecipitation experiments revealed that OPG is physically associated with vWF both within the WBP 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 reducatse, thrombospondin (tsp-1), raising the intriguing possibility that OPG may provide an important "link" between tsp-1 and vWF in regulating vWF polymer size. In summary, the intracellular localization of OPG in association with vWF, in HUVEC, together with its rapid and sustained secretory response to inflammatory stimuli, strongly support a modulatory role in vascular injury and inflammation and haemostasis.

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