Bone Abstracts (2016) 5 OC6.3 | DOI: 10.1530/boneabs.5.OC6.3

The critical biomechanical role of Lipocalin 2 in the crosstalk between endothelium and osteoblasts in unloading conditions.

Vimal Veeriah1, Mattia Capulli1, Angelo Zanniti1, Suvro Chatterjee2, Nadia Rucci1 & Anna Teti1

1University of L’Aquila, L’Aquila, Italy; 2Anna University, Chennai, India.

Angiogenesis and osteogenesis are tightly linked and dependent on each other. Lipocalin 2 (LCN2) is a mechanoresponding adipokine, strongly upregulated in osteogenic cells subjected to microgravity (0.08–0.008 g), in which it impairs osteogenesis and upregulates the osteoclastogenic cytokine, RANKL. We investigated the role of LCN2 in the crosstalk between angiogenesis and osteogenesis in simulated microgravity conditions as a model of mechanical unloading. Mouse and human endothelial cells (EC), subjected to microgravity in the NASA-developed RWV bioreactor, did not express LCN2 but secreted its upstream regulator, IL1β, in their conditioned medium, with a mechanism dependent on microgravity intensity (0.08 g 12 pg/ml; 0.008 g 36 pg/ml, P<0.001). Secreted IL1β induced LCN2 expression in mouse and human osteogenic cells (12.7-fold, P<0.001) by activation and nuclear translocation of the transcription factor NF-κB. LCN2 impaired osteoblast differentiation (−52% ALP P=0.024, −66% mineralization, P=0.010), along with the EC IL1β-induced NOS2/NO/COX2 pathway that increased osteogenic cell proliferation (1.7-fold, P=0.003) and cyclin d1 expression (5.3-fold, P=0.002). Depletion of IL1β from microgravity EC conditioned medium and deletion of LCN2 in osteogenic cells blocked the effect of microgravity on EC-to-osteogenic cell crosstalk. LCN2 was also increased in the conditioned medium of osteogenic cells directly subjected to microgravity (sixfold, P<0.001), and stimulated EC migration (twofold, P=0.027), tube formation (2.3-fold, P<0.008) and sprouting from mouse aortic rings (fourfold, P<0.001). These effects were mediated by LCN2-induced VEGF and Hif1α in EC, and were reduced by LCN2 deletion in osteogenic cells and by incubation with the VEGF receptor antagonist, avastin. Induction of EC-osteogenic cell crosstalk was mediated by these pathways also ex-vivo, in calvarias cultured in the presence of microgravity EC conditioned medium, and in vivo, in calvarias and tibias injected with microgravity EC conditioned medium, and in tibias from mice subjected to hindlimb unloading by tail suspension or treatment with botulin toxin, suggesting a pathogenic relevance of LCN2-mediated EC/osteogenic cell pathways in conditions inducing disuse osteoporosis.

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