Background HIV infection devastates the immune system but also affects other tissues and organs Bone alterations have been observed in HIV disease for nearly two decades, in particular a higher risk of low bone mineral density (BMD) and fragility fractures. Treatment with Tenofovir alone or as part of HAART, leads to changes in bone catabolism markers and significant reductions in BMD in children and young adults. Tenofovir is taken up by cells and phosphorylatedand inhibits HIV-reverse transcriptase by mimicking AMP. We have recently found that Tenofovir inhibits Pannexin-1/Connexin-43-mediated ATP release from cells and decreases extracellular adenosine levels and fibrosis in murine models. Inhibition of osteoclast formation via adenosine A2A receptor stimulation or increasing local adenosine concentration stimulates new bone formation as well as rhBMP-2.
Objectives As adenosine and ATP are key regulators of bone homeostasis, we determined whether Tenofovir directly affects bone by an adenosine- or ATP-dependent mechanism and if treatment with Dipyridamole, an agent that increases extracellular adenosine by blocking cellular adenosine uptake, may be a useful treatment to counteract Tenofovir effects.
Methods M-CSF/RANKL-induced osteoclast (OC) was studied in primary murine bone marrow culture as the number of TRAP-positive cells after challenge with Tenofovir (1nM-100μM) alone or in combination with Dipyridamole (1nM-100μM). OC markers were measured by RT-PCR. Pannexin-1 and Connexin-43 expression were permanently knocked down by lentiviral infection with appropriate shRNA or scrambled shRNA and these cells were induced to differentiate into OC by RANKL. Male C57Bl/6 mice received Tenofovir 75mg/Kg/day alone or in combination with Dipyridamole 1mg/Kg/day for 4 weeks. Double labelling of bone with calcein/Alizarin Red to analized bone formation was performed and long bones prepared for microCT and histology.
Results Tenofovir produced a dose-dependent increase in OC differentiation (EC50=44.5nM) that was reversed by Dipyridamole (IC50=0.3μM). When Pannexin-1 and Connexin-43 were absent, Tenofovir did not increase OC number. Tenofovir increases Cathepsin K and NFATc1 mRNA levels during OC differentiation, and the effect was reverted by Dipyridamole. Tenofovir reduced bone formation in vivo (19±2μm bone aposition vs 35±4μ m untreated p<0.05) and this effect was reverted in the presence of Dipyridamole (30±3μm, p<0.05 vs Tenofovir alone). microCT revealed decrease BMD and altered trabecular bone in Tenofovir-treated mice, been reverted in the presence of Dipyridamole. TRAP-staining showed increased OC in vivo in Tenofovir-treated mice (21±1 vs 16±1 OC/hpf in untreated, p<0.005) that was reverted with Dipyridamole. Similar results were obtained for Cathepsin K.
Conclusions These results indicate that Tenofovir enhances OC differentiation and inhibits osteoblast differentiation by an adenosine-dependent mechanism and suggests that treatment with agents that increase local adenosine concentrations, like Dipyridamole, might prevent bone loss following Tenofovir treatment.
Disclosure of Interest F. Conesa: None declared, P. Llamas: None declared, T. Wilder: None declared, P. Atencio: None declared, A. Cabello: None declared, M. Gόrgolas: None declared, B. Cronstein: None declared, R. Largo: None declared, G. Herrero-Beaumont: None declared, A. Mediero Grant/research support from: Instituto de Salud Carlos III, Fondos FEDER