Faculty Bibliography

Ecto-5'-nucleotidase (CD73) generates adenosine, an osteoblast activator and key regulator of skeletal growth. It is unknown, however, if CD73 regulates osteogenic differentiation during fracture healing in adulthood, and in particular how CD73 activity regulates intramembranous bone repair in the elderly. Monocortical tibial defects were created in 46-52-week-old wild type (WT) and CD73 knock-out mice (CD73-/-) mice. Injury repair was analyzed at post-operative days 5, 7, 14 and 21 by micro-computed tomography (micro-CT), histomorphometry, proliferating cell nuclear antigen (PCNA) immunostaining, alkaline phosphatase (ALP) and tartrate-resistant acid phosphatase (TRAP) histochemistry. Middle-aged CD73 knock-out mice exhibited delayed bone regeneration and significantly reduced bone matrix deposition detected by histomorphometry and micro-CT. Cell proliferation, ALP activity and osteoclast number were reduced in the CD73-/- mice, suggesting a combined defect in bone formation and resorption due the absence of CD73 activity in this model of intramembranous bone repair. Results from this study demonstrate that osteoblast activation through CD73 activity is essential during bone repair in aging mice, and it may present a drugable target for future biomimetic therapeutic approaches that aim at enhancing bone formation in the elderly patients.

Background/Purpose: Adenosine, generated from the catabolism of adenine nucleotides, modulates cell function by interacting with specific cell-surface receptors (A1R, A2AR, A2BR, A3R). Inhibition of osteoclast formation via adenosine A2A receptor stimulation or increasing local adenosine concentration stimulates new bone formation as well as rhBMP-2. Bone alterations have been observed in HIV (Human Immunodeficiency Virus) disease for nearly two decades, in particular a higher risk of low bone mineral density (BMD) and fragility fractures. Treatment with Tenofovir leads to changes in bone catabolism markers and significant reductions in BMD in children and young adults. Tenofovir is taken up by cells and phosphorylated and 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. Here we determined if Tenofovir directly affects bone and if Dipyridamole, may be a useful treatment to counteract the effects. Methods: M-CSF/RANKL-induced osteoclast (OC) were studied in primary murine bone marrow culture as the number of TRAPpositive cells after challenge with Tenofovir (1nM-100muM) alone or in combination with Dipyridamole (1nM-100muM). OC differentiation markers were study by RT-PCR. Male C57Bl/6 mice were divided into four groups: saline 0.9% (control), Tenofovir 75mg/Kg/day, Dipyridamole 25mg/Kg/day, combination Tenofovir/Dipyridamole for 4 weeks (n=10 each). Double labelling of bone with calcein /Alizarin Red was performed to analized bone formation 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=0.3muM). Tenofovir increases Cathepsin K and NFATc1 mRNA levels during OC differentiation, and the effect was reverted by Dipyridamole. In vivo, mice treated with Tenofovir lost nearly 10% of body weight (p<0.001) and DXA analysis revealed a decrease in % fat (p<0.05), effect reverted by Dipyridamole. Tenofovir reduced bone formation in vivo (19+/-2mum bone apposition vs 35+/-4mum in control, p<0.05) and this effect was reverted by Dipyridamole (30+/-3mum, p<0.05 vs Tenofovir alone). microCT analysis 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 (88+/-31 vs 27+/-1 OC/hpf in control, p<0.005) that was reverted inn the pressence of Dipyridamole (30+/-2OC/hpf, p<0.05 vs Tenofovir alone). Similar results were obtained for Cathepsin K and CD68. RANKL positive cells were increased in the presence of Tenofovir meanwhile OPG positive cells decreases, and both effets were reverted in the presence of Dipyridamole. Conclusion: These results indicate that Tenofovir enhances osteoclast differentiation and inhibits osteoblast differentiation by an adenosine-ATP-dependent mechanism and suggests that treatment with agents that increase local adenosine concentrations, like Dipyridamole, might prevent bone loss following Tenofovir treatment

Adenosine A2a receptor (A2aR) stimulation promotes the synthesis of collagens I and III, and we have recently demonstrated that there is crosstalk between the A2aR and WNT/beta-catenin signaling pathway. In in vitro studies, A2aR signaling for collagen III expression was mediated by WNT/beta-catenin signaling in human dermal fibroblasts; we further verified whether the crosstalk between A2aR and Wnt/beta-catenin signaling was involved in diffuse dermal fibrosis in vivo. Wnt-signaling reporter mice (Tcf/Lef:H2B-GFP) were challenged with bleomycin and treated with the selective A2aR antagonist istradefylline (KW6002) or vehicle. Dermal fibrosis was quantitated and nuclear translocation of beta-catenin in fibroblasts was assessed by double-staining for Green fluorescent protein or dephosphorylated beta-catenin or beta-catenin phosphorylated at Ser552, and vimentin. KW6002 significantly reduced skin thickness, skinfold thickness, breaking tension, dermal hydroxyproline content, myofibroblast accumulation, and collagen alignment in bleomycin-induced dermal fibrosis. Also, there was increased expression of Tcf/Lef:H2B-GFP reporter in bleomycin-induced dermal fibrosis, an effect that was diminished by treatment with KW6002. Moreover, KW6002 significantly inhibited nuclear translocation of Tcf/Lef:H2B-GFP reporter, as well as dephosphorylated beta-catenin and beta-catenin phosphorylated at Ser552. Our work supports the hypothesis that pharmacologic blockade of A2aR inhibits the WNT/beta-catenin signaling pathway, contributing to its capacity to inhibit bleomycin-induced dermal fibrosis.

Osteoarthritis (OA) is characterized by cartilage destruction and chondrocytes have a central role in this process. With age and inflammation chondrocytes have reduced capacity to synthesize and maintain ATP, a molecule important for cartilage homeostasis. Here we show that concentrations of ATP and adenosine, its metabolite, fall after treatment of mouse chondrocytes and rat tibia explants with IL-1beta, an inflammatory mediator thought to participate in OA pathogenesis. Mice lacking A2A adenosine receptor (A2AR) or ecto-5'nucleotidase (an enzyme that converts extracellular AMP to adenosine) develop spontaneous OA and chondrocytes lacking A2AR develop an 'OA phenotype' with increased expression of Mmp13 and Col10a1. Adenosine replacement by intra-articular injection of liposomal suspensions containing adenosine prevents development of OA in rats. These results support the hypothesis that maintaining extracellular adenosine levels is an important homeostatic mechanism, loss of which contributes to the development of OA; targeting adenosine A2A receptors might treat or prevent OA.

Radiation therapy (RT) induces release of ATP in the tumor micro environment (TME) triggering recruitment and activation of dendritic cells (DCs), including CD103 DCs, identi+ed as the key DC subsetresponsible for cross-presentation of tumor antigens to CD8 T cells. However, CD39 and CD73-expressing cells in the TME hydrolyze ATP into adenosine that potently inhibits DCs and CD8 T cells. Here, we tested the hypothesis that adenosine generation limits the ability of RT totrigger anti-tumor immunity. Wild type (WT) or BATF3 mice (CD103 DC-de+cient) were inoculated s.c. with TSA tumor cells (day 0) and assigned to treatment with: (1) control Ab; (2) anti-CD73 Ab (100 mug) (3) RT (20 Gy); (4) RT + anti-CD73 Ab. Antibodies were administered i.p. on day 11, 14, 17 and 20. RT was given locally as single 20 Gy dose on day 12. On day 18, tumors were analyzed by -ow cytometry for DC and T cell in+ltration. Mice were monitored for tumor progression. DCs generated in vitro by culture of bone marrow from WT mice with Flt3L (>90% CD103 ) were labeled with CFSE and intravenously injected in BATF3 recipient mice. Tumors were harvested after 48h and analyzed by -ow cytometry for in+ltration of CFSE+ DCs. In tumors of RT- but not sham-treated mice, anti-CD73 mAb increased in+ltration of CD103 DCs and enhanced CD8 T cell/Treg ratio. Importantly, CD73 blockade had no anti-tumor e

EMBASE:617353266

ISSN: 1550-6606

CID: 2645572