Faculty Bibliography

Short biologic half-lives limit the therapeutic utility of many small molecules. One approach to extending the half-life of pharmacologically active small molecules is conjugation to less degradable nanoparticles; here we report the synthesis and activity of six targeted polymeric (PEG-b-PLA) nanoparticles for use as adenosine receptor agonists. Using click chemistry, PLA-b-PEG400-N₃ and PLA-b-PEG2000 block copolymers were bound to adenosine at the 3',4'-OH, 5'-OH, and 6-NH₂ positions with an acetylene group. Activity of the conjugates as adenosine receptor ligands was tested by their capacity to stimulate cAMP increases in RAW264.7 murine macrophage cells. Only adenosine-conjugated nanoparticles (A-3',4'-OH-TPN2), in which PEG2000 was bound to adenosine on the 3',4' hydroxyl groups, stimulated cAMP increases and these increases were blocked by selective antagonists of both adenosine A2A and A2B receptors, consistent with ligation of these receptors. Adenosine nanoparticles were tested in vivo in a rat model of post-traumatic osteoarthritis; intra-articular injection of adenosine nanoparticles prevented the development of osteoarthritis in this model. These studies suggest that attachment of adenosine to biodegradable nanoparticles provides a novel approach to achieving prolonged therapeutic effects.

BACKGROUND:Autologous bone grafts remain a standard of care for the reconstruction of large bony defects, but limitations persist. The authors explored the bone regenerative capacity of customized, three-dimensionally printed bioactive ceramic scaffolds with dipyridamole, an adenosine A2A receptor indirect agonist known to enhance bone formation. METHODS:Critical-size bony defects (10-mm height, 10-mm length, full-thickness) were created at the mandibular rami of rabbits (n = 15). Defects were replaced by a custom-to-defect, three-dimensionally printed bioactive ceramic scaffold composed of β-tricalcium phosphate. Scaffolds were uncoated (control), collagen-coated, or immersed in 100 μM dipyridamole. At 8 weeks, animals were euthanized and the rami retrieved. Bone growth was assessed exclusively within scaffold pores, and evaluated by micro-computed tomography/advanced reconstruction software. Micro-computed tomographic quantification was calculated. Nondecalcified histology was performed. A general linear mixed model was performed to compare group means and 95 percent confidence intervals. RESULTS:Qualitative analysis did not show an inflammatory response. The control and collagen groups (12.3 ± 8.3 percent and 6.9 ± 8.3 percent bone occupancy of free space, respectively) had less bone growth, whereas the most bone growth was in the dipyridamole group (26.9 ± 10.7 percent); the difference was statistically significant (dipyridamole versus control, p < 0.03; dipyridamole versus collagen, p < 0.01 ). There was significantly more residual scaffold material for the collagen group relative to the dipyridamole group (p < 0.015), whereas the control group presented intermediate values (nonsignificant relative to both collagen and dipyridamole). Highly cellular and vascularized intramembranous-like bone healing was observed in all groups. CONCLUSION:Dipyridamole significantly increased the three-dimensionally printed bioactive ceramic scaffold's ability to regenerate bone in a thin bone defect environment.

Background: The most common cause of total joint replace-ment revision procedures is loosening of the implant due to bone erosion around the prosthesis. In most patients, wear particle induced osteolysis is responsible for prosthesis failure. We have previously identified CGS21680 (CGS), an A2A receptor agonist, as a possible treatment. Thus, we generated and tested a novel alendronate-CGS conjugate (MRS7216) that localizes to bone. We hypothesized that MRS7216 administration could prevent inflammatory osteolysis and bone erosion by enhancing A2A signaling, while preventing CGS associated side effects.
Method(s): Osteolysis was induced in 6-8 week-old C57BL/6J mice (5 mice per treatment group). Mice received a single dose or weekly 10 mg/kg intraperitoneal dose of MRS7216 conjugate, beginning at the time of surgery. Other treatment groups were given equivalent weekly doses of either alendronate-PEG6 (AlenP) or saline. After 2 weeks, mice were sacrificed. MicroCT and immunohistochemistry analyses were performed. The studies were approved by the NYU Institutional Animal Care and Use Committee.
Result(s): Studies revealed that mice treated with a weekly dose of MRS7216 had significant (40%) reduction in bone damage and (81%) reduction in osteoclasts compared to saline treated mice. AlenP did not prevent bone erosion (p<0.05) and had significant (55%) decrease in osteoclasts compared to saline group. Staining in MRS7216-treated group, showed an increase in osteoblasts compared to saline and AlenP (p<0.06).
Conclusion(s): MRS7216 conjugate suppresses wear particle-induced osteolysis, inhibits osteoclastogenesis and increases osteoblast number. MRS7216 could be a novel treatment in prevention of osteolysis and potentially promote bone regeneration. Supported by the Medical Student Training in Aging Research Program, National Institutes of Health, National Institute of Aging, Bethesda, Maryland to New York University School of Medicine

Purpose: In a previous study we demonstrated that adenosine plays an important role in maintaining homeostasis in the joint. Adenosine has a short half-life (1-4 seconds in whole blood) limiting its therapeutic potential. Previously we showed that intrarticular injection of adenosine in a liposomal formulation prevents and reverses osteoarthritis (OA) progression in a post-traumatic OA (PTOA) animal model. Here we adopt a different strategy in prolonging the therapeutic half-life of adenosine by conjugating adenosine to the biodegradable carrier poly(lactic acid)-poly(ethylene glycol) (PLA-PEG) nanoparticles. Method(s): Six targeted polymeric (PEG-b-PLA) nanoparticles were synthetized by binding PLA-b-PEG400-N3 and PLA-b-PEG2000 block copolymers to adenosine at the 3',4'-OH, 5'-OH, and 6-NH2 positions with an acetylene group. The efficacy of the new molecules to stimulate cAMP production was tested in RAW264.7 murine macrophage cells. PTOA was induced in Sprague Dawley rats (n=3 for each group) following non-surgical rupture of anterior cruciate ligament (ACL). Rats were treated with intra-articular injections of 100 ul of a suspension containing PLA-PEG2000-3,4-OH-adenosine (Nano-Ade; 1 mg/kg), or PLA-PEG2000 nanoparticles (Nano) or with saline for 6 weeks. The animals received the first injection 7 days after ACL rupture and injections were performed every 10 days. Knee swelling in the rats was measured before every injection. At the end of the experiment rats were sacrificed and both legs were collected for immunohistochemistry and microcomputed tomography (muCT) analysis. The capacity of the various nanoparticle preparations to bind to and activate adenosine receptors was studied as the capacity to stimulate cAMP accumulation and inhibit IL-1-stimulated IL-6, MMP13, col10a1 and NFkB translocation to the nucleus in primary murine chondrocytes. Result(s): Only adenosine-conjugated nanoparticles in which PEG2000 was bound to adenosine on the 3',4' hydroxyl groups stimulated cAMP increase (163% vs control; p<0.05) and this increase were blocked by selective antagonists of both adenosine A2A and A2B receptors. Intra-articular injection of the Nano-Ade in PTOA rats diminished swelling in affected knees (p<0.001 vs Nano). We observed that Nano-Ade diminished the IL-1beta-stimulated expression of IL-6 mRNA expression (from 214+/-107 to 79+/-89 vs Control group), an effect that was partially reversed by A2AR (ZM241385 and SCH58261, 1muM each) and A2BR (PSB1115) antagonists. In contrast, the Nano-Ade diminished IL-1beta-stimulated MMP13 and Collagen-10 mRNA and protein expression via stimulation of A2BR since the effect was reversed by the selective A2B antagonist but not by the A2AR-selective antagonist. Nano-Ade also diminished IL-1beta stimulated activation of NF-kB, a central intracellular signal for inflammation (67% decrease vs IL-1beta treated cells; p<0.001). In rats treated with Nano-Ade there was markedly reduced fibrillation of the cartilage surface (H&E staining) and less proteoglycan loss (Safranin-O staining) resulting in a significantly decreased OARSI score (Nano=2.99+/-1.20, Nano-Ade=0.88+/-0.69; p<0.03 vs Nano). Analysis of muCT scanned images showed an increase in cartilage volume in Nano-Ade treated rats (143% vs Nano; p=0.023). We also found that Nano-Ade reduced RNA expression for MMP-13 (83% reduction compare to IL-1beta stimulated cells; p<0.001) and Collagen-10 (81% reduction compare to IL-1beta stimulated cells; p<0.01) in primary murine chondrocytes stimulated with Il-1. Conclusion(s): Taken together these results demonstrate that the adenosine-functionalized particles bind to and activate adenosine A2A and A2B receptors on murine cells. Activation of adenosine receptors by PLA-PEG2000-3,4-OH-adenosine prevents OA progression and reduce pro-inflammatory mediators in murine chondrocytes.

Purpose: Many studies have been shown that obesity along with joint injury is one of the most common risk factor in the development of osteoarthritis (OA). In a previous study we described that intra-articular injections of liposomal preparations of adenosine completely prevent progression and reverse cartilage loss in post-traumatic OA. TGF-beta signaling plays dual and opposing roles in cartilage health and chondrocyte life depending on the signals activated downstream. Activation of downstream signaling pathways for TGF-beta leading to localization of phospho-SMAD2/3 associated with maintenance of cartilage. In contrast nuclear localization of phospho-SMAD1/5/8 results in chondrocyte hypertrophy. Here we report that intraarticular injections of liposomal adenosine and A2AR agonist reverses OA in a post-traumatic OA model in rat and in an obesity related mice model. We moreover explore the role of TGF-beta signaling in this phenomenon. Method(s): Obesity-induced OA model: C57Bl6 mice (5-6 for each group, 12 weeks old) were fed a 60% fat diet (HFF mice) for 3months, after which received intraarticular injections (10 mul) of empty liposomes (Lipo) or liposomes containing the A2AR agonist CGS21680 (Lipo-CGS) or Adenosine (Lipo-Ado) into the knee every 10 days for 4 injections. Post-traumatic OA (PTOA) was induced in Sprague Dawley rats following non-surgical rupture of anterior cruciate ligament (ACL). Four weeks later rats were injected in the knee with 100ul of saline, Lipo or Lipo-CGS every 10 days (6 injections). RNA was isolated from chondrocytes in knee cartilage of rats treated as described above (3 from each group X 3 replicates) and subjected to RNAseq analysis. TC28a2 human chondrocyte cell line was used for in vitro experiments. Result(s): Lipo-CGS and Lipo-Ado reversed OA in the obesity and post traumatic OA model. Mouse knees had an OARSI score of 5.17+/-1.84 before treatment. Treatment with LIPO-Ado and lipo-CGS decreased OA severity (OARSI score 1.33+/-0.81 and 1.83+/-0.98, respectively, p<0.001 vs pre-treatment; figure 1). In the PTOA model the OARSI score significantly decreases after Lipo-CGS and Lipo-Ado treatment compare to the saline group (OARSI: 1.28+/-0.39; 1.42+/-0.69; 2.97+/-0.0.75 respectively; p<0.05). Analysis of the transcriptome suggests that the treatment of Lipo-CGS promotes the up-regulation of genes involved in proliferation process and downregulations of genes responsible of apoptosis, cartilage catabolism and chondrocyte hypertrophy (Figure 2). TGF-beta expression was increased in deep layers of cartilage in the Lipo-CGS-treated rats and there was notable nuclear localization of phospho-SMAD2/3 in these chondrocytes. In contrast, phospho-SMAD1/5/8 was expressed in the nuclei of chondrocytes in the saline and LIPO-treated rats but not in the LIPO-CGS treated rats. Identical changes were observed in the knees of obese mice. To determine whether the effect of A2AR stimulation on TGF-beta signaling was direct or indirect we studied the effect of CGS21680 on nuclear phospho-SMAD expression in TC28a2 cells and found that CGS21680 increased nuclear phospho-SMAD2/3 and reduced nuclear phospho-SMAD1/5/8, as detected by immunofluorescence. Conclusion(s): Administration of an A2AR agonist to established OA knees reverses OA in rats and mice and shifts TGF-beta signaling from ALK1/SMAD1/5/8 to ALK5/SMAD2/3 in OA chondrocytes after activation of A2AR in 2 OA animal models.

Osteoblast differentiation and proliferation are regulated by several modulators, among which are adenosine A_2A receptors (A2ARs) and Wingless/Integrated-β-catenin pathways. Cytosolic β-catenin stabilization promotes its nuclear translocation and transcriptional activity. In the present study, we seek to determine whether there is a connection between A2AR stimulation and cellular β-catenin levels in osteoblasts. Osteoblast precursor cell line (MC3T3-E1) and primary murine osteoblasts were treated with CGS21680, a highly selective A2AR agonist. We analyzed cellular content and nuclear translocation of phosphorylated (p)-serine 552 (S552) β-catenin in response to A2AR stimulation in MC3T3-E1 cells, in both wild-type and A2AR knockout (A2AKO) mice. Moreover, we measured cellular β-catenin levels in MC3T3-E1 cells transfected with scrambled or protein kinase B (Akt) small interfering RNA following A2AR activation. CGS21680 (1 μM) stimulated an increase in both the cellular content and nuclear translocation of p-S552 β-catenin after 15 min of incubation. A2AR activation had no tangible effect on the cellular β-catenin level either in A2AKO mice or in osteoblasts with diminished Akt content. Our findings demonstrate an interaction between A2AR, β-catenin, and Akt signaling in osteoblasts. The existence of such a crosstalk has significant repercussions in the development of novel therapeutic approaches targeting medical conditions associated with reduced bone density.-Borhani, S., Corciulo, C., Larranaga-Vera, A., Cronstein, B. N. Adenosine A_2A receptor (A2AR) activation triggers Akt signaling and enhances nuclear localization of β-catenin in osteoblasts.

To develop a comprehensive listing of the greatest unmet scientific and clinical needs in rheumatology. The 20th annual international Targeted Therapies meeting brought more than 100 leading basic scientists and clinical researchers in rheumatology, immunology, epidemiology, molecular biology and other specialties. During the meeting, breakout sessions were convened, consisting of five disease-specific groups with 20-30 experts assigned to each group based on expertise. Specific groups included rheumatoid arthritis, psoriatic arthritis, axial spondyloarthritis, systemic lupus erythematosus, connective tissue diseases and a basic science immunology group spanning all of these clinical domains. In each group, experts were asked to consider recent accomplishments within their clinical domain in the last year and update the unmet needs in three categorical areas: basic/translational science, clinical science and therapeutic development, and clinical care. While progress was noted among some of previously identified needs, both new needs were identified and themes from prior meetings were re-iterated: the need for better understanding the heterogeneity within each disease, and for identifying preclinical states of disease allowing treatment and prevention of disease in those at risk, and the elusive ability to cure disease. Within the clinical care realm, improved comorbidity management and patient-centred care continue to be unmet needs, and the need for new and affordable therapeutics was highlighted. Unmet needs for new and accessible targeted therapies, disease prevention and ultimately cure remain a priority in rheumatology.

Osteopenia and fragility fractures have been associated with HIV infection. Tenofovir, a common antiviral in HIV treatment, also leads to increases in bone catabolism markers and decreased bone mineral density (BMD) in children and young adults. In murine models and human cell lines, tenofovir inhibits ATP release and decreases extracellular adenosine levels. Adenosine, and adenosine A2A receptor, inhibits osteoclast formation, and increasing local adenosine concentration with dipyridamole, an agent that blocks adenosine cellular uptaken, stimulates new bone formation as well as rhBMP-2. We hypothesized that tenofovir regulates bone resorption by diminishing endogenous adenosine levels and determined whether dipyridamole may be a useful treatment to counteract the deleterous bone effects of tenofovir. M-CSF/RANKL-induced-primary murine osteoclast was studied as the number of TRAP-positive-cells after challenge with tenofovir alone or in combination with dipyridamole. Differentiation markers were study by RT-PCR, and MAPK/NFkB expression by WesternBlot. Male C57Bl/6 mice were treated as follow: saline 0.9% (control), tenofovir 75mg/Kg/day, dipyridamole 25mg/Kg/day, combination tenofovir/dipyridamole (n = 10, 4weeks). Calcein/AlizarinRed-labelling of newly formed bone was used, and long bones were prepared for microCT/histology. Tenofovir produced a dose-dependent increase in osteoclast differentiation (EC₅₀  = 44.5nM) that was reversed by dipyridamole (IC₅₀  = 0.3µM). Tenofovir increased Cathepsin K and NFATc1 mRNA levels and dipyridamole reversed the effect. Dipyridamole reversed the effect of tenofovir on pERK1/2, pp38 and NFkB nuclear translocation. Mice treated with tenofovir lost nearly 10% of body weight (p < 0.001). MicroCT revealed decrease BMD and altered trabecular bone in tenofovir-treated mice, reversed by dipyridamole. TRAP-staining showed increased osteoclasts in tenofovir-treated mice (p < 0.005) an effect reversed by dipyridamole. Similar results were obtained for Cathepsin K and CD68. RANKL-positive-cells were increased in tenofovir-treated mice whereas OPG-positive-cells decreased, and both effects were reversed by dipyridamole. These results suggest that treatment with agents that increase local adenosine concentrations, like dipyridamole, might prevent bone loss following tenofovir treatment.

Methotrexate has been used in treatment of rheumatoid arthritis (RA) since the 1980s and to this day is often the first line medication for RA treatment. In this review, we examine multiple hypotheses to explain the mechanism of methotrexate efficacy in RA. These include folate antagonism, adenosine signaling, generation of reactive oxygen species (ROS), decrease in adhesion molecules, alteration of cytokine profiles, and polyamine inhibition amongst some others. Currently, adenosine signaling is probably the most widely accepted explanation for the methotrexate mechanism in RA given that methotrexate increases adenosine levels and on engagement of adenosine with its extracellular receptors an intracellular cascade is activated promoting an overall anti-inflammatory state. In addition to these hypotheses, we examine the mechanism of methotrexate in RA from the perspective of its adverse effects and consider some of the newer genetic markers of methotrexate efficacy and toxicity in RA. Lastly, we briefly discuss the mechanism of additive methotrexate in the setting of TNF-α inhibitor treatment of RA. Ultimately, finding a clear explanation for the pathway and mechanism leading to methotrexate efficacy in RA, there may be a way to formulate more potent therapies with fewer side effects.

Adenosine receptor activation has been investigated as a potential therapeutic approach to heal bone. Bone has enhanced regenerative potential when influenced by either direct or indirect adenosine receptor agonism. As investigators continue to elucidate how adenosine influences bone cell homeostasis at the cellular and molecular levels, a small but growing body of literature has reported successful in vivo applications of adenosine delivery. This review summarizes the role adenosine receptor ligation plays in osteoblast and osteoclast biology and remodeling/regeneration. It also reports on all the modalities described in the literature at this point for delivery of adenosine through in vivo models for bone healing and regeneration.