Faculty Bibliography

Background: Patients with inflammatory arthritis and psoriasis are at greater risk for developing atherosclerotic plaques and associated cardiovascular diseases. Apremilast (APR), a PDE4 inhibitor used to treat psoriasis and psoriatic arthritis, was tested for effects in models of cholesterol efflux, foam cell formation, and atherosclerosis.
Method(s): A RAW264.7 murine macrophage cell line was infected with lentiviruses expressing short hairpin RNA (shRNA) to silence PKA, EPAC1, or EPAC2. The effect of APR on foam cell formation was tested following treatment with interferon-gamma (0.5 U/muL) for 24 hours and then treatment with an LDL (50 mug/mL) with/without APR (10 mumol/L) for another 48 hours. Cells were stained with Oil Red O and then cells containing lipid droplets were counted. Cholesterol efflux was measured after treatment with bodipy-cholesterol for 1 hour, equilibration buffer for 18 hours, and HDL (20 mug/mL) and ApoA1 (10 mug/mL with/without APR) for 4 hours.
Result(s): APR treatment reduced foam cell formation in RAW264.7 cells stably expressing EPAC1 and EPAC2 shRNA by 43 +/- 5% and 42 +/- 1%, respectively, while in cells expressing PKA shRNA, foam cell formation increased slightly (14 +/- 6%; P <.001, analysis of variance [ANOVA]). APR treatment enhanced HDL- and ApoA1-induced cholesterol efflux from RAW264.7 cells (2.35 +/- 0.03% vs. 1.73 +/- 0.08% [P <.001; n = 4] and 9.74 +/- 0.32% vs. 3.96 +/- 0.77% [P <.05; n = 3], respectively). APR treatment also enhanced HDL-induced efflux from RAW264.7 cells expressing EPAC1 and PKA but not EPAC2 shRNA (0.737 +/- 0.6% vs. 1.19 +/- 0.04% of control; P <.05, ANOVA); APR treatment enhanced ApoA1-induced cholesterol efflux from RAW264.7 cells stably expressing scrambled shRNA but not EPAC1, EPAC2, and PKA shRNA (2.62 +/- 0.15%, 1.55 +/- 0.22%, and 1.44 +/- 0.04%, respectively, vs. 0.95 +/- 0.15% of control; P <.01, P <.05, and P <.01, ANOVA). To determine the relevance of these findings to atherosclerosis, LDLR-/- mice were fed a Western diet +/- APR. A reduction in Oil Red O-stained lipid and CD68+ macrophages (P <.05, n = 3 or n = 4 per group) was seen in the plaques of animals treated with APR.
Conclusion(s): These results suggest that APR treatment: 1) promotes HDL-induced cholesterol efflux through an EPAC2-dependent mechanism, and ApoA1-induced cholesterol efflux through EPAC1-, EPAC2-, PKA-dependent mechanisms; 2) inhibits foam cell formation only through PKA-dependent mechanisms; and 3) reduces atherosclerotic plaque macrophages in a murine model of atherosclerosis.
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Background/Purpose: 3D-printed bioactive ceramic (3DPBC) scaffolds composed of beta-tricalcium phosphate (b-TCP) and coated in the osteogenic agent dipyridamole have been previously shown to heal critically sized calvarial defects in an adult animal model. This bone tissue engineering construct has yet to be applied in a pediatric craniofacial model and there has been evidence that other osteogenic agents such as BMP-2 can prematurely fuse growing sutures. The purpose of this study is to apply the described bone tissue engineering construct in a pediatric growing animal model and 1) quantify osteogenic potential in a growing calvarium; 2) maximize the scaffold design and dipyridamole (DIPY) concentration for the growing calvarium; and 3) characterize the effects of this bone tissue engineering construct on the growing suture. Methods/Description: Bilateral calvarial defects (10 mm) were created in 5-week-old New Zealand White rabbits (n = 14) 2 mm posterior and lateral to the coronal suture and sagittal sutures, respectively. 3DPBC scaffolds were constructed in quadrant form composed by varying pore dimensions (220, 330, and 500 mum). Each scaffold was collagen coated and soaked in varying concentrations of DIPY (100, 1000, and 10 000 muM). Controls comprised empty defects and collagen-coated scaffolds. Scaffolds were then placed into the calvarial defects to fill the bone space. Animals were euthanized 8 weeks postoperatively. Calvaria were analyzed using micro-computed tomography and 3D reconstruction.Mixed model analyses were conducted considering pore size and dosage effects on bone growth (a = 0.05).
Result(s): Scaffold group healing presented bone formation throughout the scaffold structure (defect marginal and central regions) while bone healing in empty sites was restricted to the defect margins, confirming its critical size dimension at 8 weeks in vivo. No significant difference in bone formation was detected when experimental groups were collapsed over pore size (P > .40). When pore size was collapsed over DIPY concentration, higher mean values were observed for the DIPYimmersed groups, and significance was shown between the 1000-muM and collagen groups (P < .05). Pore size and DIPY interaction was more pronounced for the 330-mum pore size where both the 100-and 1000-mum dosages presented significantly higher bone formation compared to collagen (P < .05). Across all concentrations of DIPY, including 10 000 mM (10 times greater than the experimental concentration, yielding the highest bone formation), sutures remained patent.
Conclusion(s):We present an effective bone tissue engineering scaffold design and dipyridamole concentration that significantly improve bone growth in a pediatric growing calvarial model and preserves cranial suture patency

Background/Purpose: The most common cause of total joint replacement revision surgeries is loosening of the implant due to loss of bone around the prosthesis.Wear particles shed from the prosthesis plays a critical role by increasing local inflammation and osteoclast number and activity, ultimately causing osteolysis.We have previously reported that an A2A adenosine receptor selective agonist (CGS21680, CGS) prevents osteolysis in wear particle-induced osteolysis model in mice. Frequent administration requirements and potential toxicity make it a less than optimal treatment for inflammatory osteolysis.We therefore generated and tested a novel alendronate-CGS conjugate (MRS7216) that specifically localizes to bone targeting the agonist to the site of tissue injury and thereby diminishing the frequency of administration and curtailing systemic side effects.
Method(s): The conjugate was synthesized from CGS by sequential activation of the carboxylic acid moiety and reacting with the appropriate amino acid under basic conditions. A PEG6 linker was incorporated to alendronic acid by direct coupling. Osteolysis in 6-8-week-old C57BL/6J mice was induced by surgical implantation of 3mg of ultrahigh-molecular-weightpolyethylene particles over the calvaria. Mice received a weekly 10mg/kg intraperitoneal dose of MRS7216 conjugate, starting at the time of surgery. Other groups of mice were treated with equivalent weekly doses of alendronate-PEG6 (AlenP) or saline respectively. An additional control group underwent sham surgery. After 2 weeks, animals were sacrificed and microCT and histology analyses were performed. The studies were approved by the Institutional Animal Care and Use Committee of NYU School of Medicine.
Result(s): Receptor binding studies demonstrate that the Ki for CGS, 7216 conjugates and the control AlenP molecules were 21.5 nM, 69.2 nMand >10,000 nM respectively, indicating thatMRS7216 efficiently binds the A2A adenosine receptor. MicroCT studies showed that mice treated with weekly doses of 7216 had a significant reduction in bone damage of 40% (p=0.04) compared to saline treated mice. In contrast, AlenP molecules did not prevent bone erosion. Histological analysis of TRAP stained samples showed a significant decrease of osteoclast number/high-power field (HPF) of 55% (p=0.03) in AlenP treated mice compared to the saline treated group. The osteoclast depletion was more dramatic inMRS7216 treated group with an 81% reduction of osteoclasts number/HPF (p= 0.002). Additionally alkaline phosphatase staining in MRS7216 treated group, showed a significant increase in osteoblast number/HPF compared to saline (55%, p=0.01) and to AlenP group (45%, p=0.03).
Conclusion(s): Alendronate-CGS conjugates represent a novel therapeutic approach to prevent osteolysis and prosthetic failure in patients with prosthetic joints

Background/Purpose: Human Immunodeficiency Virus (HIV) infection devastates the immune system but also affects tissues and organs such as kidney, liver, central nervous system, heart and bone. 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 of patients with Tenofovir alone or in combination (as part of HAART), leads to further changes in bone catabolism markers and significant reductions in BMD in children and young adults. Tenofovir is taken up by cells and phosphorylated; tenofovir-phosphate 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. As adenosine and ATP are key regulators of bone homeostasis, we determined whether Tenofovir directly affects bone by an adenosine- or ATP-dependent mechanism.
Method(s): M-CSF/RANKL-induced osteoclast (OC) and stimulated osteoblast (OB) differentiation were studied in primary murine bone marrow culture as the number of TRAP-positive or Alizarin Red-positive cells, respectively, after challenge with Tenofovir (1nM-100muM) alone or in combination with Dipyridamole (1nM-100muM), an agent that increases extracellular adenosine by blocking cellular adenosine uptake. Pannexin-1 and Connexin-43 expression were permanently knocked down in RAW264.7cells by lentiviral infection with appropriate shRNA or scrambled shRNA and these cells were induced to differentiate into OC by RANKL. OC/OB differentiation markers were study by RT-PCR, and intracellular pathways by Western Blot.
Result(s): 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 reversed by Dipyridamole. When both Pannexin-1 and Connexin-43 were absent, Tenofovir did not increase OC number. Dipyridamole reversed the effect of Tenofovir on pERK1/2, pp38 and NFkB nuclear translocation. Tenofovir inhibits OB differentiation in a dose-dependent manner (IC50=0.4muM) and treatment with Dipyridamole reversed this effect (EC50=10nM). Tenofovir increases RANKL mRNA expression and decreases OPG mRNA expression during OB differentiation; these effects are reversed by Dipyridamole. We have also found alterations in beta catenin signaling pathway due to Tenofovir treatment.
Conclusion(s): Tenofovir enhances osteoclast differentiation and inhibits osteoblast differentiation by an adenosine-dependent mechanism, a finding that suggests that treatment with agents that increase local adenosine concentrations, like Dipyridamole, might prevent bone loss due to Tenofovir treatment

Background/Purpose: Osteopenia and fragility fractures have been associated with HIV infection. Tenofovir, one of the most commonly used antivirals in HIV, 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, acting at its adenosine A2A and A2B receptors, 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 by an A2A receptor-dependent effect. We hypothesized that Tenofovir regulates bone resoprtion by diminishing endogenous adenosine levels and determined whether Dipyridamole could counteract the deleterious effects of Tenofovir on bone.
Method(s): Male C57Bl/6 mice were treated as follows: IP injection of saline (control), Tenofovir 75mg/Kg/day, Dipyridamole 25mg/Kg/day, combination Tenofovir/Dipyridamole (n=10, 4 weeks). Female C57Bl/6 mice were ovariectomized and treated as follow: sham (no surgery), saline (control), Tenofovir 75mg/Kg/day, Dipyridamole 25mg/Kg/ day, combination Tenofovir/Dipyridamole (n=10, 5 weeks). Weekly weight was annotated. DXA scanning was performed before sacrifice. Calcein/AlizarinRed-labelling of newly formed bone was used, and long bones were prepared for microCT/ histology.
Result(s): Male mice treated with Tenofovir lost nearly 10% of body weight (p<0.001). DXA scanning showed a decrease in BMD in mice treated with Tenofovir that was reversed with Dipyridamole. microCT revealed decreased BMD and diminished trabecular bone in Tenofovir-treated mice and reversal by Dipyridamole treatment. 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. In the case of female OVX mice, Tenofovir treatment also produced a decreased in body weight (p<0.05) that was reversed with Dipyridamole. DXA scanning showed decreased BMD in Tenofovir-treated mice and microCT revealed diminished trabecular bone, similar to findings in male mice. Similar results were found for Cathepsin K, CD68, RANKL and OPG-positive-cells.
Conclusion(s): These results suggest that treatment with agents that increase local adenosine concentrations, like Dipyridamole, might prevent bone loss following Tenofovir treatment

Background/Purpose: The Wnt/beta-catenin signaling pathway plays a key role in regulating bone formation and maintaining bone hemostasis. Wnt activates a pathway that leads to stabilization of beta-catenin and its translocation to the nucleus. Osteoblast differentiation and proliferation are also regulated by adenosine receptors, among other signals. We recently reported that A2aR signaling promotes Wnt/b-catenin signaling in fibroblasts via activation of Akt and p38MAPK. In the present study we sought to determine whether there is a similar interaction between these pathways in osteoblasts.
Method(s): We studied murine osteoblast cell line (MC3T3-E1) and primary osteoblasts derived from bone marrow-derived mesenchymal stem cells of mice. The cells were treated with CGS21680, a selective A2aR agonist, at doses ranging from 0 to 10muM, and for varying incubation periods up to 240 minutes. Levels of phosphorylated beta-catenin at Ser552 (p-Ser552), a beta-catenin isoform with enhanced transcriptional activity, were measured by Western Blot assays before and after A2aR activation. We also analyzed nuclear translocation of p-Ser552 beta-catenin in the osteoblastoid cell line and primary cell cultures using immunofluorescence (IF) staining. Cellular levels of activated AKT were measured by immunoblotting assays before and following administration of CGS21680.
Result(s): We observed a significant increase in p-Ser552 beta-catenin levels in the osteoblastoid cells treated with 1 muM CGS21680 compared to the control, starting at 15 minutes following A2aR activation (253+/-122%, p<0.05, n=5). Western blot analysis showed a significant increase in nuclear translocation of p-Ser552 beta-catenin at 15 minutes after treatment with A2aR agonist in MC3T3-E1 cells (153+/-37%, p<0.05, n=4), and primary osteoblasts (148+/-31%, p<0.05, n=4). Similarly, immunofluorescence revealed approximately a 40% increase in nuclear accumulation of p-Ser552 beta-catenin in CGS21680- treated MC3T3-E1 cells as well as in primary osteoblasts. We also found a significant increase in the levels of phosphorylated AKT at Ser473 among osteoblastoid cells following A2aR stimulation (203+/-47%, p<0.05, n=4).
Conclusion(s): These findings demonstrate cross-talk between A2aR and Wnt/b-catenin signaling pathways in osteoblasts. Moreover, our results suggest that A2aR activation can bypass blockade of Wnt ligands at the cell surface and thereby maintain bone homeostasis

Background/Purpose: Low dose weekly MTX remains the anchor drug for treatment of Rheumatoid Arthritis. The principal mechanism by which MTX suppresses inflammation in Rheumatoid Arthritis is thought to be enhanced adenosine release from cells which suppresses inflammation by stimulating adenosine receptors on T cells, macrophages and other inflammatory cells (Nature Rev Rheumatol 13:41, 2017). Many patients do not respond to low dose MTX and studies in mice suggest that MTX resistance may be due to inadequately increased adenosine release (Clin Exp Rheumatol 31:433, 2013). Because adenosine is primarily taken up by cells from the extracellular space via the nucleoside transporter ent1 we asked whether an agent that blocks adenosine uptake could enhance the effect of MTX in the treatment of RA. We therefore carried out an open label 1 month study, adding an inhibitor of adenosine uptake via ent1, ticagrelor (a P2Y12 inhibitor that is approved for inhibition of platelet aggregation to prevent severe cardiovascular events) (Nat Rev Cardiology12:156,2014), to patients who were poorly controlled with low dose methotrexate therapy for RA. (NCT02874092) Methods: Patients (5 female/1 male, mean age 49.6 years) who all met ACR criteria for RA and had active disease, as defined by DAS28 (ESR) > 3.6 and who were on stable doses of MTX monotherapy (for a minimum of 12 weeks), were recruited from the Bellevue Hospital Center Arthritis Clinic. Patients had no known contraindication to ticagrelor and had no history of coronary artery disease. After giving informed consent patients entered an open label protocol in which they were administered Ticagrelor (90mg) twice daily for one month in addition to their stable dose of MTX. Disease activity was reassessed and change in activity from the start of the trial was noted. This study was approved by the NYULMCBellevue IRB.
Result(s): Five of six patient achieved an improvement in their DAS28(ESR) >0.6. Half of the patients (3 patients) achieved a reduction in DAS28 (ESR) >1.2, 2 patients achieved a reduction in DAS28 (ESR) >0.6 but less than 1.2 and 1 patient showed no improvement. Four of six patients had a reduction in their tender joints and all had a reduction in swollen joints. No patients reported any adverse reactions, including excessive bleeding.
Conclusion(s): The results of this small open label trial suggest that treatment with ticagrelor enhances the effect of MTX on RA and may be a useful addition to the therapeutic armamentarium. Moreover, these results offer further support for the hypothesis that enhanced adenosine release at inflamed sites mediates the anti-inflammatory effects of MTX therapy. The limitations of this trial include the fact that it was an open label trial in a small group of patients but the results support further study of ticagrelor in combination with MTX. (Table Presented)

Background/Purpose: Osteoarthritis (OA) is the most common form of arthritis, affecting nearly 10% of the US population. With age and injury, chondrocytes have diminished mitochondrial content and mitochondrial production of ATP contributing to OA pathogenesis. We have previously reported that chondrocytes release ATP, which is converted extracellularly to adenosine and maintains chondrocyte homeostasis via endogenous stimulation of the A2AR. Injured/ inflamed chondrocytes have lower ATP levels and release less ATP resulting in diminished extracellular adenosine and A2AR stimulation. Mice and humans lacking the capacity to convert extracellular ATP to adenosine (ecto-5'nucleotidase deficient) develop spontaneous OA as do mice lacking A2AR (A2ARKO). We therefore studied the effect of A2AR stimulation on mitochondrial health and function in chondrocytes from WTand A2ARKO mice and in a human chondrocytic cell line.
Method(s): A human chondrocyte cell line, T/C28-a2, or neonatal chondrocytes isolated from WTand A2ARKO mice (C57Bl6 background) were grown in culture, treated with IL-1beta (5ng/ml) or medium (4h, 37oC) and during the last hour of incubation, with either medium or the selective A2AR agonist (CGS21680, 1mM). Mitochondrial function was then analyzed by Seahorse Mito Stress Kits using the Seahorse apparatus. Mitochondrial health was assessed by analyzing mean pixel intensity (MPI) of tetramethylrhodamine (TMRM) live cell staining which correlates with reduced collapsibility of mitochondrial membrane potential. Mitochondrial content and ROS burden were assessed in live cell confocal imaging (MitoTracker; MitoSox) and by immunohistochemistry (anti-ATPase Ab; anti-8hydroxyguanosine Ab, 8OHg) in paraffinembedded tissue from WTand A2ARKO mice.
Result(s): The mitochondrial membrane potential and mitochondrial content were reduced in A2ARKO chondrocytes compared to WT. In WT chondrocytes mitochondrial content increased after IL-1beta treatment and A2AR stimulation increased mitochondrial membrane potential as well. Histologic staining of knee cartilage for 8OHg, a marker of ROSinduced oxidation in mitochondria, of age-matched WTand A2ARKO mice revealed increased ROS burden in OA (A2AR KO) cartilage. In T/C28-a2 cells, neither IL-1beta nor CGS21680 affected basal O2 consumption rates (OCR), maximal respiratory rate or ATP production but IL-1beta-treated T/C28-a2 cells stimulated by CGS21680 increased all three measures of mitochondrial function (p<0.03, one-way ANOVA). Membrane potential (measured by TMRM MPI) decreased in T/ C28-a2 cells after IL-1beta or CGS treatment alone, but IL-1beta + CGS21680 treatment significantly increased MPI, indicating enhanced mitochondrial health. Mitochondrial content is not significantly modulated by IL-1beta, CGS or IL-1beta+CGS in vitro, but IL-1beta treatment significantly increased ROS burden (p<0.0001) and IL-1beta+CGS did not affect ROS-burden in T/C28-a2 cells.
Conclusion(s): Mitochondrial function and biomass decline with age and after injury and diminished mitochondrial function contributes to the development of OA. A2AR stimulation enhances the function of mitochondria in inflamed chondrocytes and contributes to the maintenance of healthy chondrocytes and cartilage