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Purines and Adenosine Receptors in Osteoarthritis

Cronstein, Bruce N; Angle, Siddhesh R
OA is a common and debilitating condition that restricts mobility and diminishes the quality of life. Recent work indicates that the generation of adenosine at the cell surface is an important mediator of chondrocyte homeostasis, and topical application of adenosine in a slow-release form (liposomes) can halt the progression of OA and diminish the pain associated with OA. Here, we review the evidence indicating that adenosine, acting at A2A receptors, plays a critical role in endogenous and exogenous treatment and reversal of OA.
PMID: 38136631
ISSN: 2218-273x
CID: 5611982

"Bone Tissue Engineering in the Growing Calvaria: A 3D Printed Bioceramic Scaffold to Reconstruct Critical-Sized Defects in a Skeletally Immature Pig Model"

DeMitchell-Rodriguez, Evellyn M; Shen, Chen; Nayak, Vasudev V; Tovar, Nick; Witek, Lukasz; Torroni, Andrea; Yarholar, Lauren M; Cronstein, Bruce N; Flores, Roberto L; Coelho, Paulo G
BACKGROUND:3D-printed bioceramic scaffolds composed of 100% beta(β)-tricalcium phosphate augmented with dipyridamole (3DPBC-DIPY) can regenerate bone across critically sized defects in skeletally mature and immature animal models. Prior to human application, safe and effective bone formation should be demonstrated in a large translational animal model. This study evaluated the ability of 3DPBC-DIPY scaffolds to restore critically sized calvarial defects in a skeletally immature, growing minipig. METHODS:Unilateral calvarial defects (~1.4cm) were created in six-week-old Göttingen minipigs (n=12). Four defects were filled with a 1000µ M 3DPBC-DIPY scaffold with a cap (a solid barrier on the ectocortical side of the scaffold to prevent soft tissue infiltration), four defects were filled with a 1000µM 3DPBC-DIPY scaffold without a cap, and four defects served as negative controls (no scaffold). Animals were euthanized 12-weeks post-operatively. Calvaria were subjected to micro-computed tomography, 3D-reconstruction with volumetric analysis, qualitative histologic analysis, and nanoindentation. RESULTS:Scaffold-induced bone growth was statistically greater than negative controls (p≤0.001) and the scaffolds with caps produced significantly more bone generation compared to the scaffolds without caps (p≤0.001). Histological analysis revealed woven and lamellar bone with the presence of haversian canals throughout the regenerated bone. Additionally, cranial sutures were observed to be patent and there was no evidence of ectopic bone formation or excess inflammatory response. Reduced elastic modulus (Er) and hardness (H) of scaffold-regenerated bone were found to be statistically equivalent to native bone (p = 0.148 for Er of scaffolds with and without caps, and p = 0.228 and p = 0.902, for H of scaffolds with and without caps, respectively). CONCLUSION/CONCLUSIONS:3DPBC-DIPY scaffolds have the capacity to regenerate bone across critically sized calvarial defects in a skeletally immature translational pig model.
PMID: 36723712
ISSN: 1529-4242
CID: 5420092

Employing Indirect Adenosine 2A Receptors (A2AR) to Enhance Osseointegration of Titanium Devices: A Pre-Clinical Study

Pacheco-Vergara, Maria Jesus; Benalcázar-Jalkh, Ernesto Byron; Nayak, Vasudev V; Bergamo, Edmara T P; Cronstein, Bruce; Zétola, André Luis; Weiss, Fernando Pessoa; Grossi, João Ricardo Almeida; Deliberador, Tatiana Miranda; Coelho, Paulo G; Witek, Lukasz
The present study aimed to evaluate the effect of dipyridamole, an indirect adenosine 2A receptors (A2AR), on the osseointegration of titanium implants in a large, translational pre-clinical model. Sixty tapered, acid-etched titanium implants, treated with four different coatings ((i) Type I Bovine Collagen (control), (ii) 10 μM dipyridamole (DIPY), (iii) 100 μM DIPY, and (iv) 1000 μM DIPY), were inserted in the vertebral bodies of 15 female sheep (weight ~65 kg). Qualitative and quantitative analysis were performed after 3, 6, and 12 weeks in vivo to assess histological features, and percentages of bone-to-implant contact (%BIC) and bone area fraction occupancy (%BAFO). Data was analyzed using a general linear mixed model analysis with time in vivo and coating as fixed factors. Histomorphometric analysis after 3 weeks in vivo revealed higher BIC for DIPY coated implant groups (10 μM (30.42% ± 10.62), 100 μM (36.41% ± 10.62), and 1000 μM (32.46% ± 10.62)) in comparison to the control group (17.99% ± 5.82). Further, significantly higher BAFO was observed for implants augmented with 1000 μM of DIPY (43.84% ± 9.97) compared to the control group (31.89% ± 5.46). At 6 and 12 weeks, no significant differences were observed among groups. Histological analysis evidenced similar osseointegration features and an intramembranous-type healing pattern for all groups. Qualitative observation corroborated the increased presence of woven bone formation in intimate contact with the surface of the implant and within the threads at 3 weeks with increased concentrations of DIPY. Coating the implant surface with dipyridamole yielded a favorable effect with regard to BIC and BAFO at 3 weeks in vivo. These findings suggest a positive effect of DIPY on the early stages of osseointegration.
PMID: 37367272
ISSN: 2079-4983
CID: 5540212

Adenosine A2A receptor activation reduces chondrocyte senescence

Friedman, Benjamin; Larranaga-Vera, Ane; Castro, Cristina M; Corciulo, Carmen; Rabbani, Piul; Cronstein, Bruce N
Osteoarthritis (OA) pathogenesis is associated with reduced chondrocyte homeostasis and increased levels of cartilage cellular senescence. Chondrosenescence is the development of cartilage senescence that increases with aging joints and disrupts chondrocyte homeostasis and is associated with OA. Adenosine A2A receptor (A2AR) activation in cartilage via intra-articular injection of liposomal A2AR agonist, liposomal-CGS21680, leads to cartilage regeneration in vivo and chondrocyte homeostasis. A2AR knockout mice develop early OA isolated chondrocytes demonstrate upregulated expression of cellular senescence and aging-associated genes. Based on these observations, we hypothesized that A2AR activation would ameliorate cartilage senescence. We found that A2AR stimulation of chondrocytes reduced beta-galactosidase staining and regulated levels and cell localization of common senescence mediators p21 and p16 in vitro in the human TC28a2 chondrocyte cell line. In vivo analysis similarly showed A2AR activation reduced nuclear p21 and p16 in obesity-induced OA mice injected with liposomal-CGS21680 and increased nuclear p21 and p16 in A2AR knockout mouse chondrocytes compared to wild-type mice. A2AR agonism also increased activity of the chondrocyte Sirt1/AMPK energy-sensing pathway by enhancing nuclear Sirt1 localization and upregulating T172-phosphorylated (active) AMPK protein levels. Lastly, A2AR activation in TC28a2 and primary human chondrocytes reduced wild-type p53 and concomitantly increased p53 alternative splicing leading to increase in an anti-senescent p53 variant, Δ133p53α. The results reported here indicate that A2AR signaling promotes chondrocyte homeostasis in vitro and reduces OA cartilage development in vivo by reducing chondrocyte senescence.
PMID: 36884388
ISSN: 1530-6860
CID: 5448632

Novel alendronate-CGS21680 conjugate reduces bone resorption and induces new bone formation in post-menopausal osteoporosis and inflammatory osteolysis mouse models

Larrañaga-Vera, Ane; Toti, Kiran S; Flatow, James S; Haraczy, Alexandra J; Warnick, Eugene; Rao, Harsha; Gao, Zhan-Guo; Sussman, Sarah M; Mediero, Aranzazu; Leucht, Philipp; Jacobson, Kenneth A; Cronstein, Bruce N
Loss of bone is a common medical problem and, while it can be treated with available therapies, some of these therapies have critical side effects. We have previously demonstrated that CGS21680, a selective A2A adenosine receptor agonist, prevents bone loss, but its on-target toxicities (hypotension, tachycardia) and frequent dosing requirements make it unusable in the clinic. We therefore generated a novel alendronate-CGS21680 conjugate (MRS7216), to target the agonist to bone where it remains for long periods thereby diminishing the frequency of administration and curtailing side effects. MRS7216 was synthesized from CGS21680 by sequential activation of the carboxylic acid moiety and reacting with an appropriate amino acid (PEG, alendronic acid) under basic conditions. MRS7216 was tested on C57BL/6J (WT) mice with established osteoporosis (OP) and WT or A2A KO mice with wear particle-induced inflammatory osteolysis (OL). Mice were treated weekly with MRS7216 (10mg/kg). Bone formation was studied after in vivo labeling with calcein/Alizarin Red, and μCT and histology analyses were performed. In addition, human primary osteoblasts and osteoclasts were cultured using bone marrow discarded after hip replacement. Receptor binding studies demonstrate that MRS7216 efficiently binds the A2A adenosine receptor. MRS7216-treated OP and OL mice had significant new bone formation and reduced bone loss compared to vehicle or alendronate-treated mice. Histological analysis showed that MRS7216 treatment significantly reduced osteoclast number and increased osteoblast number in murine models. Interestingly, cultured human osteoclast differentiation was inhibited, and osteoblast differentiation was stimulated by the compound indicating that MRS7216 conjugates represent a novel therapeutic approach to treat osteoporosis and osteolysis.
PMID: 36494860
ISSN: 1478-6362
CID: 5378892

Adenosine A2A Receptor Signaling Enhances Expression of an Endogenous p53 Mouse Variant in Bone Marrow Mesenchymal Stem Cells [Meeting Abstract]

Friedman, B; Cronstein, B
Background/Purpose: Our lab has demonstrated the ability of an intra-articular liposomal formulation of an adenosine A2A receptor (A2AR), CGS21680, to regenerate cartilage in multiple rodent OA models. At the cellular level in chondrocytes, we have shown that activation of A2AR enhances chondrocyte homeostasis, increases mitophagy, and reduces chondrocyte senescence. Moreover, we identified an A2AR-mediated increase in an anti-senescent, anti-aging truncated p53 variant D133p53a in human chondrocytes. These anti-senescent effects have been recapitulated in transgenic mice overexpressing D122p53. Interestingly, a 2014 study reported an endogenous N-terminal mouse p53 variant in HeLa cells (2 separate deletions with loss of amino acids 42-89 and in-frame shift of aa 90-120) with no p53 transactivation potential and inability to upregulate p21 expression despite retention of the first p53 transactivation domain. Hence, we first hypothesized that this mouse p53 variant could be present in mesenchymal stem cells (MSCs), which have chondrogenic capabilities in vitro. Secondly, we hypothesized that A2AR ligation in mouse MSCs would increase expression of this variant.
Method(s): Bone marrow MSCs were isolated from 12-week-old mice, treated with or without 1muM CGS21680, and RTqPCR was performed to assess expression of total and variant p53 using variant specific primers. Primary and secondary gene sequence analysis was employed to evaluate the deleted/shifted region for CpG sites and G-quadruplex structures using the Sequence Manipulation Suite and the GQRS Mapper, respectively.
Result(s): We did identify this variant in mouse bone marrow MSCs. Furthermore, its expression relative to total p53 was significantly increased in the A2AR agonist treated MSCs compared to control (1.6+/-0.3 vs. 1.0+/-0.06, p=0.029, n=3). CpG site analysis revealed that the variant corresponded to a reduction in 4 CpG sites and 3 potential G-quadruplex structures if analyzed in the antisense configuration.
Conclusion(s): To our knowledge, this is the first report of this variant of mouse p53 noted in cell types aside from the initial report noted above. While more work needs to be done in differentiated cells, A2AR agonism seems to enhance formation of this mouse p53 variant has functional similarities to the D133p53a. Importantly, the deleted regions in this variant contain the key senescence residues in the protein. Further, It is possible that DNA modifications such as CpG methylation and/or alteration in G-quadruplex stability may play a role in such findings
ISSN: 2326-5205
CID: 5513142

The effects of caffeine on bone mineral density and fracture risk

Berman, N K; Honig, S; Cronstein, B N; Pillinger, M H
Caffeine is a regular part of the diet of many adults (coffee, tea, soft drinks, and energy drinks). Multiple molecular effects of caffeine suggest that it may promote bone loss. Given the extensive consumption of caffeine worldwide, any impact of caffeine consumption on bone strength and/or density would have large population health implications. The most well-established pharmacological effect of caffeine is non-specific antagonism of adenosine receptors. Adenosine regulates bone metabolism in a complex manner, with in vitro studies suggesting that direct stimulation of adenosine A2A and A2B receptors induces bone formation by activating osteoblasts and suppressing osteoclast differentiation and function. Thus, competitive inhibition of adenosine A2 receptors by caffeine may inhibit bone formation and promote bone resorption. However, antagonism of adenosine A1 receptors may have opposing effects. Caffeine has also been suggested to affect bone through derangement of calcium metabolism, alteration of vitamin D responses, and other mechanisms. In clinical and population-based studies, the impact of caffeine consumption on bone metabolism offers a mixed picture, with some but not all studies suggesting a potential link between caffeine intake and reduced bone mineral density or increased fracture risk. Differences in methodology, selected populations, and duration/timing of the studies may account for study outcome discrepancies. The in vitro effects of caffeine on cells involved in bone metabolism suggest that caffeine intake may promote osteoporosis, and some but not all clinical studies support a modest adverse caffeine impact. Herein, we describe the basic biology of caffeine as it pertains to bone, review the clinical literature to date, and consider the implications of the current data on clinical practice and future studies.
PMID: 34981132
ISSN: 1433-2965
CID: 5106942

Self-assembling human skeletal organoids for disease modeling and drug testing

Abraham, Diana M; Herman, Calvin; Witek, Lukasz; Cronstein, Bruce N; Flores, Roberto L; Coelho, Paulo G
Skeletal conditions represent a considerable challenge to health systems globally. Barriers to effective therapeutic development include a lack of accurate preclinical tissue and disease models. Most recently, work was attempted to present a novel whole organ approach to modeling human bone and cartilage tissues. These self-assembling skeletal organoids mimic the cellular milieu and extracellular organization present in native tissues. Bone organoids demonstrated osteogenesis and micro vessel formation, and cartilage organoids showed evidence of cartilage development and maturation. Skeletal organoids derived from both bone and cartilage tissues yielded spontaneous polarization of their cartilaginous and bone components. Using these hybrid skeletal organoids, we successfully generated "mini joint" cultures, which we used to model inflammatory disease and test Adenosine (A2A ) receptor agonists as a therapeutic agent. The work and respective results indicated that skeletal organoids can be an effective biological model for tissue development and disease as well as to test therapeutic agents.
PMID: 34837719
ISSN: 1552-4981
CID: 5063982

International Union of Basic and Clinical Pharmacology. CXII: Adenosine Receptors: A Further Update

IJzerman, Adriaan P; Jacobson, Kenneth A; Müller, Christa E; Cronstein, Bruce N; Cunha, Rodrigo A
Our previous International Union of Basic and Clinical Pharmacology report on the nomenclature and classification of adenosine receptors (2011) contained a number of emerging developments with respect to this G protein-coupled receptor subfamily, including protein structure, protein oligomerization, protein diversity, and allosteric modulation by small molecules. Since then, a wealth of new data and results has been added, allowing us to explore novel concepts such as target binding kinetics and biased signaling of adenosine receptors, to examine a multitude of receptor structures and novel ligands, to gauge new pharmacology, and to evaluate clinical trials with adenosine receptor ligands. This review should therefore be considered a further update of our previous reports from 2001 and 2011. SIGNIFICANCE STATEMENT: Adenosine receptors (ARs) are of continuing interest for future treatment of chronic and acute disease conditions, including inflammatory diseases, neurodegenerative afflictions, and cancer. The design of AR agonists ("biased" or not) and antagonists is largely structure based now, thanks to the tremendous progress in AR structural biology. The A2A- and A2BAR appear to modulate the immune response in tumor biology. Many clinical trials for this indication are ongoing, whereas an A2AAR antagonist (istradefylline) has been approved as an anti-Parkinson agent.
PMID: 35302044
ISSN: 1521-0081
CID: 5190562

Tissue Engineering Strategies for Craniomaxillofacial Surgery: Current Trends in 3D-Printed Bioactive Ceramic Scaffolds

Chapter by: Witek, Lukasz; Nayak, Vasudev Vivekanand; Runyan, Christopher M; Tovar, Nick; Elhage, Sharbel; Melville, James C; Young, Simon; Kim, David H; Cronstein, Bruce N; Flores, Roberto L; Coelho, Paulo G
in: Innovative Bioceramics in Translational Medicine II by Choi, Andy H; Ben-Nissan, Besim [Eds]
Cham : Springer, 2022
pp. 55-74
ISBN: 978-981-16-7438-9
CID: 5457532