Faculty Bibliography

Adenosine is generated in increased concentrations at sites of injury/hypoxia and mediates a variety of physiological and pharmacological effects via G protein-coupled receptors (A(1), A(2A), A(2B), and A(3)). Because all adenosine receptors are expressed on osteoclasts, we determined the role of A(2A) receptor in the regulation of osteoclast differentiation. Differentiation and bone resorption were studied as the macrophage colony-stimulating factor-1-receptor activator of NF-kappaB ligand formation of multinucleated tartrate-resistant acid phosphatase (TRAP)-positive cells from primary murine bone marrow-derived precursors. A(2A) receptor and osteoclast marker expression levels were studied by RT-PCR. Cytokine secretion was assayed by enzyme-linked immunosorbent assay. In vivo examination of A(2A) knockout (KO)/control bones was determined by TRAP staining, micro-computed tomography, and electron microscopy. The A(2A) receptor agonist, CGS21680, inhibited osteoclast differentiation and function (half maximal inhibitory concentration, 50 nmol/L), increased the percentage of immature osteoclast precursors, and decreased IL-1beta and tumor necrosis factor-alpha secretion, an effect that was reversed by the A(2A) antagonist, ZM241385. Cathepsin K and osteopontin mRNA expression increased in control and ZM241385-pretreated osteoclasts, and this was blocked by CGS21680. Micro-computed tomography of A(2A)KO mouse femurs showed a significantly decreased bone volume/trabecular bone volume ratio, decreased trabecular number, and increased trabecular space. A(2A)KO femurs showed an increased TRAP-positive osteoclast. Electron microscopy in A(2A)KO femurs showed marked osteoclast membrane folding and increased bone resorption. Thus, adenosine, acting via the A(2A) receptor, inhibits macrophage colony-stimulating factor-1-receptor activator of NF-kappaB ligand-stimulated osteoclast differentiation and may regulate bone turnover under conditions in which adenosine levels are elevated

Background: Our prior observations showed that the nucleoside adenosine and its receptor mediate collagen production and dermal fibrosis by human dermal fibroblasts in vitro. In addition, we showed that IL-13 expression is upregulated in tissues characterized by high levels of adenosine in the adenosine deaminase-deficient mouse model. This suggests adenosine upregulates the pro-fibrogenic cytokine IL-13. However, the receptor(s) and mechanism involved in this upregulation remained unknown. The aim of this study was to characterize further the contributions of endogenous adenosine and adenosine A2A receptors to skin fibrosis. Methods: In an in vitro study of cultured normal human dermal fibroblasts, the effect of adenosine A2A receptor modulation on IL-13 expression was studied. Using both the A2A receptor agonist (CGS21680, 1muM) and antagonist (ZM241385, 1muM), levels of IL-13 and IL-13 receptors were tested. Measurements of mRNA for IL-13 and three IL-13 receptor proteins, IL-13Ralpha1, IL-13Ralpha2 and IL-4 were assessed using real-time PCR amplification. Results: The A2A agonist induced elevated expression of message for IL-13 and two of three IL-13 receptors: IL-13Ralpha1 and IL-13Ralpha2. A 5- fold increase in expression was found for IL-13Ralpha1 and a more modest 1.8-fold increase in IL-13Ralpha2. These inducible elevations were blocked by coincubation with the A2A receptor antagonist but not by antagonists to other adenosine receptors. Incubation with the A2A receptor antagonist alone did not affect the expression of IL-13 or its receptors. Conclusion: Despite efforts at investigating the mechanisms underlying fibrogenic processes in the skin of patients with scleroderma, no effective antifibrotic therapy exists. The nucleoside adenosine induces expression of pro-fibrotic cytokine IL-13 and particularly its cognate receptor IL-13Ralpha1.These findings suggest that blockade of the A2A receptor may be useful as a novel therapeutic agent to modify dermal fibrosis in scleroderma

Various types of orthopedic procedures, including spinal fusion and repair of bone defects due to trauma, infection or metastatic disease, require formation of new bone. Adenosine, generated from the catabolism of adenine nucleotides, modulates cell function by interacting with specific cell-surface receptors (A1, A2A, A2B, A3). In previous studies we have demonstrated that adenosine A1 receptor blockade and A2A receptor stimulation diminishes osteoclast formation without directly affecting osteoblast formation or function. We therefore determined whether targeting adenosine receptors affected bone regeneration in a murine model.Male C57Bl/6 mice were anesthetized and a 3mm trephine defect was formed and covered with a collagen scaffold soaked in saline or 1muM adenosine receptor agonists/antagonists. Animals received the appropriate treatment daily until sacrifice. At 0, 2, 4, 6 and 8 weeks calvarias were harvested and prepared for microCT and histology. XenoLight Rediject Bone Probe 680 was injected intravenously in mice at different time points.Eight weeks after surgery microCT examination of mouse calvaria demonstrate that an A1R antagonist (DPCPX), A2AR agonist (CGS21680M) or dipyridamole (blocks adenosine uptake and increases extracellular adenosine levels) markedly enhances bone regeneration (77+/-0.2%, 60+/-2% and 79+/-2% bone regeneration respectively vs 32+/-4% in control mice, p<0.001, n=5 mice per condition) whereas an A3R agonist (IB-MECA) had no effect (32+/-3% regeneration, n=5). Alkaline phophatase and osteocalcin immunostaining at the sites of bone formation was increased in defects treated with DPCPX, CGS21680 and Dipyridamole. In contrast, TNFalpha, Semaphorin4D and PlexinB1 immunostaining was diminished in DPCPX, CGS21680 and Dipyridamole treated defects. TRAP staining revealed fewer osteoclasts in DPCPX, CGS21680 and Dipyridamole treated defects (14+/-1, 17+/-1 and 16+/-1 Osteoclast/Ipf respectively vs 24+/-1 Osteoclast/Ipf for control, p<0.001) and in vivo imaging with a m!

BACKGROUND: Previous work from our laboratory has uncovered a critical role of adenosine A receptor (A R) in osteoclastogenesis both in vivo and in vitro. Adenosine may be generated by hydrolysis of extracellular adenosine nucleotides including CD39, ecto-5'-nucleotidase (CD73) and nucleotide pyrophosphatase phosphodiesterase 1 (NPP-1). Interestingly selective A R agonists neither affect basal osteoclast formation nor do they reverse A R-mediated inhibition of osteoclast formation. In this study, we determined whether ectonucleotidase-mediated adenosine production was required for osteoclast formation and, when we saw no effect, determined whether the A R was constitutively activated and the antagonist was acting as an inverse agonist to mediate its effects on osteoclast formation. METHODS: Osteoclasts were generated from bone marrow mononuclear cells (BMMs) extracted from wildtype, CD39KO, CD73KO and NPP-1KO mice using differentiation factors M-CSF and RANKL. The A R specific antagonist, rolofylline, was added to the culture media. TRAP+ staining was performed and Acp5 and Ctsk mRNA expression were examined to study osteoclastogenesis. Intracellular cAMP concentration was determined by ELISA. RESULTS: A R blockade inhibits osteoclast differentiation of BMMs derived from wildtype mice in a dosedependent manner (IC =1muM p<0.05, n=3). A R blockade similarly inhibits osteoclast formation by BMMs from CD73KO, CD39KO and NPP-1KO mice in a dose-dependent manner (IC =1muM, 1muM, and 0.1muM, respectively, p<0.05 for all, n=3) for all three knockouts, although baseline osteoclast formation was significantly less (310 in CD73 KO vs 91 in wildtype, p<0.05, n=3) in BMMs from CD73KO mice. Moreover, in the absence of agonist rolofylline (1muM) caused an increase of cAMP content of BMMs by ~8.56 fold (p<0.05, n=3). Similarly, rolofylline (1muM) promotes increased cAMP production in normal human BMMs by ~22.3 fold, which is consistent with our findings that rolofylline inhibits human BMMs-derived osteoclast f!

Adenosine, a nucleoside released at sites of injury and hypoxia, mediates its physiologic and pharmacologic effects via activation of G-protein-coupled receptors (A1, A2A, A2B, A3). Previously we reported that the A2AR agonist CGS21680 inhibited osteoclast differentiation in a dose-dependent manner so here we determined which intracellular pathways are involved in A2AR-mediated regulation of osteoclast differentiation. Osteoclast morphological characterization was studied in M-CSF/RANKLstimulated murine marrow osteoclast precursors in the presence/absence of CGS21680 (A2A agonist) and ZM241385 (A2A antagonist) stained with AlexaFluor555-Phalloidin. Osteoclast differentiation in the presence/absence of CGS21680, ZM241385 and PKA/EPAC activators/inhibitors was further studied by TRAP staining. Signaling events (PKA/EPAC, NFkB and MAPK) were studied by Western Blot in PKA knockdown (lentiviral shRNA for PKA or scrambled) RAW264.7 cells. Osteoclast marker expression was studied by RT-PCR. Cytokine expression was assayed by ELISA. A2AR stimulation increases cAMP which activates protein kinase A (PKA) and direct activation of PKA inhibits osteoclast differentiation by 40.1+/-4.3% (p<0.001, n=5). Interestingly, cAMP also stimulates EPAC activation and direct EPAC activation increases osteoclast formation by 6.8+/-3.2% (p<0.001, n=5). PKA activation increased over time in the presence of CGS21680 with no change in EPAC activation. A2AR activation inhibits NFkB nuclear translocation in scrambled shRNA but not PKA knockdown cells (p<0.001, n=4). CGS21680 activates MAPK (pERK1/2, p-p38 and pJNK) and pre-treatment with ZM241385 reversed this activation; ERK1/2 was activated scrambled but not PKA-knockdown cells. A2AR activation inhibits the expression of osteoclast differentiation markers (Cathepsin K and Osteopontin) by a PKA-dependent mechanism. Finally, A2AR activation decreases IL-1beta/TNFalpha secretion during osteoclast differentiation and theses cytokines reverse the A2AR effect. Adenosine, acting !

Topical bovine thrombin has been associated with immune responses and anecdotal reports of coagulopathy. This open-label study assessed the impact on clinical hemostasis of human antibodies to bovine thrombin (aBT) or factor V/Va (aBV/Va) in response to topical bovine thrombin (THROMBIN-JMI) in patients both with and without preexisting anti-bovine antibodies. Noninferiority analysis assessed primary endpoint for mean shift from baseline activated partial thromboplastin time (aPTT) at 48 hours postsurgery; secondary endpoints included changes from baseline antibodies/titers and coagulation parameters through 8 weeks postsurgery. A total of 550 patients underwent surgery with THROMBIN-JMI utilized at investigator's discretion. Adjusted mean aPTT change in (+)aBT/(+)THROMBIN-JMI cohort was greater than (-)aBT/(-)THROMBIN-JMI cohort; 4.67-second upper confidence bound exceeded 4.5-second margin (based on assumed mean aPTT of 30 seconds) and noninferiority was not met. Post hoc analysis indicated noninferiority would have been met had noninferiority margin been set prior at relative 15% of actual baseline aPTT. Antibodies/titers were unchanged by THROMBIN-JMI exposure 48 hours postsurgery and unrelated to postsurgical changes in coagulation. Thus, THROMBIN-JMI exposure in patients with/without preexisting aBT or aBV/Va does not alter hemostasis

Adenosine regulates a wide variety of physiological processes including heart rate, vasodilation and inflammation through the activation of specific cell surface adenosine receptors. In addition to these well-established roles of adenosine, recent genetic and pharmacological research has implicated adenosine as an important regulator in bone remodeling. The secretion of adenosine and the presence of its four receptors in bone cells have been well documented. More recently, we provided the first evidence that adenosine regulates osteoclast formation and function through A1 receptor (A1R), and showed that A1R-knockout mice have significantly increased bone volume as a result of impaired osteoclast-mediated bone resorption. Moreover, adenosine A1R-knockout mice are protective from boss loss following ovariectomy further supporting the involvement of adenosine in osteoclast formation and function. This short review summarizes current knowledge related to the roles of adenosine and adenosine receptors in bone formation and remodeling. A deeper insight into the regulation of bone metabolism by adenosine receptors should assist in developing new therapies for osteoporosis