Aránzazu Mediero

Adenosine A2A Receptor Activation Prevents Wear Particle-Induced Osteolysis

An adenosine A2A receptor agonist prevents osteolysis caused by polymeric wear particles in mouse calvaria. Agonist Abates Bone Destruction Surgeons perform tens of thousands of total hip replacements per year in the United States. Despite the prevalence of hip implants, they often loosen over time, making the patient return to the surgeon for a revision procedure, typically involving implant removal. Prosthesis loosening has been associated with a breakdown of the implant materials into tiny wear particles, which leads to inflammation in the joint and destruction of the bone (“pitting”) via the small signaling molecule adenosine. Mediero and colleagues have now found that by stimulating the adenosine A2A receptor (A2AR), they can prevent wear particle–induced bone damage and inflammation at the implant site. To simulate wear particle exposure, the authors injected mice with ultrahigh–molecular weight polyethylene (UHMWPE) particles. After 2 weeks, these mouse calvaria showed pitting and increased porosity compared to particle-free mice. Giving the mice CGS21680, an adenosine A2AR agonist, at the same time as the wear particles reduced bone destruction and inflammation. Mechanism was confirmed in A2AR knockout mice, where the agonist had no effect on bone pitting. CGS21680 also inhibited differentiation of human-derived osteoclast precursor cells (from the bone marrow of four patients) into osteoclasts—the cell type that chews up bone. This suggests that the agonist will have a similar effect on human cells and could be used to prevent wear particle–induced damage in people, although only future clinical trials will confirm this. The authors indicate that this agonist could be included in bone cement or as a coating on prostheses to exert its bone-protective effects over time and thus prevent painful revision procedures. Prosthesis loosening, associated with wear particle–induced inflammation and osteoclast-mediated bone destruction, is a common cause for joint implant failure, leading to revision surgery. Adenosine A2A receptors (A2ARs) mediate potent anti-inflammatory effects in many tissues and prevent osteoclast differentiation. We tested the hypothesis that an A2AR agonist could reduce osteoclast-mediated bone resorption in a murine calvaria model of wear particle–induced bone resorption. C57BL/6 and A2AR knockout (A2AR KO) mice received ultrahigh–molecular weight polyethylene particles and were treated daily with either saline or the A2AR agonist CGS21680. After 2 weeks, micro-computed tomography of calvaria demonstrated that CGS21680 reduced particle-induced bone pitting and porosity in a dose-dependent manner, increasing cortical bone and bone volume compared to control mice. Histological examination demonstrated diminished inflammation after treatment with CGS21680. In A2AR KO mice, CGS21680 did not affect osteoclast-mediated bone resorption or inflammation. Levels of bone resorption markers receptor activator of nuclear factor κB (RANK), RANK ligand, cathepsin K, CD163, and osteopontin were reduced after CGS21680 treatment, together with a reduction in osteoclasts. Secretion of interleukin-1β (IL-1β) and tumor necrosis factor–α was significantly decreased, whereas IL-10 was markedly increased in bone by CGS21680. These results in mice suggest that site-specific delivery of an adenosine A2AR agonist could enhance implant survival, delaying or eliminating the need for revision arthroplastic surgery.

Activation of adenosine A2A receptor reduces osteoclast formation via PKA- and ERK1/2-mediated suppression of NFκB nuclear translocation

We previously reported that adenosine, acting at adenosine A2A receptors (A2AR), inhibits osteoclast (OC) differentiation in vitro (A2AR activation OC formation reduces by half) and in vivo. For a better understanding how adenosine A2AR stimulation regulates OC differentiation, we dissected the signalling pathways involved in A2AR signalling.

Adenosine and bone metabolism

Bone is a dynamic organ that undergoes continuous remodeling while maintaining a balance between bone formation and resorption. Osteoblasts, which synthesize and mineralize new bone, and osteoclasts, the cells that resorb bone, act in concert to maintain bone homeostasis. In recent years, there has been increasing appreciation of purinergic regulation of bone metabolism. Adenosine, released locally, mediates its physiologic and pharmacologic actions via interactions with G protein-coupled receptors, and recent work has indicated that these receptors are involved in the regulation of osteoclast differentiation and function, as well as in osteoblast differentiation and bone formation. Moreover, adenosine receptors also regulate chondrocyte and cartilage homeostasis. These recent findings underscore the potential therapeutic importance of adenosine receptors in regulating bone physiology and pathology.

Adenosine A2A Receptor Ligation Inhibits Osteoclast Formation

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-κB 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-1β and tumor necrosis factor-α 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-κB ligand-stimulated osteoclast differentiation and may regulate bone turnover under conditions in which adenosine levels are elevated.

Adenosine 2A receptor promotes collagen production by human fibroblasts via pathways involving cyclic AMP and AKT but independent of Smad2/3

Activation of adenosine A2A receptor (A2AR) promotes fibrosis and collagen synthesis. However, the underlying mechanism is still unclear, not least because cAMP, its principal effector, has been found to inhibit TGFβ1‐induced collagen synthesis. Here, we show that in primary normal human dermal fibroblasts, A2AR stimulation with CGS21680 elicits a modest cAMP increase (150±12% of control; EC50 54.8 nM), which stimulates collagen1 (Col1) and collagen3 (Col3), but maximal cAMP resulting from direct activation of adenylyl cyclase by forskolin (15,689±7038% of control; EC50 360.7 nM) inhibits Col1 and increases Col3. Similar to Col1 expression, fibroblast proliferation increased following physiological cAMP increases by CGS21680 but was inhibited by cAMP increases beyond the physiological range by forskolin. The A2AR‐mediated increase of Col1 and Col3 was mediated by AKT, while Col3, but not Col1, expression was dependent on p38 and repressed by ERK. TGFβ1 induced phosphorylation of Smad2/3 and increased Col3 expression, which was prevented by Smad3 depletion. In contrast, CGS21680 did not activate Smad2/3, and Smad2/3 knockdown did not prevent CGS21680‐induced Col1 or Col3 increases. Our results indicate that cAMP is a concentration‐dependent switch for collagen production via noncanonical, AKT‐dependent, Smad2/3‐independent signaling. These observations explain the paradoxical effects of cAMP on collagen expression.—Perez‐Aso, M., Fernandez, P., Mediero, A., Chan, E. S., and Cronstein, B. N. Adenosine 2A receptor promotes collagen production by human fibroblasts via pathways involving cyclic AMP and AKT but independent of Smad2/3. FASEB J. 28, 802–812 (2014). www.fasebj.org

Direct or indirect stimulation of adenosine A2A receptors enhances bone regeneration as well as bone morphogenetic protein-2

Promoting bone regeneration and repair of bone defects is a need that has not been well met to date. We have previously found that adenosine, acting via receptors (A2AR) promotes wound healing and inhibits inflammatory osteolysis and hypothesized that A2AR might be a novel target to promote bone regeneration. Therefore, we determined whether direct A2AR stimulation or increasing endogenous adenosine concentrations via purine transport blockade with dipyridamole regulates bone formation. We determined whether coverage of a 3 mm trephine defect in a mouse skull with a collagen scaffold soaked in saline, bone morphogenetic protein‐2 (BMP‐2; 200 ng), 1 μM CGS21680 (A2AR agonist, EC50 = 160 nM), or 1 μM dipyridamole (EC50 = 32 nM) promoted bone regeneration. Microcomputed tomography examination demonstrated that CGS21680 and dipyridamole markedly enhanced bone regeneration as well as BMP‐2 8 wk after surgery (60 ± 2%, 79 ± 2%, and 75 ± 1% bone regeneration, respectively, vs. 32 ± 2% in control, P < 0.001). Blockade by a selective A2AR antagonist (ZM241385, 1 μM) or deletion of A2AR abrogated the effect of CGS21680 and dipyridamole on bone regeneration. Both CGS21680 and dipyridamole treatment increased alkaline phosphatase‐positive osteoblasts and diminished tartrate resistance acid phosphatase‐positive osteoclasts in the defects. In vivo imaging with a fluorescent dye for new bone formation revealed a strong fluorescent signal in treated animals that was equivalent to BMP‐2. In conclusion, stimulation of A2AR by specific agonists or by increasing endogenous adenosine levels stimulates new bone formation as well as BMP‐2 and represents a novel approach to stimulating bone regeneration.—Mediero, A., Wilder, T., Perez‐Aso, M., Cronstein, B. N. Direct or indirect stimulation of adenosine A2A receptors enhances bone regeneration as well as bone morphogenetic protein‐2. FASEB J. 29, 1577‐1590 (2015). www.fasebj.org

Bone regeneration in critical bone defects using three-dimensionally printed β-tricalcium phosphate/hydroxyapatite scaffolds is enhanced by coating scaffolds with either dipyridamole or BMP-2

Bone defects resulting from trauma or infection need timely and effective treatments to restore damaged bone. Using specialized three-dimensional (3D) printing technology we have created custom 3D scaffolds of hydroxyapatite (HA)/beta-tri-calcium phosphate (β-TCP) to promote bone repair. To further enhance bone regeneration we have coated the scaffolds with dipyridamole, an agent that increases local adenosine levels by blocking cellular uptake of adenosine. Nearly 15% HA:85% β-TCP scaffolds were designed using Robocad software, fabricated using a 3D Robocasting system, and sintered at 1100°C for 4 h. Scaffolds were coated with BMP-2 (200 ng mL-1 ), dypiridamole 100 µM or saline and implanted in C57B6 and adenosine A2A receptor knockout (A2AKO) mice with 3 mm cranial critical bone defects for 2-8 weeks. Dipyridamole release from scaffold was assayed spectrophotometrically. MicroCT and histological analysis were performed. Micro-computed tomography (microCT) showed significant bone formation and remodeling in HA/β-TCP-dipyridamole and HA/β-TCP-BMP-2 scaffolds when compared to scaffolds immersed in vehicle at 2, 4, and 8 weeks (n = 5 per group; p ≤ 0.05, p ≤ 0.05, and p ≤ 0.01, respectively). Histological analysis showed increased bone formation and a trend toward increased remodeling in HA/β-TCP- dipyridamole and HA/β-TCP-BMP-2 scaffolds. Coating scaffolds with dipyridamole did not enhance bone regeneration in A2AKO mice. In conclusion, scaffolds printed with HA/β-TCP promote bone regeneration in critical bone defects and coating these scaffolds with agents that stimulate A2A receptors and growth factors can further enhance bone regeneration. These coated scaffolds may be very useful for treating critical bone defects due to trauma, infection or other causes.

Apremilast, a novel phosphodiesterase 4 (PDE4) inhibitor, regulates inflammation through multiple cAMP downstream effectors

IntroductionThis work was undertaken to delineate intracellular signaling pathways for the PDE4 inhibitor apremilast and to examine interactions between apremilast, methotrexate and adenosine A2A receptors (A2AR).MethodsAfter apremilast and LPS incubation, intracellular cAMP, TNF-α, IL-10, IL-6 and IL-1α were measured in the Raw264.7 monocytic murine cell line. PKA, Epac1/2 (signaling intermediates for cAMP) and A2AR knockdowns were performed by shRNA transfection and interactions with A2AR and A2BR, as well as with methotrexate were tested in vitro and in the murine air pouch model. Statistical differences were determined using one or two-way ANOVA or Student’s t test. The alpha nominal level was set at 0.05 in all cases. A P value of < 0.05 was considered significant.ResultsIn vitro, apremilast increased intracellular cAMP and inhibited TNF-α release (IC50=104nM) and the specific A2AR-agonist CGS21680 (1μM) increased apremilast potency (IC50=25nM). In this cell line, apremilast increased IL-10 production. PKA, Epac1 and Epac2 knockdowns prevented TNF-α inhibition and IL-10 stimulation by apremilast. In the murine air pouch model, both apremilast and MTX significantly inhibited leukocyte infiltration, while apremilast, but not MTX, significantly inhibited TNF-α release. The addition of MTX (1 mg/kg) to apremilast (5 mg/kg) yielded no more inhibition of leukocyte infiltration or TNF-α release than with apremilast alone.ConclusionsThe immunoregulatory effects of apremilast appear to be mediated by cAMP through the downstream effectors PKA, Epac1, and Epac2. A2AR agonism potentiated TNF-α inhibition by apremilast, consistent with the cAMP-elevating effects of that receptor. Because the A2AR is also involved in the anti-inflammatory effects of MTX, the mechanism of action of both drugs involves cAMP-dependent pathways and is therefore partially overlapping in nature.

Activation of EPAC1/2 is essential for osteoclast formation by modulating NFκB nuclear translocation and actin cytoskeleton rearrangements

Bisphosphonates inhibit osteoclast differentiation/function via inhibition of Rap1A isoprenylation. As Rap1 is the effector of exchange protein directly activated by cAMP (EPAC) proteins, we determined the role of EPAC in osteoclast differentiation. We examined osteoclast differentiation as the number of primary murine/human bone‐marrow precursors that differentiated into multinucleated TRAP‐positive cells in the presence of EPAC‐selective stimulus (8‐pCTP‐2‘‐O‐Me‐cAMP, 100 μM; 8‐pCTP‐2‘‐O‐Me‐cAMP‐AM, 1 μM) or inhibitor brefeldin A (BFA), ESI‐05, and ESI‐09 (10 μM each). Rap1 activity was assessed, and signaling events, as well as differentiation in EPAC1/2‐knockdown RAW264.7 cells, were studied. Direct EPAC1/2 stimulation significantly increased osteoclast differentiation, whereas EPAC1/2 inhibition diminished differentiation (113±6%, P<0.05, and 42±10%, P<0.001, of basal, respectively). Rap1 activation was maximal 15 min after RANKL stimulation (147±9% of basal, P< 0.001), whereas silencing of EPAC1/2 diminished activated Rap1 (43±13 and 20±15% of control, P<0.001) and NFκB nuclear translocation. TRAP‐staining revealed no osteoclast differentiation in EPAC1/2‐KO cells. Cathepsin K, NFATc1, and osteopontin mRNA expression decreased in EPAC1/2‐KO cells when compared to control. RhoA, cdc42, Rac1, and FAK were activated in an EPAC1/2‐dependent manner, and there was diminished cytoskeletal assembly in EPAC1/2‐KO cells. In summary, EPAC1 and EPAC2 are critical signaling intermediates in osteoclast differentiation that permit RANKL‐stimulated NFκB nuclear translocation and actin rearrangements. Targeting this signaling intermediate may diminish bone destruction in inflammatory arthritis.—Mediero, A., Perez‐Aso, M., Cronstein, B. N., Activation of EPAC1/2 is essential for osteoclast formation by modulating NFκB nuclear translocation and actin cytoskeleton rearrangements. FASEB J. 28, 4901–4913 (2014). www.fasebj.org

Netrin-1 Is a Critical Autocrine/Paracrine Factor for Osteoclast Differentiation

Bone metabolism is a vital process that involves resorption by osteoclasts and formation by osteoblasts, which is closely regulated by immune cells. The neuronal guidance protein Netrin‐1 regulates immune cell migration and inflammatory reactions, but its role in bone metabolism is unknown. During osteoclast differentiation, osteoclast precursors increase expression of Netrin‐1 and its receptor Unc5b. Netrin‐1 binds, in an autocrine and paracrine manner, to Unc5b to promote osteoclast differentiation in vitro, and absence of Netrin‐1 or antibody‐mediated blockade of Netrin‐1 or Unc5b prevents osteoclast differentiation of both murine and human precursors. We confirmed the functional relationship of Netrin‐1 in osteoclast differentiation in vivo using Netrin‐1‐deficient (Ntn1‐/‐) or wild‐type (WT) bone marrow transplanted mice. Notably, Ntn1‐/‐ chimeras have markedly diminished osteoclasts, as well as increased cortical and trabecular bone density and volume compared with WT mice. Mechanistic studies revealed that Netrin‐1 regulates osteoclast differentiation by altering cytoskeletal assembly. Netrin‐1 increases regulator of Rho‐GEF subfamily (LARG) and repulsive guidance molecule (RGMa) association with Unc5b, which increases expression and activation of cytoskeletal regulators RhoA and focal adhesion kinase (FAK). Netrin‐1 and its receptor Unc5b likely play a role in fusion of osteoclast precursors because Netrin‐1 and DC‐STAMP are tightly linked. These results identify Netrin‐1 as a key regulator of osteoclast differentiation that may be a new target for bone therapies.