Blocking chondrocyte hypertrophy in conditional Evc knockout mice does not modify cartilage damage in osteoarthritis.

Chondrocytes in osteoarthritic (OA) cartilage acquire a hypertrophic-like phenotype, where Hedgehog (Hh) signaling is pivotal. Hh overexpression causes OA-like cartilage lesions, whereas its downregulation prevents articular destruction in mouse models. Mutations in EVC and EVC2 genes disrupt Hh signaling, and are responsible for the Ellis-van Creveld syndrome skeletal dysplasia. Since Ellis-van Creveld syndrome protein (Evc) deletion is expected to hamper Hh target gene expression we hypothesized that it would also prevent OA progression avoiding chondrocyte hypertrophy. Our aim was to study Evc as a new therapeutic target in OA, and whether Evc deletion restrains chondrocyte hypertrophy and prevents joint damage in an Evc tamoxifen induced knockout (EvccKO ) model of OA. For this purpose, OA was induced by surgical knee destabilization in wild-type (WT) and EvccKO adult mice, and healthy WT mice were used as controls (n = 10 knees/group). Hypertrophic markers and Hh genes were measured by qRT-PCR, and metalloproteinases (MMP) levels assessed by western blot. Human OA chondrocytes and cartilage samples were obtained from patients undergoing knee joint replacement surgery. Cyclopamine (CPA) was used for Hh pharmacological inhibition and IL-1 beta as an inflammatory insult. Our results showed that tamoxifen induced inactivation of Evc inhibited Hh overexpression and partially prevented chondrocyte hypertrophy during OA, although it did not ameliorate cartilage damage in DMM-EvccKO mice. Hh pathway inhibition did not modify the expression of proinflammatory mediators induced by IL-1 beta in human OA chondrocytes in culture. We found that hypertrophic-IHH-and inflammatory-COX-2-markers co-localized in OA cartilage samples. We concluded that tamoxifen induced inactivation of Evc partially prevented chondrocyte hypertrophy in DMM-EvccKO mice, but it did not ameliorate cartilage damage. Overall, our results suggest that chondrocyte hypertrophy per se is not a pathogenic event in the progression of OA.

Energy Regulation in Inflammatory Sarcopenia by the Purinergic System.

The purinergic system has a dual role: the maintenance of energy balance and signaling within cells. Adenosine and adenosine triphosphate (ATP) are essential for maintaining these functions. Sarcopenia is characterized by alterations in the control of energy and signaling in favor of catabolic pathways. This review details the association between the purinergic system and muscle and adipose tissue homeostasis, discussing recent findings in the involvement of purinergic receptors in muscle wasting and advances in the use of the purinergic system as a novel therapeutic target in the management of sarcopenia.

Effects of Tofacitinib on Muscle Remodeling in Experimental Rheumatoid Sarcopenia.

Sarcopenia is a frequent comorbidity of rheumatoid arthritis (RA). Clinical trials have shown that JAK inhibitors (JAKi) produce an asymptomatic increase in serum creatine kinase (CK) in RA, suggesting an impact on muscle. We evaluated the effect of JAKi in muscle remodeling in an experimental RA model. Antigen-induced arthritis (experimental RA, e-RA) was performed in 14 rabbits. Seven rabbits received tofacitinib (TOFA, orally 10 mg/kg/day). Animals were euthanized one day after the last ovalbumin injection, and muscles were prepared for histology, RT-PCR, and WB. C-reactive protein (CRP) and Myostatin (MSTN) serum concentration were determined by ELISA. Creatine and creatine kinase (CK) were analyzed. An increase in body weight as well as tibialis anterior cross-sectional area and diameter was observed in e-RA+TOFA vs. e-RA. e-RA decreased type II fibers and increased the myonuclei number, with all reverted by TOFA. TOFA did not modify CRP levels, neither did MSTN. TOFA significantly reduced IL-6, atrogin-1, and MuRF-1 compared with e-RA. e-RA+TOFA showed higher CK and lower creatine levels compared with e-RA. No differences in PAX-7 were found, while TOFA prevented the increase in MyoD1 in e-RA. Our model reflects the features of rheumatoid sarcopenia in RA. JAKi increased muscle mass through attenuating IL-6/JAK/STAT activation, decreasing atrogenes, and restoring muscle differentiation markers. These data together with an increase in CK support the role of CK as a valuable marker of muscle gain following JAKi treatment.

ATP transporters in the joints.

Extracellular adenosine triphosphate (ATP) plays a central role in a wide variety of joint diseases. ATP is generated intracellularly, and the concentration of the extracellular ATP pool is determined by the regulation of its transport out of the cell. A variety of ATP transporters have been described, with connexins and pannexins the most commonly cited. Both form intercellular channels, known as gap junctions, that facilitate the transport of various small molecules between cells and mediate cell-cell communication. Connexins and pannexins also form pores, or hemichannels, that are permeable to certain molecules, including ATP. All joint tissues express one or more connexins and pannexins, and their expression is altered in some pathological conditions, such as osteoarthritis (OA) and rheumatoid arthritis (RA), indicating that they may be involved in the onset and progression of these pathologies. The aging of the global population, along with increases in the prevalence of obesity and metabolic dysfunction, is associated with a rising frequency of joint diseases along with the increased costs and burden of related illness. The modulation of connexins and pannexins represents an attractive therapeutic target in joint disease, but their complex regulation, their combination of gap-junction-dependent and -independent functions, and their interplay between gap junction and hemichannel formation are not yet fully elucidated. In this review, we try to shed light on the regulation of these proteins and their roles in ATP transport to the extracellular space in the context of joint disease, and specifically OA and RA.

Increased risk factors associated with lower BMD in antiretroviral-therapy-naïve HIV-infected adult male.

BACKGROUND: Low BMD (bone mineral density) has been described as a non-AIDS (Acquired Immune Deficiency Syndrome)-related event in HIV (human immunodeficiency virus)-patients but it is poorly studied in young HIV-infected men who have received no previous antiretroviral therapy. METHODS: A cross-sectional study of 245 naïve-HIV-infected men over 21 and under 50 years old who voluntary attended the Infectious Disease Division appointment in Hospital Fundación Jimenez Díaz in Madrid, from January 1st, 2014 to September 30th, 2017. All subjects underwent a baseline DXA scan (dual energy x-ray absorptiometry) performed prior to start antiretroviral treatment. Further, all patients who started treatment between May 1st and September 30th, 2017 were invited to participate in a substudy on bone mineral metabolism. All the information was collected through clinical history and complementary questionnaire. RESULTS: The mean age was 36.4 years, been 68% Caucasian, 29.3% Latin American and 2.7% African race. At the time of diagnosis, 91% of patients had stage-A (median CD4+ T-cell 481cells/µL, IQR, 320-659). 10% had a count below 200 CD4 cells/µL, and 40% had a CD4/CD8 cell-count-ratio below 0.4. Regarding lifestyle and risk factors, 14.1% presented underweight, 36.1% were not engage in any regular exercise, 51.9% were active smokers and 35.3% reported drug use. Low levels of vitamin D were seen in 87.6% of the study participants. Low BMD (Z-score <- 2.0) was found in 22.8% of the patients. It was only observed a significant association of Z-score in lumbar spine (LS) with CD8 and the CD4/CD8 ratio, and with alcohol for femoral neck (FN) measurement. CONCLUSIONS: We find prevalence of increased bone involvement among naïve HIV-infected men under 50 years old. Further studies are necessary to evaluate if changes in actual guidelines are needed to assess BMD measurements in HIV-infected adult male patients under 50.

Tenofovir Modulates Semaphorin 4D Signaling and Regulates Bone Homeostasis, Which Can Be Counteracted by Dipyridamole and Adenosine A2A Receptor.

Semaphorin 4D (Sema4D) is a neurotrophin that is secreted by osteoclasts and binds to its receptor PlexinB1 on osteoblasts to inhibit their differentiation and function. Adenosine A2A activation inhibits osteoclast Sema4D-mediated secretion, diminishes inflammatory osteolysis and prevents bone loss following tenofovir (one of the most used antivirals in HIV). Therefore, tenofovir might activate Sema4D signaling to alter bone turnover. Female C57Bl/6/A2AKO mice were ovariectomized and treated with saline (control), tenofovir 75 mg/Kg/day, dipyridamole 25 mg/Kg/day or a combination for 5 weeks and long bones were prepared for histology. Primary murine-induced osteoclast/osteoblast were challenged with tenofovir/dipyridamole 1 µM each, and the expression of Sema4D/PlexinB1, RhoA/ROCK/IGF1R was studied by RT-PCR, Western blot and immunostaining. In vivo tenofovir showed an increased expression of Sema4D when compared to control mice, and dipyridamole reverted the expression in an A2A-dependent manner. In vitro, tenofovir increases Sema4D expression and secretion in osteoclast precursors, and pre-treatment with dipyridamole reverted this effect. pRhoA and ROCK1 activation were increased and IRS1/IGF1R expression was diminished by tenofovir in the Vav3/ARHGAP18 mechanism in osteoblast precursors and reverted by dipyridamole in an A2A-dependent manner. This suggests that tenofovir increases bone loss by activation of Sema4D/PlexinB1 signaling, which inhibits osteoblast differentiation. Agents that increase local adenosine concentrations, such as dipyridamole, might prevent bone loss following the inhibition of this pathway.

Adenosine Deaminase as a Biomarker of Tenofovir Mediated Inflammation in Naïve HIV Patients.

Plasma levels of adenosine deaminase (ADA), an enzyme that deaminates adenosine to inosine, are increased during inflammation. An increase in ADA activity occurs with lower human immunodeficiency virus (HIV) viral load and higher CD4+ T cell counts. We aimed to investigate the role of plasma ADA as a biomarker of inflammation in treatment-naïve HIV patients who received tenofovir or another nucleoside analog for comparison. Ninety-two treatment-naïve patients were included in the study and grouped by treatment, i.e., tenofovir disoproxil fumarate (TDF), tenofovir alafenamide (TAF) or Triumeq. ADA activity was measured in plasma and cytokines were analyzed by MILLIPLEX® MAP-Luminex® Technology. Plasma concentration of monocytes and neutrophils was measured at 0, 3, and 12 months post-treatment. Treatment-naïve HIV patients had increased ADA concentrations (over 15 U/L) that decreased after treatment with TAF and Triumeq, though this did not occur in TDF-treated patients. However, all groups exhibited a pro-inflammatory systemic profile at 12 months of treatment. Plasma GM-CSF levels decreased after 12 months of treatment in the TDF group, with a concomitant decrease in blood monocyte count, and a negative correlation with ADA values was found. In conclusion, ADA levels may be modulated by antiretroviral therapy in HIV patients, possibly affecting inflammatory status.

Microbiological and Cellular Evaluation of a Fluorine-Phosphorus-Doped Titanium Alloy, a Novel Antibacterial and Osteostimulatory Biomaterial with Potential Applications in Orthopedic Surgery.

Joint prosthesis failure is mainly related to aseptic loosening and prosthetic joint infections, both of which are associated with high morbidity and substantial costs for patients and health systems. The development of a biomaterial that is capable of stimulating bone growth while minimizing bacterial adhesion would reduce the incidence of prosthetic failure. We report antibacterial and osteostimulatory effects in a novel fluorine-phosphorus (F-P)-doped TiO2 oxide film grown on Ti-6Al-4V alloy with a nanostructure of bottle-shaped nanotubes (bNT) using five bacterial species (Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, Pseudomonas aeruginosa, and Stenotrophomonas maltophilia) and MCT3T3-E1 osteoblastic cells. The interaction between the bacteria and bNT Ti-6Al-4V was complex, as the adhesion of four bacterial species decreased (two staphylococcus species, E. coli, and S. maltophilia), and the viability of staphylococci and S. maltophilia also decreased because of the aluminum (Al) released by bNT Ti-6Al-4V. This released Al can be recruited by the bacteria through siderophores and was retained only by the Gram-negative bacteria tested. P. aeruginosa showed higher adhesion on bNT Ti-6Al-4V than on chemically polished (CP) samples of Ti-6Al-4V alloy and an ability to mobilize Al from bNT Ti-6Al-4V. The cell adhesion and proliferation of MCT3T3-E1 osteoblastic cells significantly increased at 48 and 168 h, as did the matrix mineralization of these cells and the gene expression levels of three of the most important markers related to bone differentiation. According to our results, the bNT Ti-6Al-4V alloy could have clinical application, preventing infection and stimulating bone growth and thus preventing the two main causes of joint prosthesis failure.IMPORTANCE This work evaluates F-P-doped bNT Ti-6Al-4V from microbiological and cellular approaches. The bacterial results highlight that the antibacterial ability of bNT Ti-6Al-4V is the result of a combination of antiadhesive and bactericidal effects exerted by Al released from the alloy. The cell results highlight that F-P bNT Ti-6Al-4V alloy increases osseointegration due to modification of the chemical composition of the alloy resulting from P incorporation and not due to the nanostructure, as reported previously. A key finding was the detection of Al release from inside the bNT Ti-6Al-4V nanostructures, a result of the nanostructure growth during the anodizing process that is in part responsible for its bactericidal effect.

Adenosine A2A receptor (A2AR) stimulation modulates expression of semaphorins 4D and 3A, regulators of bone homeostasis.

The axonal guidance proteins semaphorin (Sema)4D and Sema3A play important roles in communication between osteoclasts and osteoblasts. As stimulation of adenosine A2A receptors (A2AR) regulates both osteoclast and osteoblast function, we asked whether A2AR regulates both osteoclast and osteoblast expression of Semas. In vivo bone formation and Sema3A/PlexinA1/Neuropilin-1, Sema4D/PlexinB1 protein expression were studied in a murine model of wear particle-induced osteolysis. Osteoclast/osteoblast differentiation were studied in vitro as the number of tartrate-resistant acid phosphatase+/Alizarin Red+ cells after challenge with CGS21680 (A2AR agonist, 1 µM) or ZM241385 (A2AR antagonist, 1 µM), with or without Sema4D or Sema3A (10 ng/ml). Sema3A/PlexinA1/Neuropilin-1, Sema4D/PlexinB1, and receptor activator of NF-κB ligand/osteoprotegerin (RANKL/OPG) expression was studied by RT-PCR and Western blot. ß-Catenin activation and cytoskeleton changes were studied by fluorescence microscopy and Western blot. In mice with wear particles implanted over the calvaria, CGS21680 treatment increased bone formation in vivo, reduced Sema4D, and increased Sema3A expression compared with mice with wear particle-induced osteolysis treated with vehicle alone. During osteoclast differentiation, CGS21680 abrogated RANKL-induced Sema4D mRNA expression (1.3 ± 0.3- vs. 2.5 ± 0.1-fold change, P < 0.001, n = 4). PlexinA1, but not Neuropilin-1, mRNA was enhanced by CGS21680 treatment. CGS21680 enhanced Sema3A mRNA expression during osteoblast differentiation (8.7 ± 0.2-fold increase, P < 0.001, n = 4); PlexinB1 mRNA was increased 2-fold during osteoblast differentiation and was not altered by CGS21680. Similar changes were observed at the protein level. CGS21680 decreased RANKL, increased OPG, and increased total/nuclear ß-catenin expression in osteoblasts. Sema4D increased Ras homolog gene family, member A phosphorylation and focal adhesion kinase activation in osteoclast precursors, and CGS21680 abrogated these effects. In summary, A2AR activation diminishes secretion of Sema4D by osteoclasts, inhibits Sema4D-mediated osteoclast activation, and enhances secretion of Sema3A by osteoblasts, increasing osteoblast differentiation and diminishing inflammatory osteolysis.-Mediero, A., Wilder, T., Shah, L., Cronstein, B. N. Adenosine A2A receptor (A2AR) stimulation modulates expression of semaphorins 4D and 3A, regulators of bone homeostasis.

Adenosine A2A receptor plays an important role in radiation-induced dermal injury.

Ionizing radiation is a common therapeutic modality and following irradiation dermal changes, including fibrosis and atrophy, may lead to permanent changes. We have previously demonstrated that occupancy of A2A receptor (A2AR) stimulates collagen production, so we determined whether blockade or deletion of A2AR could prevent radiation-induced fibrosis. After targeted irradiation (40 Gy) of the skin of wild-type (WT) or A2AR knockout (A2ARKO) mice, the A2AR antagonist ZM241385 was applied daily for 28 d. In irradiated WT mice treated with the A2AR antagonist, there was a marked reduction in collagen content and skin thickness, and ZM241385 treatment reduced the number of myofibroblasts and angiogenesis. After irradiation, there is an increase in loosely packed collagen fibrils, which is significantly diminished by ZM241385. Irradiation also induced an increase in epidermal thickness, prevented by ZM241385, by increasing the number of proliferating keratinocytes. Similarly, in A2ARKO mice, the changes in collagen alignment, skin thickness, myofibroblast content, angiogenesis, and epidermal hyperplasia were markedly reduced following irradiation. Radiation-induced changes in the dermis and epidermis were accompanied by an infiltrate of T cells, which was prevented in both ZM241385-treated and A2ARKO mice. Radiation therapy is administered to a significant number of patients with cancer, and radiation reactions may limit this therapeutic modality. Our findings suggest that topical application of an A2AR antagonist prevents radiation dermatitis and may be useful in the prevention or amelioration of radiation changes in the skin.

Netrin-1 and its receptor Unc5b are novel targets for the treatment of inflammatory arthritis.

Rheumatoid arthritis is an autoimmune disease that is characterized by chronic inflammation and destruction of joints. Netrin-1, a chemorepulsant, laminin-like matrix protein, promotes inflammation by preventing macrophage egress from inflamed sites and is required for osteoclast differentiation. We asked whether blockade of Netrin-1 or its receptors [Unc5b and DCC (deleted in colorectal carcinoma)] may be useful therapeutic targets for treatment of inflammatory arthritis. Arthritis was induced in 8-wk-old C57Bl/6 mice by intraperitoneal injection of K/BxN serum. Murine monoclonal antibodies against Netrin-1, Unc5b, or DCC (10 µg/mouse) were injected weekly for 4 wk (n = 10). Paw swelling and thickness were assessed and following euthanasia 2-4 wk after serum transfer, paws were prepared for micro-computed tomography and histology. Paw inflammation was maximal 2 wk after injection. Anti-Netrin-1 or anti-Unc5b, but not anti-DCC, antibodies significantly reduced paw inflammation (clinical score: 9.8 ± 0.8, 10.4 ± 0.9, and 13.5 ± 0.5, respectively vs 16 ± 0 for control; P < 0.001). Micro-computed tomography showed bony erosions in untreated or anti-DCC-treated mice, whereas there were no erosions in anti-Netrin-1/anti-Unc5b-treated-animals. Tartrate-resistant acid phosphatase staining demonstrated a marked decrease in osteoclasts in anti-Netrin-1/anti-Unc5b-treated animals. Immunofluorescence staining revealed a decrease in cathepsin K+ and CD68+ cells in anti-Netrin-1/anti-Unc5b-treated animals. Blockade of Netrin-1/Unc5b by monoclonal antibodies prevents bone destruction and reduces the severity of K/BxN serum transfer-induced arthritis. Netrin-1 may be a novel therapeutic target for treatment of inflammatory bone destruction.-Mediero, A., Wilder, T., Ramkhelawon, B., Moore, K. J., Cronstein, B. N. Netrin-1 and its receptor Unc5b are novel targets for the treatment of inflammatory arthritis.

Ticagrelor regulates osteoblast and osteoclast function and promotes bone formation in vivo via an adenosine-dependent mechanism.

As many as 10% of bone fractures heal poorly, and large bone defects resulting from trauma, tumor, or infection may not heal without surgical intervention. Activation of adenosine A2A receptors (A2ARs) stimulates bone formation. Ticagrelor and dipyridamole inhibit platelet function by inhibiting P2Y12 receptors and platelet phosphodiesterase, respectively, but share the capacity to inhibit cellular uptake of adenosine and thereby increase extracellular adenosine levels. Because dipyridamole promotes bone regeneration by an A2AR-mediated mechanism we determined whether ticagrelor could regulate the cells involved in bone homeostasis and regeneration in a murine model and whether inhibition of P2Y12 or indirect A2AR activation via adenosine was involved. Ticagrelor, dipyridamole and the active metabolite of clopidogrel (CAM), an alternative P2Y12 antagonist, inhibited osteoclast differentiation and promoted osteoblast differentiation in vitro. A2AR blockade abrogated the effects of ticagrelor and dipyridamole on osteoclast and osteoblast differentiation whereas A2BR blockade abrogated the effects of CAM. Ticagrelor and CAM, when applied to a 3-dimentional printed resorbable calcium-triphosphate/hydroxyapatite scaffold implanted in a calvarial bone defect, promoted significantly more bone regeneration than the scaffold alone and as much bone regeneration as BMP-2, a growth factor currently used to promote bone regeneration. These results suggest novel approaches to targeting adenosine receptors in the promotion of bone regeneration.-Mediero, A., Wilder, T., Reddy, V. S. R., Cheng, Q., Tovar, N., Coelho, P. G., Witek, L., Whatling, C., Cronstein, B. N. Ticagrelor regulates osteoblast and osteoclast function and promotes bone formation in vivo via an adenosine-dependent mechanism.

Netrin-1 is highly expressed and required in inflammatory infiltrates in wear particle-induced osteolysis.

OBJECTIVE: Netrin-1 is a chemorepulsant and matrix protein expressed during and required for osteoclast differentiation, which also plays a role in inflammation by preventing macrophage egress. Because wear particle-induced osteolysis requires osteoclast-mediated destruction of bone, we hypothesised that blockade of Netrin-1 or Unc5b, a receptor for Netrin-1, may diminish this pathological condition. METHODS: C57BL/6 mice, 6-8 weeks old, had 3 mg of ultrahigh-molecular-weight polyethylene particles implanted over the calvaria and then received 10 µg of monoclonal antibodies for Netrin-1 or its receptors, Unc5b and deleted in colon cancer (DCC), injected intraperitoneally on a weekly basis. After 2 weeks, micro-computed tomography and histology analysis were performed. Netrin-1 expression was analysed in human tissue obtained following primary prosthesis implantation or after prosthesis revision for peri-implant osteolysis and aseptic implant loosening. RESULTS: Weekly injection of anti-Netrin-1 or anti-Unc5b-antibodies significantly reduced particle-induced bone pitting in calvaria exposed to wear particles (46±4% and 49±3% of control bone pitting, respectively, p<0.001) but anti-DCC antibody did not affect inflammatory osteolysis (80±7% of control bone pitting, p=ns). Anti-Netrin-1 or anti-Unc5b, but not anti-DCC, antibody treatment markedly reduced the inflammatory infiltrate and the number of tartrate resistance acid phosphatase (TRAP)-positive osteoclasts (7±1, 4±1 and 14±1 cells/high power field (hpf), respectively, vs 12±1 cells/hpf for control, p<0.001), with no significant changes in alkaline phosphatase-positive osteoblasts on bone-forming surfaces in any antibody-treated group. Netrin-1 immunostaining colocalised with CD68 staining for macrophages. The peri-implant tissues of patients undergoing prosthesis revision surgery showed an increase in Netrin-1 expression, whereas there was little Netrin-1 expression in soft tissues removed at the time of primary joint replacement. CONCLUSIONS: These results demonstrate a unique role for Netrin-1 in osteoclast biology and inflammation and may be a novel target for prevention/treatment of inflammatory osteolysis.

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 A2A 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.

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 NFkB 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 NFkB nuclear translocation and actin rearrangements. Targeting this signaling intermediate may diminish bone destruction in inflammatory arthritis.

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 TGFß1-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. TGFß1 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.

Adenosine A(2A) 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-1ß 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 A2A receptor (A2AR) is a fine-tune regulator of the collagen1:collagen3 balance.

Adenosine is a potent endogenous anti-inflammatory and immunosuppressive metabolite that is a potent modulator of tissue repair. However, the adenosine A2A receptor (A2AR)-mediated promotion of collagen synthesis is detrimental in settings such as scarring and scleroderma. The signaling cascade from A2AR stimulation to increased collagen production is complex and obscure, not least because cAMP and its downstream molecules PKA and Epac1 have been reported to inhibit collagen production. We therefore examined A2AR-stimulated signaling for collagen production by normal human dermal fibroblasts (NHDF). Collagen1 (Col1) and collagen3 (Col3) content after A2AR activation by CGS21680 was studied by western blotting. Contribution of PKA and Epac was analyzed by the PKA inhibitor PKI and by knockdowns of the PKA-Cα, -Cß, -Cγ, Epac1, and Epac2. CGS21680 stimulates Col1 expression at significantly lower concentrations than those required to stimulate Col3 expression. A2AR stimulates Col1 expression by a PKA-dependent mechanism since PKA inhibition or PKA-Cα and -Cß knockdown prevents A2AR-mediated Col1 increase. In contrast, A2AR represses Col3 via PKA but stimulates both Col1 and Col3 via an Epac2-dependent mechanism. A2AR stimulation with CGS21680 at 0.1 µM increased Col3 expression only upon PKA blockade. A2AR activation downstream signaling for Col1 and Col3 expression proceeds via two distinct pathways with varying sensitivity to cAMP activation; more highly cAMP-sensitive PKA activation stimulates Col1 expression, and less cAMP-sensitive Epac activation promotes both Col1 and Col3 expression. These observations may explain the dramatic change in Col1:Col3 ratio in hypertrophic and immature scars, where adenosine is present in higher concentrations than in normal skin.

The Antimicrobial Activity of Micron-Thin Sol-Gel Films Loaded with Linezolid and Cefoxitin for Local Prevention of Orthopedic Prosthesis-Related Infections.

Orthopedic prosthesis-related infections (OPRI) are an essential health concern. OPRI prevention is a priority and a preferred option over dealing with poor prognosis and high-cost treatments. Micron-thin sol-gel films have been noted for a continuous and effective local delivery system. This study aimed to perform a comprehensive in vitro evaluation of a novel hybrid organic-inorganic sol-gel coating developed from a mixture of organopolysiloxanes and organophosphite and loaded with different concentrations of linezolid and/or cefoxitin. The kinetics of degradation and antibiotics release from the coatings were measured. The inhibition of biofilm formation of the coatings against Staphylococcus aureus, S. epidermidis, and Escherichia coli strains was studied, as well as the cell viability and proliferation of MC3T3-E1 osteoblasts. The microbiological assays demonstrated that sol-gel coatings inhibited the biofilm formation of the evaluated Staphylococcus species; however, no inhibition of the E. coli strain was achieved. A synergistic effect of the coating loaded with both antibiotics was observed against S. aureus. The cell studies showed that the sol-gels did not compromise cell viability and proliferation. In conclusion, these coatings represent an innovative therapeutic strategy with potential clinical use to prevent staphylococcal OPRI.

MSC therapy ameliorates experimental gouty arthritis hinting an early COX-2 induction.

Objective: The specific effect of Adipose-Derived Mesenchymal Stem Cells (Ad-MSC) on acute joint inflammation, where the response mostly depends on innate immunity activation, remains elusive. The pathogenesis of gouty arthritis, characterized by the deposition of monosodium urate (MSU) crystals in the joints, associated to acute flares, has been associated to NLRP3 inflammasome activation and subsequent amplification of the inflammatory response. Our aim was to study the effect of human Ad-MSC administration in the clinical inflammatory response of rabbits after MSU injection, and the molecular mechanisms involved. Methods: Ad-MSC were administered by intraarterial route shortly after intraarticular MSU crystal injections. Joint and systemic inflammation was sequentially studied, and the mechanisms involved in NLRP3 inflammasome activation, and the synthesis of inflammatory mediators were assessed in the synovial membranes 72h after insult. Ad-MSC and THP-1-derived macrophages stimulated with MSU were co-cultured in transwell system. Results: A single systemic dose of Ad-MSC accelerated the resolution of local and systemic inflammatory response. In the synovial membrane, Ad-MSC promoted alternatively M2 macrophage presence, inhibiting NLRP3 inflammasome and inducing the production of anti-inflammatory cytokines, such as IL-10 or TGF-ß, and decreasing nuclear factor-κB activity. Ad-MSC induced a net anti-inflammatory balance in MSU-stimulated THP-1 cells, with a higher increase in IL-10 and IDO expression than that observed for IL-1ß and TNF. Conclusion: Our in vivo and in vitro results showed that a single systemic dose of Ad-MSC decrease the intensity and duration of the inflammatory response by an early local COX-2 upregulation and PGE2 release. Ad-MSCs suppressed NF-kB activity, NLRP3 inflammasome, and promoted the presence of M2 alternative macrophages in the synovium. Therefore, this therapeutic approach could be considered as a pharmacological alternative in patients with comorbidities that preclude conventional treatment.