Adenosine A2A receptor activation reduces chondrocyte senescence.

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.

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

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.

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.

Modulation of the Inflammatory Process by Hypercholesterolemia in Osteoarthritis.

Objective: Several studies have linked metabolic syndrome to the development of osteoarthritis (OA) through hypercholesterolemia, one of its components. However, epidemiological studies showed contradictory results, and it is not clear how hypercholesterolemia itself, or oxidized LDL (oxLDL)-a pathological molecule potentially involved in this relationship-could be affecting OA. The objectives of this study were to investigate the effect of hypercholesterolemia induced by high-fat diet (HFD) in cartilage from OA rabbits, and how oxLDL affect human chondrocyte inflammatory and catabolic responses. Design: New Zealand rabbits were fed with HFD for 18 weeks. On week 6, OA was surgically induced. At the end of the study, cartilage damage and IL-1ß, IL-6, MCP-1, MMP-13, and COX-2 expression in articular cartilage were evaluated. In addition, cultured human OA articular chondrocytes were treated with oxLDL at concentrations equivalent to those expected in synovial fluid from HFD rabbits, in the presence of IL-1ß and TNFα. The effect of oxLDL on cell viability, nitric oxide production and catabolic and pro-inflammatory gene expression was evaluated. Results: HFD intake did not modify cartilage structure or pro-inflammatory and catabolic gene expression and protein presence, both in healthy and OA animals. OxLDL did not affect human chondrocyte viability, ADAMTS5 and liver X receptor (LXR) α gene expression, but decreased the induction of IL-1ß, IL-6, MCP-1, MMP-13, iNOS, and COX-2 gene expression and MMP-13 and COX-2 protein presence, evoked by cytokines. Conclusions: Our data suggest that cholesterol intake per se may not be deleterious for articular cartilage. Instead, cholesterol de novo synthesis and altered cholesterol metabolism could be involved in the associations observed in human disease.

Adenosine A2A receptor (A2AR) activation triggers Akt signaling and enhances nuclear localization of ß-catenin in osteoblasts.

Osteoblast differentiation and proliferation are regulated by several modulators, among which are adenosine A2A receptors (A2ARs) and Wingless/Integrated-ß-catenin pathways. Cytosolic ß-catenin stabilization promotes its nuclear translocation and transcriptional activity. In the present study, we seek to determine whether there is a connection between A2AR stimulation and cellular ß-catenin levels in osteoblasts. Osteoblast precursor cell line (MC3T3-E1) and primary murine osteoblasts were treated with CGS21680, a highly selective A2AR agonist. We analyzed cellular content and nuclear translocation of phosphorylated (p)-serine 552 (S552) ß-catenin in response to A2AR stimulation in MC3T3-E1 cells, in both wild-type and A2AR knockout (A2AKO) mice. Moreover, we measured cellular ß-catenin levels in MC3T3-E1 cells transfected with scrambled or protein kinase B (Akt) small interfering RNA following A2AR activation. CGS21680 (1 µM) stimulated an increase in both the cellular content and nuclear translocation of p-S552 ß-catenin after 15 min of incubation. A2AR activation had no tangible effect on the cellular ß-catenin level either in A2AKO mice or in osteoblasts with diminished Akt content. Our findings demonstrate an interaction between A2AR, ß-catenin, and Akt signaling in osteoblasts. The existence of such a crosstalk has significant repercussions in the development of novel therapeutic approaches targeting medical conditions associated with reduced bone density.-Borhani, S., Corciulo, C., Larranaga-Vera, A., Cronstein, B. N. Adenosine A2A receptor (A2AR) activation triggers Akt signaling and enhances nuclear localization of ß-catenin in osteoblasts.

Tenofovir Causes Bone Loss via Decreased Bone Formation and Increased Bone Resorption, Which Can Be Counteracted by Dipyridamole in Mice.

Osteopenia and fragility fractures have been associated with human immunodeficiency virus (HIV) infection. Tenofovir, a common antiviral in HIV treatment, also leads to increases in bone catabolism markers and decreased BMD in children and young adults. In murine models and human cell lines, tenofovir inhibits adenosine triphosphate release and decreases extracellular adenosine levels. Adenosine and adenosine A2A receptor inhibit osteoclast formation, and increase local adenosine concentration with dipyridamole, an agent that blocks adenosine cellular uptake and stimulates new bone formation as well as bone morphogenic protein 2. We hypothesized that tenofovir regulates bone resorption by diminishing endogenous adenosine levels and questioned whether dipyridamole may be a useful treatment to counteract the deleterous bone effects of tenofovir. Primary murine osteoclasts were induced by M-CSF/RANKL, and the number of TRAP-positive-cells was studied after challenge with tenofovir alone or in combination with dipyridamole. Differentiation markers were studied by RT-PCR and MAPK/NFkB expression by Western blot. Male C57Bl/6 mice were treated as follows: saline 0.9% (control), tenofovir 75 mg/kg/day, dipyridamole 25 mg/kg/day, combination tenofovir/dipyridamole (n = 10, 4 weeks). Calcein/Alizarin Red-labeling of newly formed bone was used, and long bones were prepared for micro-computed tomography (µCT)/histology. Tenofovir produced a dose-dependent increase in osteoclast differentiation (EC50 = 44.5nM) that was reversed by dipyridamole (IC50 = 0.3 µM). Tenofovir increased cathepsin K and NFATc1 mRNA levels and dipyridamole reversed the effect. Dipyridamole reversed the effect of tenofovir on pERK1/2, pp38, and NFkB nuclear translocation. Mice treated with tenofovir lost nearly 10% of their body weight (p < 0.001). µCT revealed decreased BMD and altered trabecular bone in tenofovir-treated mice, reversed by dipyridamole. 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 osteoprotegerin-positive cells were decreased; both effects were reversed by dipyridamole. These results suggest that treatment with agents that increase local adenosine concentrations, like dipyridamole, might prevent bone loss following tenofovir treatment. © 2019 American Society for Bone and Mineral Research.

Increased synovial lipodystrophy induced by high fat diet aggravates synovitis in experimental osteoarthritis.

BACKGROUND: Metabolic syndrome (MetS) may be associated with knee osteoarthritis (OA), but the association between the individual components and OA are not well-understood. We aimed to study the effect of hypercholesterolemia on synovial inflammation in knee OA. METHODS: OA was surgically induced in rabbits fed with standard diet (OA group, n = 10) or in rabbits fed with high fat diet (OA-HFD, n = 10). Healthy rabbits receiving standard diet (Control, n = 10) or fed with HFD (HFD, n = 6) were also monitored. Twelve weeks after OA induction, synovial membranes were isolated and processed for studies. RESULTS: Animals fed HFD showed higher levels of total serum cholesterol, triglycerides and C-reactive protein than control rabbits. Twelve weeks after OA induction, synovial membrane inflammation and macrophage infiltration were increased in rabbits with OA, particularly in the OA-HFD group. Extensive decrease of synovial adipose tissue area, adipocyte size and perilipin-1A synthesis were observed in the OA-HFD group in comparison to the OA and control groups. The HFD further increased the proinflammatory mediators IL-1ß, IL-6 and TNF in the OA synovium. However, the synovial gene expression of adipokines, such as leptin and adiponectin, were markedly decreased in the rabbits with OA, especially in the OA-HFD group, in correlation with adipose tissue loss. However, circulating leptin was upregulated in the HFD and OA-HFD groups. CONCLUSION: Our results indicate that a HFD is an aggravating factor worsening synovial membrane inflammation during OA, guided by increased infiltration of macrophages and removal of the adipose tissue, together with a remarkable presence of proinflammatory factors. Synovial adipocytes and dyslipemia could probably play pivotal roles in OA joint deterioration in patients with MetS, supporting that the link between obesity and OA transcends mechanical loading.

The increase in O-linked N-acetylglucosamine protein modification stimulates chondrogenic differentiation both in vitro and in vivo.

Insulin is an inducer of chondrocyte hypertrophy and growth plate chondrogenesis, although the specific molecular mechanisms behind these effects are mostly unknown. Our aim was to investigate whether insulin-induced chondrocyte hypertrophy occurs through a modification in the amount of O-linked N-acetylglucosamine (O-GlcNAc)-modified proteins and in the expression of the key enzymes of this pathway, O-GlcNAc transferase and O-GlcNAcase (OGA). We also studied if O-GlcNAc accumulation per se, induced by an OGA inhibitor, was able to induce pre-hypertrophic chondrocyte differentiation both in vitro and in vivo. Insulin-induced differentiation of ATDC5 pre-chondrocytes occurred alongside a gradual increase in the accumulation of O-GlcNac-modified proteins (O-GlcNAcylated proteins), as well as an increase in the expression of O-GlcNAc transferase and OGA. In the absence of insulin, O-GlcNAc accumulation induced by thiamet-G, a specific OGA inhibitor, was able to increase the gene expression of differentiation markers, as well as the activity of MMP-2 and -9. Thiamet-G also activated pERK, p-JNK, and p-p38 and the O-GlcNAcylation of Akt. Thiamet-G administration to C57/bl mice induced a significant expansion in the growth plate height and in the hypertrophic zone height. Therefore, our results show that O-GlcNAc glycosylation has chondromodulating activity.