Faculty Bibliography

The joint is a complex anatomical structure consisting of different tissues, each with a particular feature, playing together to give mobility and stability at the body. All the joints have a similar composition including cartilage for reducing the friction of the movement and protecting the underlying bone, a synovial membrane that produces synovial fluid to lubricate the joint, ligaments to limit joint movement, and tendons for the interaction with muscles. Direct or indirect damage of one or more of the tissues forming the joint is the foundation of different pathological conditions. Many molecular mechanisms are involved in maintaining the joint homeostasis as well as in triggering disease development. The molecular pathway activated by the purinergic system is one of them.The purinergic signaling defines a group of receptors and intermembrane channels activated by adenosine, adenosine diphosphate, adenosine 5'-triphosphate, uridine triphosphate, and uridine diphosphate. It has been largely described as a modulator of many physiological and pathological conditions including rheumatic diseases. Here we will give an overview of the purinergic system in the joint describing its expression and function in the synovium, cartilage, ligament, tendon, and bone with a therapeutic perspective.

Introduction: Tenofovir is anti-retroviral agent prescribed to human immunodeficiency virus (HIV) patients as part of the drug regimen known as highly active anti-retroviral therapy (HAART). Tenofovir also causes osteopenia resulting in pathological fractures and hospitalisation in these patients. This is now increasingly significant since tenofovir is one of the components of HIV pre-exposure prophylaxis, wich is available for the population at risk. Currently there are not many studies in this regard but several authors has reported that this population also suffers a decrease in BMD. A recently published study suggests that this is caused by a reduction of extracellular adenosine produced by Tenofovir mediated blockage of Pannexin-1 ATP transporter and this effect can be reversed by dipyridamole that blocks adenosine re-uptake. To further confirm this study Pannexin-1 KO (PANX1KO) mice were studied and compared to C57BL/6J (WT) mice.
Method(s): PANX1KO animals were treated daily with 75mg/Kg of tenofovir, 25 mg/kg tenofovir or both during 4 weeks, after that bone mineral density (BMD) was measured. Additionally primary osteoclasts were differentiated and treated with different concentrations of Tenofovir and dipyridamole. The differentiation stage and extracellular ATP levels were studied.
Result(s): In WT mice Tenofovir caused a reduction bone mineral density and this was reversed in the animals who received both drugs. However PANX1KO mice that receive Tenofovir did not present a lower BMD. In WT mice Dipyridamole inhibited osteoclast differentiation (p= 0.0068). The dipyridamole induced inhibition was reverted with Tenofovir in a dose dependent manner (0.0055). PANX1KO mice osteoclast differentiation was also inhibited by dipyridamole (p=0.0005), however Tenofovir was unable to revert dipyridamole induced inhibition (p= 0.9756). Additionally, opposed to the WT osteoclasts, PAX1KO mice did not decrease ATP release when treated with Tenofovir (p= 0.3292).
Conclusion(s): In the absence of pannexin-1 transporter tenofovir is not able to influence BMD or osteoclast differentiation

Aging and injury often lead to chronic low-grade inflammation, a condition known as Inflammaging, which contributes to the development of disease in the musculoskeletal system and is also thought to contribute to the development of diabetes and other diseases of aging. Previous studies demonstrate that chondrocytes from osteoarthritic cartilage have lower cellular ATP levels, lower biomass and diminished mitochondrial function and we have discovered that this leads to diminished ATP transport into the extracellular space resulting in lower extracellular adenosine levels. In other experiments we found that reduced extracellular adenosine levels or loss of A2A receptors (A2AR) lead to the development of premature osteoarthritis (OA). Intra-articular injection of liposomal adenosine or CGS21680 (A2AR agonist) both prevents development of OA after trauma in rats, an effect completely reversed by intra-articular injection of an A2AR antagonist (ZM241385), and reverses post-traumatic OA in rats and obesity-induced OA in mice. A2AR stimulation promotes chondrocyte homeostasis and reverses OA by multiple mechanisms which will be reviewed

Background or Introduction: Osteopenia and fragility fractures have been associated with HIV infection and Tenofovir, a common antiviral in HIV treatment. In murine models and human cell lines, tenofovir inhibits ATP release and decreases extracellular adenosine levels. Adenosine, and adenosine A2A receptor, inhibits osteoclast formation, and increasing local adenosine concentration with dipyridamole, an agent that blocks adenosine cellular uptaken, stimulates new bone formation as well as rhBMP-2. We hypothesized that tenofovir regulates bone resorption by diminishing endogenous adenosine levels and determined whether dipyridamole and A2A receptor may counteract the effects.
Material(s) and Method(s): M-CSF/RANKL-induced-primary murine osteoclasts were studied as the number of TRAP-positive-cells after challenge with tenofovir alone or in combination with dipyridamole. Differentiation markers were study by RT-PCR, and MAPK/NFkB expression by Western Blot. Male C57Bl/6 mice and A2AKO littermates were treated as follow: saline 0.9% (control), tenofovir 75mg/Kg/day, dipyridamole 25mg/Kg/day, combination tenofovir/dipyridamole (n=10, 4 weeks). Calcein/AlizarinRed-labelling of newly formed bone was used, and long bones were prepared for microCT/histology.
Result(s): Tenofovir produced a dose-dependent increase in osteoclast differentiation (EC50=44.5nM) that was reversed by dipyridamole (IC50=0.3muM). 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. WT mice treated with tenofovir lost nearly 10% of body weight (p<0.001). MicroCT revealed decrease 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 and RANKL-positive-cells, whereas OPG-positive-cells decreased with tenofovir. Similar results were obtained for tenofovir in A2AKO mice, but dipyridamole was not able to reverse the effect.
Conclusion(s): These results suggest that treatment with agents that increase local adenosine concentrations, like dipyridamole, and activate adenosine A2A receptor might prevent bone lost following tenofovir treatment

Background:We have previously reported that endogenously produced adenosine, interacting with A2AR, is a critical autocrine factor for maintenance of chondrocyte and cartilage homeostasis and intra-articular injections of liposomal preparations of adenosine inhibit progression of OA in a posttraumatic OA (PTOA) model in rats. We therefore determined whether intra-articular injection of a more selective A2AR agonist could also prevent progression and possibly reverse OA in this model and in the obesity related OA model in mice.
Method(s): PTOA was induced in SD rats following rupture of the anterior cruciate ligament (ACL) by application of external force to the knee. Starting 4 weeks after injury, when OA has already progressed, knees were injected with 100ul of saline, empty liposomes (LIPO) or liposomes containing CGS21680 (LIPO-CGS) every 10 days (6 injections) before sacrifice. The cartilage volume in OA and normal knees was measured by microCTafter staining with hexabrix (40%). Chondrocytes were isolated from neonatal mice and cultured, only first passage chondrocytes were studied. For the obesity-OA model, C57Bl6 mice (5-6/group, 12 weeks old) were fed a 60%fat diet (HFF mice). After 3 months, when OAwas present, mice received intrarticular knee injection (10 mul) of LIPO, LIPOCGS or liposomal adenosine (LIPO-Ado) every 10 days for 4 injections before sacrifice.
Result(s): Injection of LIPO-CGS but not saline or LIPO, significantly reduced swelling of affected rat knees (p<0.001). Surprisingly, there was an increase in tibial and femoral cartilage volume in normal knees treated with intra-articular injections of LIPO-CGS but not LIPO or saline (47% increase in tibia and 22% in femur). More importantly, intra-articular injections of LIPO-CGS, but not LIPO or saline, increased tibial and femoral cartilage volume inOAknees, as compared to normal knees and completely abrogated the histologic evidence of OA as well (OARSI score for CGS21680 0.66+/-0.33 vs 4.55+/-0.82 in the vehicle group and 3.90+/-0.89 in the saline group). There wasmarked chondrocyte proliferation in the deep cartilage of knees of rats treated with LIPO-CGS (Ki67 immunofluorescence). Similarly, LIPO-CGS reversed the OA changes in the obesity related OA model. HFF mice had an OARSI score of 5.17+/-1.84. Treatments with LIPO-Ado and lipo-CGS decreased OA severity (OARSI score 1.33+/-0.81 and 1.83+/-0.98, respectively, p<0.001 vs untreated). A2AR stimulation increased TGF-beta immunostaining in LIPO-CGS-injected joints and increased TGF-beta production by cultured neonatal murine chondrocytes with increased SMAD2/3 phosphorylation and diminished RUNX2 expression.
Conclusion(s): These results demonstrate that intra-articular injection of a long-acting A2AR agonist stimulates chondrocyte and cartilage regeneration, likely by a TGF-beta-dependent mechanism. More importantly, these results indicate that treatment with an A2AR agonist can reverse OA in both traumatic and obesity-related OA