Intraarticular injection of liposomal adenosine reduces cartilage damage in established murine and rat models of osteoarthritis

Abstract

Osteoarthritis (OA) affects nearly 10% of the population of the United States and other industrialized countries and, at present, short of surgical joint replacement, there is no therapy available that can reverse the progression of the disease. Adenosine, acting at its A2A receptor (A2AR), is a critical autocrine factor for maintenance of cartilage homeostasis and here we report that injection of liposomal suspensions of either adenosine or a selective A2AR agonist, CGS21680, significantly reduced oA cartilage damage in a murine model of obesity-induced oA. the same treatment also improved swelling and preserved cartilage in the affected knees in a rat model of established posttraumatic OA (PTOA). Differential expression analysis of mRNA from chondrocytes harvested from knees of rats with PTOA treated with liposomal A2AR agonist revealed downregulation of genes associated with matrix degradation and upregulation of genes associated with cell proliferation as compared to liposomes alone. Studies in vitro and in affected joints demonstrated that A2AR ligation increased the nuclear P-SMAD2/3/P-SMAD1/5/8 ratio, a change associated with repression of terminal chondrocyte differentiation. These results strongly suggest that targeting the A2AR is an effective approach to treat oA. Osteoarthritis (OA) is a common disease affecting 151 million people worldwide and its incidence is expected to increase in industrialized countries due to aging and increased obesity of the population, a condition that together with previous joint injury represent the most common risk factors 1. OA can affect any joint, but most commonly affects the knee, hip and hand. The prevalence of OA is greatest in the knee joint, in both women (47%) and men (40%), and there is no therapy currently available that can reverse or halt the progression of OA 2,3 short of total joint replacement. Total knee replacements are the most common joint surgeries and it has been estimated that there will be a fivefold increase in the number of patients undergoing this surgical procedure up to 3.5 million by 2030 4,5. In search of effective therapies, a number of different approaches have been taken including a focus on both growth factors, such as transforming growth factor-beta (TGFβ), and other molecular pathways involved in regulating cartilage development and homeostasis. TGFβ molecular signaling exerts dual and opposing roles in cartilage and chondrocyte health depending on the receptor and signal activated downstream. It has been shown that inhibition of high levels of systemic TGFβ attenuates anterior cruciate ligament rupture-induced OA in mice by preventing loss of proteoglycan from the cartilage and protecting the subchondral bone from structural alteration 6. Moreover, the effect of TGFβ on joint health depends on which receptor it binds. Activation of

Adenosine A2A receptor signaling promotes FoxO associated autophagy in chondrocytes

“…4 E). Injections with empty liposomes alone also reduced apoptosis, as has been observed previously 25 , although the reduction was not nearly as marked as that induced by liposomes containing either adenosine or CGS21680. Thus our results from in vitro and in vivo studies demonstrate that A2AR signaling reduces chondrocyte apoptosis in the setting of OA.…”

“…The obese mouse OA model employed in this study as mentioned develop early, severe arthritis and are especially prone to metabolic defects, inflammation, and mitochondrial dysfunction 25 , 31 . It is also important to understand that while FoxO1 and FoxO3 often play similar roles, they do have specific functions.…”

“…With respect to possible regulation of FoxO levels, localization, and activity, although AKT is a major inhibitory regulator of FoxOs by phosphorylation at three sites, the inhibition of AKT by A2AR signaling is unlikely to explain the observed FOXO changes given the fact that A2AR actually leads to early activation of AKT observed in osteoblasts 38 , fibroblasts 39 , and recently chondrocytes 25 . Furthermore, there was an A2AR specific effect with moderately higher cytoplasmic phospho-FoxO1 and phospho-FoxO3 in mice treated with liposomal-CGS21680 compared to liposomal-adenosine, which may result from an A2AR-specific known elevation in active AKT in chondrocytes and other cell types 38 , 40 .…”

“…The effect of liposomal adenosine on development of OA is mediated through ligation of the A2AR since an A2AR antagonist, but not A2BR or A3R antagonists, completely reverses the effect of adenosine on OA in this model. Importantly, the beneficial, cartilage regenerative changes observed with A2AR ligation in PTOA rats was paralleled in knee joints from an obesity (high fat diet induced) OA mouse model treated with injections of liposomal adenosine or an A2AR-specific agonist 25 . Furthermore, results from our lab have highlighted A2AR signaling as essential for mitochondrial structure and function as well as reducing IL-1ß induced chondrocyte oxidative stress in human primary cells with very similar effects observed in cells from the TC28α2 human chondrocyte cell line.…”

“…Recent differential expression studies from our lab comparing chondrocytes from A2AR knockout and WT mice (unpublished observation) and cartilage from post-traumatic OA (PTOA) treated with or without joint injections of liposomal A2AR agonist suggest that A2AR ligation might prevent OA progression by stimulating reparative mechanisms, including autophagy, that collectively promote chondrocyte homeostasis 25 . We therefore tested the hypothesis that A2AR stimulation increases autophagy through FoxO signaling in chondrocytes and thereby maintains chondrocyte homeostasis.…”