Faculty Bibliography

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.

Background/Purpose: Our lab has demonstrated the ability of an intra-articular liposomal formulation of an adenosine A2A receptor (A2AR), CGS21680, to regenerate cartilage in multiple rodent OA models. At the cellular level in chondrocytes, we have shown that activation of A2AR enhances chondrocyte homeostasis, increases mitophagy, and reduces chondrocyte senescence. Moreover, we identified an A2AR-mediated increase in an anti-senescent, anti-aging truncated p53 variant D133p53a in human chondrocytes. These anti-senescent effects have been recapitulated in transgenic mice overexpressing D122p53. Interestingly, a 2014 study reported an endogenous N-terminal mouse p53 variant in HeLa cells (2 separate deletions with loss of amino acids 42-89 and in-frame shift of aa 90-120) with no p53 transactivation potential and inability to upregulate p21 expression despite retention of the first p53 transactivation domain. Hence, we first hypothesized that this mouse p53 variant could be present in mesenchymal stem cells (MSCs), which have chondrogenic capabilities in vitro. Secondly, we hypothesized that A2AR ligation in mouse MSCs would increase expression of this variant.
Method(s): Bone marrow MSCs were isolated from 12-week-old mice, treated with or without 1muM CGS21680, and RTqPCR was performed to assess expression of total and variant p53 using variant specific primers. Primary and secondary gene sequence analysis was employed to evaluate the deleted/shifted region for CpG sites and G-quadruplex structures using the Sequence Manipulation Suite and the GQRS Mapper, respectively.
Result(s): We did identify this variant in mouse bone marrow MSCs. Furthermore, its expression relative to total p53 was significantly increased in the A2AR agonist treated MSCs compared to control (1.6+/-0.3 vs. 1.0+/-0.06, p=0.029, n=3). CpG site analysis revealed that the variant corresponded to a reduction in 4 CpG sites and 3 potential G-quadruplex structures if analyzed in the antisense configuration.
Conclusion(s): To our knowledge, this is the first report of this variant of mouse p53 noted in cell types aside from the initial report noted above. While more work needs to be done in differentiated cells, A2AR agonism seems to enhance formation of this mouse p53 variant has functional similarities to the D133p53a. Importantly, the deleted regions in this variant contain the key senescence residues in the protein. Further, It is possible that DNA modifications such as CpG methylation and/or alteration in G-quadruplex stability may play a role in such findings

Adenosine signaling plays a critical role in the maintenance of articular cartilage and may serve as a novel therapeutic for osteoarthritis (OA), a highly prevalent and morbid disease without effective therapeutics in the current market. Mice lacking adenosine A2A receptors (A2AR) develop spontaneous OA by 16 weeks of age, a finding relevant to human OA since loss of adenosine signaling due to diminished adenosine production (NT5E deficiency) also leads to development of OA in mice and humans. To better understand the mechanism by which A2AR and adenosine generation protect from OA development, we examined differential gene expression in neonatal chondrocytes from WT and A2AR null mice. Analysis of differentially expressed genes was analyzed by KEGG pathway analysis, and oPOSSUM and the flatiron database were used to identify transcription factor binding enrichment, and tissue-specific network analyses and patterns were compared to gene expression patterns in chondrocytes from patients with OA. There was a differential expression of 2211 genes (padj<0.05). Pathway enrichment analysis revealed that pro-inflammatory changes, increased metalloprotease, reduced matrix organization, and homeostasis are upregulated in A2AR null chondrocytes. Moreover, stress responses, including autophagy and HIF-1 signaling, seem to be important drivers of OA and bear marked resemblance to the human OA transcriptome. Although A2AR null mice are born with grossly intact articular cartilage, we identify here the molecular foundations for early-onset OA in these mice, further establishing their role as models for human disease and the potential use of adenosine as a treatment for human disease.

Background/UNASSIGNED:Presence of ethanol (EtOH) may alter the relationship between blood lactate concentrations and mortality. This study compares lactate-associated mortality risk in the presence and absence of EtOH. Methods/UNASSIGNED:We performed a retrospective cohort study including all patients, age >17 years, presenting from January 2012-December 2018, to an urban, academic emergency department, with a clinically measured lactate. Data were electronically abstracted from the medical record. The primary outcome was 28-day in-hospital mortality. Patients were grouped by EtOH test results as follows: 1) present (any EtOH detected), 2) absent (EtOH concentration measured and not detected), or 3) not ordered. Marginal analysis was used to calculated probability of mortality for fixed values of lactate and model covariates. Results/UNASSIGNED:Of 40,956 adult emergency department patients with measured lactate, we excluded 768 (1.89%) for lactate >10.0 mmol/L, leaving 40,240 for analysis: 4,066 (10.1%) EtOH present, 10,819 (26.9%) EtOH absent, 25,355 (63%) EtOH not ordered. Of these, 1790 (4.4%) had 28-day in-hospital mortality. Marginal probability of mortality calculated for specific lactate values found less risk for EtOH Present patients versus EtOH absent patients at lactate 0.0 mmol/L (0.8% [95%CI: 0.5-1.2%] vs 3.2% [2.8-3.6%]), 2.0 mmol/L (1.5% [1.1-1.9%] vs 4.0% [3.7-4.3%]), 4.0 mmol/L (2.6% [2.2-3.1%] vs 5.0% [4.6-5.4%]), until 6.0 mmol/L (4.5% [3.7-5.4%] vs 6.2% [5.4-7.0%]). Conclusion/UNASSIGNED:EtOH presence significantly alters lactate-associated mortality risk when lactate <6.0 mmol/L. Emergency department clinicians should interpret these lactate values with caution and consider other data for risk stratification when EtOH is present.

Autophagy, a homeostatic pathway upregulated during cellular stress, is decreased in osteoarthritic chondrocytes and this reduction in autophagy is thought to contribute to the development and progression of osteoarthritis (OA). The adenosine A2A receptor (A2AR) is a potent anti-inflammatory receptor and deficiency of this receptor leads to the development of OA in mice. Moreover, treatment using liposomally conjugated adenosine or a specific A2AR agonist improved joint scores significantly in both rats with post-traumatic OA (PTOA) and mice subjected to a high fat diet obesity induced OA. Importantly, A2AR ligation is beneficial for mitochondrial health and metabolism in vitro in primary and the TC28a2 human cell line. An additional set of metabolic, stress-responsive, and homeostatic mediators include the Forkhead box O transcription factors (FoxOs). Data has shown that mouse FoxO knockouts develop early OA with reduced cartilage autophagy, indicating that FoxO-induced homeostasis is important for articular cartilage. Given the apparent similarities between A2AR and FoxO signaling, we tested the hypothesis that A2AR stimulation improves cartilage function through activation of the FoxO proteins leading to increased autophagy in chondrocytes. We analyzed the signaling pathway in the human TC28a2 cell line and corroborated these findings in vivo in a metabolically relevant obesity-induced OA mouse model. We found that A2AR stimulation increases activation and nuclear localization of FoxO1 and FoxO3, promotes an increase in autophagic flux, improves metabolic function in chondrocytes, and reduces markers of apoptosis in vitro and reduced apoptosis by TUNEL assay in vivo. A2AR ligation additionally enhances in vivo activation of FoxO1 and FoxO3 with evidence of enhanced autophagic flux upon injection of the liposome-associated A2AR agonist in a mouse obesity-induced OA model. These findings offer further evidence that A2AR may be an excellent target for promoting chondrocyte and cartilage homeostasis.

Le méthotrexate est utilisé dans le traitement de la polyarthrite rhumatoïde (PR) depuis les années 1980 et est souvent à ce jour le médicament de première intention pour le traitement de la PR. Dans cette revue, nous examinons plusieurs hypothèses pour expliquer le mécanisme à l'origine de l'efficacité du méthotrexate dans la PR. Celles-ci comprennent l'antagonisme du folate, la signalisation par l'adénosine, la génération d'espèces réactives de l'oxygène (ROS), la diminution des molécules d'adhérence, la modification des profils cytokiniques et l'inhibition des polyamines, entre autres. Actuellement, la signalisation par l'adénosine est probablement l'explication la plus largement acceptée du mécanisme du méthotrexate dans la PR, car le méthotrexate augmente les taux d'adénosine et suite à l'engagement de l'adénosine avec ses récepteurs extracellulaires, une cascade intracellulaire est activée et favorise un état antiinflammatoire global. Outre ces hypothèses, nous examinons le mécanisme du méthotrexate dans la PR sous l'angle de ses effets indésirables et considérons certains des nouveaux marqueurs génétiques de l'efficacité et de la toxicité du méthotrexate dans la PR. Enfin, nous discutons brièvement du mécanisme du méthotrexate en association avec un traitement de la PR par un inhibiteur du TNF-. En fin de compte, en trouvant une explication claire de la voie et du mécanisme conduisant à l'efficacité du méthotrexate dans la PR, il pourrait exister un moyen de formuler des thérapies plus puissantes avec moins d'effets secondaires.

Methotrexate has been used in treatment of rheumatoid arthritis (RA) since the 1980s and to this day is often the first line medication for RA treatment. In this review, we examine multiple hypotheses to explain the mechanism of methotrexate efficacy in RA. These include folate antagonism, adenosine signaling, generation of reactive oxygen species (ROS), decrease in adhesion molecules, alteration of cytokine profiles, and polyamine inhibition amongst some others. Currently, adenosine signaling is probably the most widely accepted explanation for the methotrexate mechanism in RA given that methotrexate increases adenosine levels and on engagement of adenosine with its extracellular receptors an intracellular cascade is activated promoting an overall anti-inflammatory state. In addition to these hypotheses, we examine the mechanism of methotrexate in RA from the perspective of its adverse effects and consider some of the newer genetic markers of methotrexate efficacy and toxicity in RA. Lastly, we briefly discuss the mechanism of additive methotrexate in the setting of TNF-α inhibitor treatment of RA. Ultimately, finding a clear explanation for the pathway and mechanism leading to methotrexate efficacy in RA, there may be a way to formulate more potent therapies with fewer side effects.