M. Ribeiro

Anti-inflammatory and Chondroprotective Activity of (+)-α-Pinene: Structural and Enantiomeric Selectivity

Previous studies have suggested that α-pinene, a common volatile plant metabolite, may have anti-inflammatory effects in human chondrocytes, thus exhibiting potential antiosteoarthritic activity. The objective of this study was to further characterize the potential antiosteoarthritic activity of selected pinene derivatives by evaluating their ability to modulate inflammation and extracellular matrix remodeling in human chondrocytes and to correlate the biological and chemical properties by determining whether the effects are isomer- and/or enantiomer-selective. To further elucidate chemicopharmacological interactions, the activities of other naturally occurring monoterpenes with the pinane nucleus were also investigated. At noncytotoxic concentrations, (+)-α-pinene (1) elicited the most potent inhibition of the IL-1β-induced inflammatory and catabolic pathways, namely, NF-κB and JNK activation and the expression of the inflammatory (iNOS) and catabolic (MMP-1 and -13) genes. (-)-α-Pinene (2) was less active than the (+)-enantiomer (1), and β-pinene (3) was inactive. E-Pinane (4) and oxygenated pinane-derived compounds, pinocarveol (5), myrtenal (6), (E)-myrtanol (7), myrtenol (8), and (Z)-verbenol (9), were less effective or even completely inactive and more cytotoxic than the pinenes tested (1-3). The data obtained show isomer- and enantiomer-selective anti-inflammatory and anticatabolic effects of α-pinene in human chondrocytes, (+)-α-pinene (1) being the most promising for further studies to determine its potential value as an antiosteoarthritic drug.

Evaluation of the anti-inflammatory, anti-catabolic and pro-anabolic effects of E-caryophyllene, myrcene and limonene in a cell model of osteoarthritis

Osteoarthritis is a progressive joint disease and a major cause of disability for which no curative therapies are yet available. To identify compounds with potential anti-osteoarthritic properties, in this study, we screened one sesquiterpene, E-caryophyllene, and two monoterpenes, myrcene and limonene, hydrocarbon compounds for anti-inflammatory, anti-catabolic and pro-anabolic activities in human chondrocytes. At non-cytotoxic concentrations, myrcene and limonene inhibited IL-1β-induced nitric oxide production (IC50=37.3μg/ml and 85.3µg/ml, respectively), but E-caryophyllene was inactive. Myrcene, and limonene to a lesser extent, also decreased IL-1β-induced NF-κB, JNK and p38 activation and the expression of inflammatory (iNOS) and catabolic (MMP-1 and MMP-13) genes, while increasing the expression of anti-catabolic genes (TIMP-1 and -3 by myrcene and TIMP-1 by limonene). Limonene increased ERK1/2 activation by 30%, while myrcene decreased it by 26%, relative to IL-1β-treated cells. None of the compounds tested was able to increase the expression of cartilage matrix-specific genes (collagen II and aggrecan), but both compounds prevented the increased expression of the non-cartilage specific, collagen I, induced by IL-1β. These data show that myrcene has significant anti-inflammatory and anti-catabolic effects in human chondrocytes and, thus, its ability to halt or, at least, slow down cartilage destruction and osteoarthritis progression warrants further investigation.

Assessment of cell line competence for studies of pharmacological GPR30 modulation

Abstract Context/objective: Cell lines used to study the role of the G protein-coupled receptor 30 (GPR30) or G protein-coupled estrogen receptor (GPER) as a mediator of estrogen responses have yielded conflicting results. This work identified a simple assay to predict cell line competence for pharmacological studies of GPR30. Materials and methods: The phosphorylation or expression levels of ERK1/2, Akt, c-Fos and eNOS were evaluated to assess GPR30 activation in response to known agonists (17β-estradiol and G-1) in MCF-7 and T-47D breast cancer cell lines and in bovine aortic endothelial cells. GPR30 expression was analyzed by qRT-PCR and Western blot with two distinct antibodies directed at its carboxy and amino terminals. Results: None of the agonists, at any of the concentrations tested, activated any of those target proteins. Additional experiments excluded the disruption of the signaling pathway, interference of phenol red in the culture medium and constitutive proteasome degradation of GPR30 as possible causes for the lack of response of the three cell lines. Analysis of receptor expression showed the absence of clearly detectable GPR30 species of 44 and 50–55 kDa previously identified in cell lines that respond to 17β-estradiol and G-1. Discussion and conclusion: Cells that do not express the 44 and 50–55 kDa species do not respond to GPR30 agonists. Thus, the presence or absence of these GPR30 species is a simple and rapid manner to determine whether a given cell line is suitable for pharmacological or molecular studies of GPR30 modulation.

Diabetes-induced osteoarthritis: role of hyperglycemia in joint destruction

Recent epidemiologic and experimental data reinforced the concept that diabetes mellitus (DM) is an independent risk factor for osteoarthritis (OA). Besides a systemic inflammatory response that can affect joint tissues and contribute to OA pathogenesis, direct effects of hyperglycaemia have been shown to cause cell damage and induce inflammation by various mechanisms in several tissues associated to diabetic complications. Whether and how glucose directly affects joint tissues and cells is just beginning to be unraveled. Indirect effects of high glucose can result from enhanced formation of advanced glycation end products (AGEs) which accumulate in OA cartilage in an age-dependent manner and play a pro-inflammatory and pro-catabolic role mediated by activation of their specific receptor, RAGE, on chondrocytes and synovial cells. Some direct effects of high glucose have also been demonstrated, namely induction of IGF-1 resistance[1] and inhibition of dehydroascorbate transport which can compromise collagen synthesis[2]. Our studies have been aimed at determining whether and how hyperglycemia affects chondrocyte functions and contributes to OA development and progression. The results obtained showed that high and low glucose concentrations regulate the availability of facilitative glucose transporter (GLUT) isoforms and the glucose transport capacity of human chondrocytes. High glucose concentrations decrease the transport capacity and GLUT-1 protein content without affecting its mRNA levels, but this ability to adjust glucose transport capacity as a function of its availability is compromised in aged/OA chondrocytes leading to its intracellular accumulation[3]. The consequences of this are increased and prolonged ROS production[3] and expression of metalloproteinases (MMP)-1 and -13[4], IL-1β, TNF-α, inducible nitric oxide (NO) synthase (iNOS) and NO production, mediated by high glucose-induced NF-κB activation[5], as well as decreased responsiveness to TGF-β[4] and impaired autophagy[5]. High glucose is thus sufficient to induce an inflammatory and catabolic response in human OA chondrocytes. Furthermore, it potentiates pro-inflammatory effects of IL-1β, namely IL-6, cyclooxygenase 2 (Cox)-2, prostaglandin E2 (PGE2) and NO production[6]. The pro-inflammatory effects of high glucose in human chondrocytes and diabetic mice, namely induction of Cox-2, IL-6 and MMP-13 and production of PGE2, as well as decreased production of Collagen II, have also been shown to involve impairment of anti-inflammatory pathways, namely by decreasing PPAR-γ expression[7]. Elucidating how high glucose modulates joint tissue homeostasis will identify novel targets for development of innovative strategies both to identify diagnostic and prognostic biomarkers of OA and to effectively modify disease progression.

A5.1 Culture OF human chondrocytes in high glucose induces inflammatory markers and impairs autophagy

Background and Objectives Accumulating evidence indicates that Diabetes Mellitus is an independent risk factor for severe osteoarthritis (OA). Understanding the mechanisms involved is essential for designing preventive strategies and targeted therapies that can halt OA progression. Our previous studiesshowed that culture of human chondrocytes under excess glucose favours catabolic responses and oxidative stress. This study aims at elucidating whether exposure of chondrocytes to high glucose favours inflammatory responses and impairs autophagy, a crucial mechanism for the elimination of damaged molecules and organelles. Materials and Methods Human chondrocytes isolated from knee cartilage obtained from multi-organ donors at the Orthopaedics Department of the University and Hospital Center of Coimbra and the human chondrocytic cell line, C28/I2, were cultured in medium containing regular (10 mM) or excess (30 mM) glucose for various periods. The expression of pro-inflammatory and pro-catabolic markers (iNOS, IL-1β and TNF-α) was evaluated by qRT-PCR. iNOS protein levels and the nuclear translocation of p65, a marker of NF-κB activation, were evaluated by western blot. NO production was assessed by the Griess reaction. Autophagy was assessed by determining the protein levels of LC3-I and II in the presence and absence of the lysosome inhibitor, chloroquine. To rule out possible osmotic effects, parallel experiments were performed in the presence of the cell-impermeable polyol, mannitol. Results Culture of human chondrocytes in high glucose (30 mM) for 24h significantly increased iNOS mRNA and protein levels, as well as NO production relative to cells maintained in regular glucose. IL-1β and TNF-α mRNA levels were also significantly increased, while nuclear levels of NF-κB p65 increased in a time-dependent manner, peaking after 1h treatment. On the other hand, LC3-II levels were significantly reduced by culture of C28/I2 cells in high glucose for 24h, either in the presence or absence of chloroquine, indicating that its synthesis was decreased relative to cells maintained in regular glucose medium. Conclusions Hyperglycemia-like glucose concentrations are sufficient to induce the inflammatory process and to impair autophagy in human chondrocytes which can contribute to the development and progression of OA. Work supported by grants PEst-C/SAU/LA0001/2011 and PTDC/EME-TME/113039/2009 from FEDER through COMPETE and FCT.

OP0312 Diabetes-Accelerated Experimental Osteoarthritis Is Prevented by Autophagy Activation

Background Type 2 Diabetes (T2D) is a risk factor for Osteoarthritis (OA) (1). Autophagy, an essential mechanism in articular cartilage, is defective in T2D and OA (2). However, how T2D may influence OA progression is still unknown. Objectives We aimed to determine how diabetes affects cartilage integrity and whether pharmacological activation of autophagy has disease-modifying efficacy in diabetic mice with OA. Methods Experimental OA was performed in the right knee of 9 weeks-old C57Bl/6 male mice (Lean group, N=8) and of 9 weeks-old B6.BKS (D)-Leprdb male mice (db/db group, N=16) by transection of medial meniscotibial and medial collateral ligaments. Left knee was employed as control knee. Rapamycin (2 mg/kg weight/day) or Vehicle (dimethyl sulphoxide) were administered intraperitoneally 3 times a week for 10 weeks. Body weight and fasting blood glucose levels were monitorized weekly. Insulin levels in plasma were determined by ELISA at the end of the study. Histopathology in articular cartilage and synovium was evaluated by using semiquantitative scoring and synovitis grading systems, respectively. Immunohistochemistry was employed to evaluate the effect of diabetes and Rapamycin on cartilage integrity and OA biomarkers. Results Diabetic mice have increased body weight, fasting blood glucose levels and insulin levels compared to lean mice. Importantly, Rapamycin treated mice have significantly reduced body weight and blood glucose and insulin compared to control db/db mice. Moreover, rapamycin treatment significantly reduced the phosphorylation of rpS6, a direct target of mTOR and increased phosphorylation of AMPK. The results indicated that cartilage damage was significantly increased in db/db mice compared to lean group after surgical induction of OA, while no differences are observed in the control knee. mTOR inhibition protects against surgical-induced OA in db/db mice joints. Cartilage damage and OA severity was significantly reduced in the treated group. This reduction was accompanied with a significant reduction in synovium inflammation and in the expression of OA biomarkers, such as MMP-13 and ADAMTS-5 and decreased IL-12 levels. Furthermore, LC3 lipidation, a marker of autophagosome formation, was increased and cartilage cellularity was maintained after treatment, suggesting that autophagy activation protects chondrocytes against death. Conclusions Our findings indicate that diabetic mice exhibit accelerated-joint damage after experimental OA, and that autophagy activation might be an effective therapy for diabetes-accelerated OA. References Berenbaum F. Diabetes-induced osteoarthritis: from a new paradigm to a new phenotype. Postgrad Med J 2012.88:240–2. Levine B and Kroemer G. Autophagy in the pathogenesis of disease. Cell 2008. 132:27–42. Acknowledgement This study was supported by Instituto de Salud Carlos III-Ministerio de Economía y Competitividad, Spain-CP11/00095 and Fondo Europeo de Desarrollo Regional (FEDER). Madalena Ribeiro was supported by Fundo Social Europeu (FSE), through “Programa Operacional Potencial Humano” (POPH), and national funds via FCT – Fundaçao Portuguesa para a Ciencia e a Tecnologia under the PhD fellowship, SFRH/BD/78188/2011. Disclosure of Interest None declared

SAT0039 Insulin-Induced Cartilage Degradation in Osteoarthritis is Associated to Defective Autophagy

Background Autophagy, a key cellular quality control mechanism, is defective in Osteoarthritis (OA) and Type 2 Diabetes (T2D) (1,2). T2D has been proposed as a risk factor for OA. Although epidemiological studies suggest a strong association between these diseases (3), how T2D may have an effect on the deterioration of articular cartilage is still unknown. Objectives The objective of this study is to understand the role of autophagy in the articular cartilage function under diabetic conditions. Methods Human chondrocyte cell line (TC28a2) and primary human chondrocytes (HC) were cultivated in DMEM high glucose (25 mM) and treated with Insulin (10, 100, 500 nM) for 2, 6 and 24 hours. Activity of LC3-II, Akt and rpS6 was evaluated by Western blotting (WB). To investigate whether autophagy activation protects from diabetic conditions, autophagy was induce by Rapamycin (10 μM). Human cartilage explants were cultivated in DMEM 25mM glucose and insulin (100, 500, 1000nM) for 24 hours to evaluate histopathological changes. MMP-13 and IL-1β expression was determined by immunohistochemistry and WB, respectively. Expression of LC3 and p-rpS6 was determined by WB in human chondrocytes from Non Diabetic-OA and Diabetic-OA patients. Results In the presence of high glucose and increased doses of insulin autophagy was decreased in a dose dependent-manner in human chondrocytes, as indicated by LC3II expression, the main marker of autophagy activation (TC28-a2; p<0.05 at 6 hours post-treatment; HC; p<0.01 at 24 hours post-treatment). To investigate the mechanism by which autophagy is reduced by insulin, Akt and rpS6 phosphorylation was analyzed. We observed a significant increase in p-AKT and p-rpS6 activity, suggesting that insulin effect is mediated by AKT/mTOR pathway (TC28-a2 p<0.05 at 6 hours; HC; p<0.01 at 2 hours). Autophagy activation by Rapamycin reversed insulin effects on LC3 and p-rbS6 expression (Tc28a2 and HC:p<0.05), indicating that autophagy induction prevents insulin-mediated autophagy signaling downregulation. To evaluate the impact of insulin-mediated autophagy regulation in the context of articular cartilage biology, cartilage explants were treated with insulin (100, 500 and 1000 nM) for 24 hours. Histological analysis indicated a loss of proteoglycans and increased MMP-13 and IL-1β expression (p<0.01) after insulin treatment. Remarkably, chondrocytes from OA-diabetic patients showed decreased LC3 and increased p-rpS6 expression compared to Non-Diabetic OA patients. Conclusions Our findings demonstrate that diabetic conditions decrease autophagy by an AKT/mTOR dependent mechanism. Pharmacological activation of autophagy might protect against T2D in human chondrocytes. Our data also indicate that chondrocytes from OA-diabetic patients exhibit a deficient autophagy. Taking together, these results suggest that impaired autophagy might be one of the mechanisms by which T2D diabetes accelerates cartilage degradation. References Caramés, B., et al., Arthritis Rheum, 2010. 62(3): p. 791-801 Murrow, L., et al. Annu Rev Pathol, 2013. 8: p. 105-37. Berenbaum, F., Ann Rheum Dis, 2011. 70(8): p. 1354-6. Acknowledgements This study was supported by Instituto de Salud Carlos III- Ministerio de Economía y Competitividad, Spain-CP11/00095. M.R is supported by FCT (SFRH/BD/78188/2011). Disclosure of Interest None declared