Effects of florfenicol on LPS-induced nitric oxide and prostaglandin E₂ production in RAW 264.7 macrophages

Florfenicol, an antibiotic commonly used to treat infections, has previously been shown to modulate lipopolysaccharide (LPS)‐induced early cytokine responses by blocking the nuclear factor‐κB (NF‐κB) pathway. In this study, we investigated the effects of florfenicol on nitric oxide (NO) and prostaglandin E₂ (PGE₂) production as well as on inducible nitric oxide synthase (iNOS) and cyclooxygenase‐2 (COX‐2) expression in LPS‐stimulated murine RAW 264.7 macrophages. We also analysed the effects of florfenicol on mitogen‐activated protein kinase (MAPK) pathways. Florfenicol significantly inhibited LPS‐induced NO and PGE₂ production. Consistent with these observations, mRNA and protein expression of iNOS and COX‐2 were also inhibited by florfenicol in a dose‐dependent manner. Furthermore, phosphorylation of p38 and extracellular signal‐regulated kinase 1/2 (ERK1/2) in LPS‐stimulated RAW 264.7 cells was suppressed by florfenicol. However, c‐Jun N‐terminal kinase (JNK) phosphorylation remained unaffected. Using specific inhibitors of ERK and p38, we found that florfenicol may inhibit NO and PGE₂ mostly through ERK and p38 pathway. These results suggest that florfenicol inhibits NO and PGE₂ production in conjunction with an inhibition of iNOS and COX‐2 expression, at least partially via suppression of ERK1/2 and p38 MAPK phosphorylation.     

Detrimental arterial inflammatory effect of microparticles circulating in preeclamptic women: ex vivo evaluation in human arteries

Elevated plasmatic levels of lympho‐monocyte and platelet microparticles (MPs) have been reported in preeclampsia. Previous studies suggest that MPs could participate in preeclampsia vascular impairment. In this study, we investigated the ex vivo vascular effects of MPs from preeclamptic women on arteries from normotensive pregnant women. Omental arteries were collected from normal pregnant women undergoing cesarean section and incubated during 24 h with MPs from normal pregnant or preeclamptic women. Vascular contraction to serotonin and phenylephrine was studied on a wire myograph with or without pharmacological selective inhibitors of inducible nitric oxide synthase (iNOS) and/or cyclo‐oxygenase‐2 (COX‐2). Expression of iNOS, COX‐2, and NF‐κB and production of superoxide anion and 8‐isoprostane were also assessed by immunohistological or biochemical staining and/or Western blot or ELISA assay, respectively. Microparticles from preeclamptic women, but not those from normal pregnant women, induced hyporeactivity to vasocontracturant agonists in omental arteries. Selective inhibitor of iNOS partially restored this arterial contraction, suggesting that nitric oxide (NO) is involved in vascular contractility alteration. Conversely, COX‐2 induced 8‐isoprostane release, a vasoconstricting metabolite modulating the agonist‐induced contraction. COX‐2 selective inhibitor almost abolished the arterial contraction in the same vessels. Interestingly, the association of iNOS and COX‐2 selective inhibitors restored the contraction to control levels. Moreover, iNOS, COX‐2, and NF‐κB expressions are upregulated and superoxide anion levels increased in vessels incubated with MPs from preeclamptic women. In conclusion, circulating MPs from preeclamptic women induce vascular inflammation and enhance oxidative stress. These results suggest a possible role of MPs during preeclampsia‐induced arterial dysfunction.     

The use of mechano growth factor to prevent cartilage degeneration in knee osteoarthritis

Previous studies have attached more importance to growth factors in treating cartilage degeneration and osteoarthritis (OA). Here, the capability of mechano growth factor‐C24E (MGF) to prevent osteoarthritic cartilage degeneration was evaluated in vitro and in vivo. Using in vitro cultured human OA chondrocytes treated with 10–60‐ng/ml MGF for 12 hr, we detected the cell proliferation, migration, and anabolism of OA chondrocytes. The unfolded protein response and the protein characteristic of OA pathology, such as transforming growth factor β, SMAD family member 3, and hypoxia‐inducible factor 2α of OA chondrocytes, were also detected by western blotting. Furthermore, protein kinase RNA‐like endoplasmic reticulum kinase was knocked down via small interfering RNA to illuminate the potential mechanism of MGF's treatment of OA. In a rabbit knee joint OA model, cartilage degeneration was inhibited after 2 weeks of treatment with 0.1–10‐μg/ml MGF. This study demonstrated that MGF treatment can inhibit the pathological apoptosis of OA chondrocytes and promote the proliferation, migration, and matrix synthesis of the chondrocytes. The results also demonstrate that the degeneration of OA cartilage can be delayed by MGF treatment partially via unfolded protein response regulated by protein kinase RNA‐like endoplasmic reticulum kinase and suggest a potential therapeutic application of MGF for OA treatment.     

Inhibitors of Microsomal Prostaglandin E2 Synthase‐1 Enzyme as Emerging Anti‐Inflammatory Candidates

Cyclooxygenases (COX‐1 and COX‐2) catalyze the conversion of arachidonic acid (AA) into PGH₂ that is further metabolized by terminal prostaglandin (PG) synthases into biologically active PGs, for example, prostaglandin E₂ (PGE₂), prostacyclin I₂ (PGI₂), thromboxane A₂ (TXA₂), prostaglandin D₂ (PGD₂), and prostaglandin F₂ alpha (PGF_2α). Among them, PGE₂ is a widely distributed PG in the human body, and an important mediator of inflammatory processes. The successful modulation of this PG provides a beneficial strategy for the potential anti‐inflammatory therapy. For instance, nonsteroidal anti‐inflammatory agents (NSAIDs), both classical nonselective (cNSAIDs) and the selective COX‐2 inhibitors (coxibs) attenuate the generation of PGH₂ from AA that in turn reduces the synthesis of PGE₂ and modifies the inflammatory conditions. However, the long‐term use of these agents causes severe side effects due to the nonselective inhibition of other PGs, such as PGI₂ and TXA₂, etc. Microsomal prostaglandin E₂ synthase‐1 (mPGES‐1), a downstream PG synthase, specifically catalyzes the biosynthesis of COX‐2‐derived PGE₂ from PGH₂, and describes itself as a valuable therapeutic target for the treatment of acute and chronic inflammatory disease conditions. Therefore, the small molecule inhibitors of mPGES‐1 would serve as a beneficial anti‐inflammatory therapy, with reduced side effects that are usually associated with the nonselective inhibition of PG biosynthesis.     

5-Lipoxygenase Metabolic Contributions to NSAID-Induced Organ Toxicity

Cyclooxygenase (COX)-1, COX-2, and 5-lipoxygenase (5-LOX) enzymes produce effectors of pain and inflammation in osteoarthritis (OA) and many other diseases. All three enzymes play a key role in the metabolism of arachidonic acid (AA) to inflammatory fatty acids, which contribute to the deterioration of cartilage. AA is derived from both phospholipase A₂ (PLA₂) conversion of cell membrane phospholipids and dietary consumption of omega-6 fatty acids. Nonsteroidal antiinflammatory drugs (NSAIDs) inhibit the COX enzymes, but show no anti-5-LOX activity to prevent the formation of leukotrienes (LTs). Cysteinyl LTs, such as LTC₄, LTD₄, LTE₄, and leukoattractive LTB₄ accumulate in several organs of mammals in response to NSAID consumption. Elevated 5-LOX-mediated AA metabolism may contribute to the side-effect profile observed for NSAIDs in OA. Current therapeutics under development, so-called “dual inhibitors” of COX and 5-LOX, show improved side-effect profiles and may represent a new option in the management of OA.     

Roles of COX inhibition in pathogenesis of NSAID-induced small intestinal damage

Nonsteroidal anti-inflammatory drugs (NSAIDs) such as indomethacin decrease mucosal PGE₂ content by inhibiting cyclooxygenase (COX) activity and produce damage in the small intestine. The development of intestinal lesions induced by indomethacin was accompanied by increases in intestinal motility, enterobacterial invasion, and myeloperoxidase (MPO) as well as inducible nitric oxide synthase (iNOS) activity, together with the up-regulation of COX-2 and iNOS mRNA expression. Neither SC-560, a selective COX-1 inhibitor, nor rofecoxib, a selective COX-2 inhibitor, alone caused intestinal damage, but their combined administration provoked lesions in the small intestine. SC-560, but not rofecoxib, caused intestinal hypermotility, bacterial invasion and the expression of COX-2 as well as iNOS mRNA, yet the iNOS and MPO activity was increased only when rofecoxib was administered together with SC-560. Although SC-560 inhibited PG production, the level of PGE₂ recovered in a rofecoxib-dependent manner. The intestinal hypermotility in response to indomethacin was prevented by both 16,16-dimethyl PGE₂ and atropine but not by ampicillin, yet all these agents inhibited not only the bacterial invasion but also the expression of COX-2 as well as the iNOS activity in the intestinal mucosa following indomethacin treatment, thereby preventing the intestinal damage. These results suggest that inhibition of COX-1, despite causing intestinal hypermotility, bacterial invasion and iNOS expression, up-regulates the expression of COX-2, and the PGE₂ derived from COX-2 counteracts the deleterious events caused by COX-1 inhibition and maintains mucosal integrity. These sequences of events explain why intestinal damage occurs when both COX-1 and COX-2 are inhibited.     

Human osteoarthritic chondrons outnumber patient‐ and joint‐matched chondrocytes in hydrogel culture—Future application in autologous cell‐based OA cartilage repair?

Autologous chondrocyte implantation (ACI) is used in 34–60% for osteoarthritic (OA) cartilage defects, although ACI is neither recommended nor designed for OA. Envisioning a hydrogel‐based ACI for OA that uses chondrons instead of classically used chondrocytes, we hypothesized that human OA chondrons may outperform OA chondrocytes. We compared patient‐ and joint surface‐matched human OA chondrons with OA chondrocytes cultured for the first time in a hydrogel, using a self‐assembling peptide system. We determined yield, viability, cell numbers, mRNA expression, GAPDH mRNA enzyme activity, Collagen II synthesis (CPII) and degradation (C2C), and sulfated glycosaminoglycan. Ex vivo, mRNA expression was comparable. Over time, significant differences in survival led to 3.4‐fold higher OA chondron numbers in hydrogels after 2 weeks (p = .002). Significantly, more enzymatically active GAPDH protein indicated higher metabolic activity. The number of cultures that expressed mRNA for Collagen Types I and VI, COMP, aggrecan, VEGF, TGF‐β1, and FGF‐2 (but not Collagen Types II and X) was different, resulting in a 3.5‐fold higher number of expression‐positive OA chondron cultures (p < .05). Measuring CPII and C2C per hydrogel, OA chondron hydrogels synthesized more than they degraded Collagen Type II, the opposite was true for OA chondrocytes. Per cell, OA chondrons but not OA chondrocytes displayed more synthesis than degradation. Thus, OA chondrons displayed superior biosynthesis and mRNA expression of tissue engineering and phenotype‐relevant genes. Moreover, human OA chondrons displayed a significant survival advantage in hydrogel culture, whose presence, drastic extent, and timescale was novel and is clinically significant. Collectively, these data highlight the high potential of human OA chondrons for OA ACI, as they would outnumber and, thus, surpass OA chondrocytes.     

Loganin attenuates interleukin-1β-induced chondrocyte inflammation,cartilage degeneration,and rat synovial inflammation by regulating TLR4/MyD88/NF-κB

ObjectiveInflammation plays a crucial part in osteoarthritis (OA) development. This work aimed to explore loganin’s role and molecular mechanism in inflammation and clarify its anti-inflammatory effects in OA treatment.MethodsChondrocytes were stimulated using interleukin (IL)-1β and loganin at two concentrations (1 μM and 10 μM). Nitric oxide (NO) and prostaglandin E2 (PGE2) expression was assessed. Real-time polymerase chain reaction was used to evaluate inducible NO synthase (iNOS), cyclooxygenase (COX)-2, IL-6, and tumor necrosis factor (TNF)-α mRNA levels. Western blot was used to investigate TLR4, MyD88, p-p65, and IκB-α expression. p65 nuclear translocation, synovial inflammatory response, and cartilage degeneration were also assessed.ResultsLoganin significantly reduced IL-1β-mediated PGE2, NO, iNOS, and COX-2 expression compared with that of the IL-1β stimulation group. The TLR4/MyD88/NF-κB pathway was suppressed by loganin, which decreased inflammatory cytokine (TNF-α and IL-6) levels compared with those of the IL-1β stimulation group. Loganin inhibited IL-1β-mediated NF-κB p65 nuclear translocation compared with that of the IL-1β stimulation group. Loganin partially suppressed cartilage degeneration and the synovial inflammatory response in vivo.ConclusionThis work demonstrated that loganin inhibited IL-1β-mediated inflammation in rat chondrocytes through TLR4/MyD88/NF-κB pathway regulation, thereby reducing rat cartilage degeneration and the synovial inflammatory response.     

Human chondrocyte migration behaviour to guide the development of engineered cartilage

Tissue‐engineering techniques have been successful in developing cartilage‐like tissues in vitro using cells from animal sources. The successful translation of these strategies to the clinic will likely require cell expansion to achieve sufficient cell numbers. Using a two‐dimensional (2D) cell migration assay to first identify the passage at which chondrocytes exhibited their greatest chondrogenic potential, the objective of this study was to determine a more optimal culture medium for developing three‐dimensional (3D) cartilage‐like tissues using human cells. We evaluated combinations of commonly used growth factors that have been shown to promote chondrogenic growth and development. Human articular chondrocytes (AC) from osteoarthritic (OA) joints were cultured in 3D environments, either in pellets or encapsulated in agarose. The effect of growth factor supplementation was dependent on the environment, such that matrix deposition differed between the two culture systems. ACs in pellet culture were more responsive to bone morphogenetic protein (BMP2) alone or combinations containing BMP2 (i.e. BMP2 with PDGF or FGF). However, engineered cartilage development within agarose was better for constructs cultured with TGFβ3. These results with agarose and pellet culture studies set the stage for the development of conditions appropriate for culturing 3D functional engineered cartilage for eventual use in human therapies. Copyright © 2015 John Wiley & Sons, Ltd.     

Activated protein C protects against myocardial ischemic/reperfusion injury through AMP‐activated protein kinase signaling

Summary. Background: Activated protein C (APC) is a vitamin K‐dependent plasma serine protease that down‐regulates clotting and inflammatory pathways. It is known that APC exerts a cardioprotective effect by decreasing apoptosis of cardiomyocytes and inhibiting expression of inflammatory mediators after myocardial ischemia. Objectives: The objective of this study was to understand the mechanism of the APC‐mediated cardioprotection against ischemic injury. Methods: Cardioprotective activities of wild‐type APC and two derivatives, having either dramatically reduced anticoagulant activity or lacking signaling activity, were monitored in an acute ischemia/reperfusion injury model in which the left anterior descending coronary artery (LAD) was occluded. Results: APC reduced the myocardial infarct size by a mechanism that was largely independent of its anticoagulant activity. Thus, the non‐anticoagulant APC‐2Cys mutant, but not the non‐signaling APC‐E170A mutant, attenuated myocardial infarct size by EPCR and PAR‐1‐dependent mechanisms. Further studies revealed that APC acts directly on cardiomyocytes to stimulate the AMP‐activated protein kinase (AMPK) signaling pathway. The activation of AMPK by APC ameliorated the post‐ischemic cardiac dysfunction in isolated perfused mouse hearts. Moreover, both APC and APC‐2Cys inhibited production of TNFα and IL‐6 in vivo by attenuating the ischemia/reperfusion‐induced JNK and NF‐κB signaling pathways. Conclusions: APC exerts a cardioprotective function in ischemic/reperfusion injury through modulation of AMPK, NF‐κB and JNK signaling pathways.     

Attenuation of osteoarthritis progression in mice following intra‐articular administration of simvastatin‐conjugated gelatin hydrogel

Simvastatin is clinically used for the treatment of hypercholesterolaemia. This study investigated the effects of the intra‐articular administration of a simvastatin‐conjugated gelatin hydrogel on osteoarthritis (OA) progression in a murine model. The medial meniscus of the knee joint was de‐stabilized to induce OA in 10‐week‐old mice. Mice were then intra‐articularly injected with dimethyl sulfoxide (control), drug‐free gelatin hydrogel, simvastatin, or simvastatin‐conjugated gelatin hydrogel. At 8 weeks postsurgery, OA progression was significantly attenuated in the simvastatin‐conjugated gelatin hydrogel‐treated mice compared with the other three groups. Attenuation of OA in mice treated with simvastatin‐conjugated gelatin hydrogel was associated with decreased expression of cartilage‐degrading enzymes and interleukin (IL)‐1β and increased expression of type II collagen and an autophagic marker, LC3, in the articular cartilage. The effects of simvastatin on gene expression and autophagy in the mouse primary chondrocytes were also examined in the presence or absence of IL‐1β. Treatment with simvastatin down‐regulated Mmp‐13 and Il‐1β and up‐regulated Col2a1 and autophagic activity. Our findings suggest that the intra‐articular administration of simvastatin‐conjugated gelatin hydrogel could be used as a new potential therapy for OA.     

Cartilage regeneration by selected chondrogenic clonal mesenchymal stem cells in the collagenase‐induced monkey osteoarthritis model

Osteoarthritis (OA) is the most common form of arthritis, in which cartilage is irreversibly degraded, causing severe pain and disability. Current therapeutic strategies cannot repair damaged cartilage. We evaluated the repair potential of selected chondrogenic clonal MSCs (sC‐MSCs) by delivering them into the injured cartilage site in a collagenase‐induced OA model in Cynomolgus monkeys. In vitro characterization showed that the isolated monkey sC‐MSCs and polyclonal MSCs (P‐MSCs) expressed mesenchymal stem cell markers and could differentiate into chondrocytes. The articular cartilage lesions in animals were treated with normal saline (NS), autologous P‐MSCs and sC‐MSCs, respectively, by direct delivery. The clinical parameters, radiographic images, histological and immunohistochemical examinations at weeks 8, 16 and 24 post‐treatment demonstrated that the abrasions of articular cartilage were significantly improved and repaired by MSC‐based treatment, particularly in the sC‐MSC‐treated group, which displayed consistently higher histological scores than those of other groups. In summary, treatment with sC‐MSCs can effectively improve the healing of cartilage lesions in the Cynomolgus monkey collagenase‐induced OA model. Due to the genetic proximity of monkey and human, the therapeutic strategy presented in this study will have broad applications in clinical practice. Copyright © 2013 John Wiley & Sons, Ltd.     

Sub-toxic levels of Co2+ are anti-inflammatory and protect cartilage from degradation caused by IL-1β

BackgroundCobalt ions from some orthopaedic implants induce a dose-dependent cytotoxic and pro-inflammatory response. Recent studies show that sub-toxic levels of cobalt influence actin organisation regulating fibroblasts and macrophages behaviour. However little is known about the influence of sub-toxic levels of cobalt on articular cartilage biology and biomechanics. Previously, we have reported that IL-1β signalling in chondrocytes, is regulated by primary cilia and associated intraflagellar transport. Since primary cilia expression is modulated by actin organisation, we set out to test the hypothesis that sub-toxic levels of cobalt regulate cilia expression and IL-1β signalling thereby influencing articular cartilage degradation.MethodsIsolated chondrocytes and bovine cartilage explants were subjected to Co²⁺ in the presence and absence of IL-1β. Primary cilia were monitored by confocal immunofluorescence. Nitric oxide and PGE₂ release were used to monitor IL-1β signalling. Degradation of cartilage matrix was assessed by the release of sGAG and the biomechanical properties of the tissue in uniaxial unconfined compression.FindingsSub-toxic levels of Co²⁺ (50 μM) blocked IL-1β-induced primary cilia elongation in isolated chondrocytes. This was associated with disruption of pro-inflammatory signalling in both isolated chondrocytes and cartilage explants, and inhibition of cartilage matrix degradation and loss of biomechanical properties.InterpretationThis study reveals that low levels of cobalt ions are anti-inflammatory, preventing cartilage degradation in response to IL-1β. This mechanism is associated with regulation of primary cilia elongation. These observations provide new insight into the potential beneficial role of cobalt and may lead to novel mechanisms for controlling cartilage inflammation.     

Passaged human chondrocytes accumulate extracellular matrix when induced by bovine chondrocytes

A source of sufficient number of cells is a major limiting factor for cartilage tissue engineering. To circumvent this problem, we developed a co‐culture method to induce redifferentiation in bovine articular chondrocytes, which had undergone dedifferentiation following serial passage in monolayer culture. In this study we determine whether human osteoarthritic (OA) and non‐diseased passaged dedifferentiated chondrocytes will respond similarly. Human passaged chondrocytes were co‐cultured for 4 weeks with primary bovine chondrocytes and their redifferentiation status was determined. Afterwards the cells were cultured either independently or in co‐culture with cryopreserved passaged cells for functional analysis. The co‐culture of passaged cells with primary chondrocytes resulted in reversion of their phenotype towards articular chondrocytes, as shown by increased gene expression of type II collagen and COMP, decreased type I collagen expression and extracellular matrix formation in vitro. Furthermore, this redifferentiation was stable, as those cells not only formed hyaline‐like cartilage tissue when grown on their own but also they could induce redifferentiation of passaged chondrocytes in co‐culture. These data suggest that it may be possible to use autologous chondrocytes obtained from osteoarthritic cartilage to form tissue suitable to use for cartilage repair. Copyright © 2009 John Wiley & Sons, Ltd.     

Cyclooxygenase-independent mechanism of ibuprofen-induced antipyresis: the role of central vasopressin V₁ receptors

This study compared the antipyretic effects of ibuprofen and indomethacin regarding the efficacy in blocking fevers induced by lipopolysaccharide (LPS from E. coli) or pyrogenic mediators that act on prostaglandin (PG)‐dependent and PG‐independent pathways. The content of PGE₂ in the cerebrospinal fluid (CSF) and the dependence on central arginine vasopressin (AVP) release by both antipyretics were also compared during the reduction of LPS‐induced fever. Finally, we investigated the effect of ibuprofen on hypothalamic cytokine content during LPS‐induced fever. Ibuprofen (intraperitoneally, i.p.) dose‐dependently inhibited the fever induced by LPS (intravenously, i.v.). Indomethacin (2 mg/kg) and ibuprofen (10 mg/kg) reduced the fever induced by i.c.v. injection of interleukin (IL)‐1β, IL‐6, tumor necrosis factor (TNF)‐α, or arachidonic acid (AA). Ibuprofen, but not indomethacin, inhibited i.c.v. endothelin‐1‐ and pre‐formed pyrogenic factor (PFPF)‐induced fever. Neither ibuprofen nor indomethacin affected fever by PGE₂, PGF_2α, or corticotrophin‐releasing factor (CRF); however, both reduced the CSF PGE₂ content after LPS. Bilateral injection of the AVP V₁ receptor antagonist d(CH2)₅Tyr(Me)AVP into the ventral septal area blocked both ibuprofen‐ and indomethacin‐induced antipyresis. Ibuprofen did not modify the hypothalamic increase in either IL‐1β or IL‐6 induced by LPS. In conclusion, although the antipyretic effect of ibuprofen involves the blockage of central production of PGE₂ and the endogenous release of AVP, differently from low dose of indomethacin, ibuprofen not only reduced the fever induced by PGE₂‐dependent, but also, that induced by PGE₂‐independent endogenous pyrogens. Moreover, ibuprofen does not affect the hypothalamic synthesis/release of IL‐1β and IL‐6.     

Differences in the sensitivity of behavioural measures of pain to the selectivity of cyclo‐oxygenase inhibitors

Objectives: Freund's complete adjuvant (FCA) is an animal model of inflammatory pain commonly used in the screening of COX‐inhibitors. However, there is little understanding of how behavioural measures of the anti‐inflammatory effect in the FCA model correlate to differences in mechanism of action and whether such endpoints equally reflect drug activity in humans. In the current investigation we evaluate the time course of the analgesic effect for different endpoints after treatment with drugs with varying degrees of selectivity for COX‐1 and COX‐2. We also assess prostaglandin (PGE₂) and thromboxane (TXB₂) inhibition to establish the correlation between behavioural measures and the degree of selectivity for COX‐1 and COX‐2. Methods: Sprague–Dawley rats were treated with FCA by intra‐plantar injection. On post‐inoculation day (PID) 7, rats received a single oral dose of naproxen, diclofenac, ketorolac or rofecoxib. Drug treatment continued until PID 21. A control group received placebo only. Behavioural endpoints for inflammatory pain and blood samples for biomarkers were obtained at various time points before and after dosing to characterise the time course of drug effect and disease progression. Results: COX‐inhibitors showed no effect on the dynamic plantar test. In contrast, full analgesia was observed after drug administration for weight bearing capacity (WBC) and paw pressure (PP), with varying duration of the effect for each of the endpoints. No tolerance to drug effect was observed up to 14 days of chronic treatment. Rofecoxib showed an increase in baseline pain threshold values after chronic treatment, which may be related to its pharmacokinetic characteristics. Conclusions: Changes in paw pressure threshold seem to best reflect the anti‐hyperalgesic properties of COX‐inhibitors with enough sensitivity to enable estimation of the dose‐exposure‐response curve.     

Bioenergetic reprogramming of articular chondrocytes by exposure to exogenous and endogenous reactive oxygen species and its role in the anabolic response to low oxygen

Monolayer culture is integral to many cell‐based cartilage repair strategies, but chondrocytes lose regenerative potential with increasing duration in vitro. This coincides with elevated reactive oxygen species (ROS) levels and a bioenergetic transformation characterized by increasing mitochondrial function. This study investigates ROS as stimuli for bioenergetic reprogramming and the effect of antioxidants on the propensity of chondrocytes to regenerate a cartilaginous matrix. Articular chondrocytes were cultured in monolayer under a 2% O₂ atmosphere. Oxidative stress was increased using 50 μm H₂O₂ or a 20% O₂ culture atmosphere, or decreased using the antioxidant N‐acetyl‐cysteine (NAC). Mitochondrial function was characterized using 200 nm Mitotracker green and an oxygen biosensor. After two population doublings ± NAC, chondrocytes were encapsulated in alginate beads (1 × 10⁷ cells/ml) for an additional 10 days before DMB assay of glycosaminoglycan content. The beads were cultured under both 20% O₂ and the more physiological 5% O₂ condition. Chondrocytes expanded in 20% O₂ exhibited elevated mitochondrial mass and functional capacity, which was partially mimicked by the exogenous ROS, H₂O₂. Oligomycin treatment revealed that the increased oxygen consumption was coupled to oxidative phosphorylation. NAC limited these markers of bioenergetic reprogramming during culture‐expansion with no significant effect on subsequent GAG production under 20% O₂. However, NAC treatment in monolayer abolished the hypoxic induction of GAG in alginate beads. This supports the hypothesis of a causal relationship between exposure to ROS and acquired mitochondrial function in chondrocytes. Additionally, mitochondrial function may be required for the hypoxic induction of GAG synthesis by chondrocytes. © 2015 The Authors. Journal of Tissue Engineering and Regenerative Medicine Published by John Wiley & Sons, Ltd.     

The carbon monoxide-releasing molecule tricarbonyldichlororuthenium(II) dimer protects human osteoarthritic chondrocytes and cartilage from the catabolic actions of interleukin-1beta

We have investigated the effects of a carbon monoxide-releasing molecule, tricarbonyldichlororuthenium(II) dimer (CORM-2), on catabolic processes in human osteoarthritis (OA) cartilage and chondrocytes activated with interleukin-1beta. In these cells, proinflammatory cytokines induce the synthesis of matrix metalloproteinases (MMPs) and aggrecanases, including members of a disintegrin and metalloproteinase with thrombospondin domain (ADAMTS) family, which may contribute to cartilage loss. CORM-2 down-regulated MMP-1, MMP-3, MMP-10, MMP-13, and ADAMTS-5 in OA chondrocytes, and it inhibited cartilage degradation. These effects were accompanied by increased aggrecan synthesis and collagen II expression in chondrocytes. Our results also indicate that the inhibition of extracellular signal-regulated kinase 1/2 and p38 activation by CORM-2 may contribute to the maintenance of extracellular matrix homeostasis. These observations suggest that CORM-2 could exert chondroprotective effects due to the inhibition of catabolic activities and the enhancement of aggrecan synthesis.     

Phenyl N-tert-butylnitrone down-regulates interleukin-1 beta-stimulated matrix metalloproteinase-13 gene expression in human chondrocytes: suppression of c-Jun NH2-terminal kinase,p38-mitogen-activated protein kinase and activating protein-1

Cytokine-mediated induction and overexpression of matrix metalloproteinases (MMPs) is recognized as an important factor in the pathogenesis of arthritis. Interleukin (IL)-1 beta is a proinflammatory cytokine that is known to superinduce the expression and production of MMP-13 in many cell types. Phenyl N-tert-butylnitrone (PBN), a spin trap agent, inhibited the IL-1 beta-induced expression of MMP-13 in human osteoarthritis (OA) chondrocytes. Down-regulation of MMP-13 expression correlated with the inhibition of mitogen-activated protein kinase (MAPK) subgroups c-Jun NH2-terminal kinase (JNK) and p38-MAPK activation, accumulation of phospho-c-jun, and the DNA binding activity of activating protein-1 (AP-1). Results of in vitro kinase assays showed that exogenously added PBN completely blocked the c-Jun phosphorylating activity of JNK. Interestingly, using in vitro kinase assay, we also found that chondrocyte p38-MAPK phosphorylate c-Jun and that PBN was not very effective in inhibiting c-Jun phosphorylating activity of p38-MAPK. In addition, PBN did not block the ATF-2 phosphorylating activity of p38-MAPK and Elk-1 phosphorylating activity of extracellular regulated kinase p44/p42 in vitro, indicating that PBN may act selectively to inhibit the phosphorylation of c-Jun in OA chondrocytes. Together, our results for the first time demonstrate that PBN suppresses the IL-1 beta-stimulated expression of MMP-13 in OA chondrocytes and that this was achieved by inhibiting the activation of JNK and AP-1. These results suggest that use of PBN or compounds derived from it may be of potential benefit in inhibiting signaling events associated with cartilage degradation in arthritis.