Kousuke Kumagai

Regulatory role of tyrosine phosphorylation in the swelling-activated chloride current in isolated rabbit articular chondrocytes

Articular chondrocytes are exposed in vivo to the continually changing osmotic environment and thus require volume regulatory mechanisms. The present study was designed to investigate (i) the functional role of the swelling‐activated Cl− current (ICl,swell) in the regulatory volume decrease (RVD) and (ii) the regulatory role of tyrosine phosphorylation in ICl,swell, in isolated rabbit articular chondrocytes. Whole‐cell membrane currents were recorded from chondrocytes in isosmotic, hyposmotic and hyperosmotic external solutions under conditions where Na+, K+ and Ca2+ currents were minimized. The cell surface area was also measured using microscope images from a separate set of chondrocytes and was used as an index of cell volume. The isolated chondrocytes exhibited a RVD during sustained exposure to hyposmotic solution, which was mostly inhibited by the ICl,swell blocker 4‐(2‐butyl‐6,7‐dichloro‐2‐cyclopentyl‐indan‐1‐on‐5‐yl)oxobutyric acid (DCPIB) at 20 μm. Exposure to a hyposmotic solution activated ICl,swell, which was also largely inhibited by 20 μm DCPIB. ICl,swell in rabbit articular chondrocytes had a relative taurine permeability (Ptau/PCl) of 0.21. Activation of ICl,swell was significantly reduced by the protein tyrosine kinase (PTK) inhibitor genistein (30 μm) but was only weakly affected by its inactive analogue daidzein (30 μm). Intracellular application of protein tyrosine phosphatase (PTP) inhibitor sodium orthovanadate (250 and 500 μm) resulted in a gradual activation of a Cl− current even in isosmotic solutions. This Cl− current was almost completely inhibited by 4,4′‐diisothiocyanatostilbene‐2,2′‐disulfonate (DIDS, 500 μm) and was also largely suppressed by exposure to hyperosmotic solution, thus indicating a close similarity to ICl,swell. Pretreatment of chondrocytes with genistein significantly prevented the activation of the Cl− current by sodium orthovanadate, suggesting that the basal activity of endogenous PTK is required for the activation of this Cl− current. Our results provide evidence to indicate that activation of ICl,swell is involved in RVD in isolated rabbit articular chondrocytes and is facilitated by tyrosine phosphorylation.

Improved quality of cartilage repair by bone marrow mesenchymal stem cells for treatment of an osteochondral defect in a cynomolgus macaque model

Background and purpose — Integration of repaired cartilage with surrounding native cartilage is a major challenge for successful tissue-engineering strategies of cartilage repair. We investigated whether incorporation of mesenchymal stem cells (MSCs) into the collagen scaffold improves integration and repair of cartilage defects in a cynomolgus macaque model. Methods — Cynomolgus macaque bone marrow-derived MSCs were isolated and incorporated into type-I collagen gel. Full-thickness osteochondral defects (3 mm in diameter, 5 mm in depth) were created in the patellar groove of 36 knees of 18 macaques and were either left untreated (null group, n = 12), had collagen gel alone inserted (gel group, n = 12), or had collagen gel incorporating MSCs inserted (MSC group, n = 12). After 6, 12, and 24 weeks, the cartilage integration and tissue response were evaluated macroscopically and histologically (4 null, 4 gel, and 4 MSC knees at each time point). Results — The gel group showed most cartilage-rich reparative tissue covering the defect, owing to formation of excessive cartilage extruding though the insufficient subchondral bone. Despite the fact that a lower amount of new cartilage was produced, the MSC group had better-quality cartilage with regular surface, seamless integration with neighboring naïve cartilage, and reconstruction of trabecular subchondral bone. Interpretation — Even with intensive investigation, MSC-based cell therapy has not yet been established in experimental cartilage repair. Our model using cynomolgus macaques had optimized conditions, and the method using MSCs is superior to other experimental settings, allowing the possibility that the procedure might be introduced to future clinical practice.

17β‐Oestradiol inhibits doxorubicin‐induced apoptosis via block of the volume‐sensitive Cl‐ current in rabbit articular chondrocytes

BACKGROUND AND PURPOSE Chondrocyte apoptosis contributes to disruption of cartilage integrity in osteoarthritis. Recent evidence suggested that the volume‐sensitive organic osmolyte/anion channel [volume‐sensitive (outwardly rectifying) Cl‐ current (ICl,vol)] plays a functional role in the development of cell shrinkage associated with apoptosis (apoptotic volume decrease) in several cell types. In this study, we investigated the cellular effects of 17β‐oestradiol on doxorubicin‐induced apoptotic responses in rabbit articular chondrocytes.

Lidocaine induces ROCK-dependent membrane blebbing and subsequent cell death in rabbit articular chondrocytes

Local anesthetics are administered intraarticularly for pain control in orthopedic clinics and surgeries. Although previous studies have shown that local anesthetics can be toxic to chondrocytes, the underlying cellular mechanisms remain unclear. The present study investigates acute cellular responses associated with lidocaine‐induced toxicity to articular chondrocytes. Rabbit articular chondrocytes were exposed to lidocaine and their morphological changes were monitored with live cell microscopy. The viability of chondrocytes was evaluated using a fluorescence based LIVE/DEAD assay. Acute treatment of chondrocytes with lidocaine (3–30 mM) induced spherical protrusions on the cell surface (so called “membrane blebbing”) in a time‐ and concentration‐dependent manner. The concentration‐response relationship for the lidocaine effect was shifted leftward by elevating extracellular pH, as expected for the non‐ionized lidocaine being involved in the bleb formation. ROCK (Rho‐kinase) inhibitors Y‐27632 and fasudil completely prevented the lidocaine‐induced membrane blebbing, suggesting that ROCK activation is required for bleb formation. Caspase‐3 levels were unchanged by 10 mM lidocaine (p = 0.325) and a caspase inhibitor z‐VAD‐fmk did not affect the lidocaine‐induced blebbing (p = 0.964). GTP‐RhoA levels were significantly increased (p < 0.001), but Rho inhibitor‐1 failed to suppress the membrane blebbing (p = 0.875). Lidocaine (30 mM) reduced the cell viability of isolated chondrocytes (p < 0.001) and in situ chondrocytes (p < 0.001). The chondrotoxicity was attenuated by pretreatment of cells with ROCK inhibitors or a myosin‐II inhibitor blebbistatin (p < 0.001). These findings suggest that lidocaine induces ROCK‐dependent membrane blebbing and thereby produces a cytotoxic effect on chondrocytes.

The COX-2 Selective Blocker Etodolac Inhibits TNFα-Induced Apoptosis in Isolated Rabbit Articular Chondrocytes

Chondrocyte apoptosis contributes to the disruption of cartilage integrity in osteoarthritis (OA). Recently, we reported that activation of volume-sensitive Cl− current (ICl,vol) mediates cell shrinkage, triggering apoptosis in rabbit articular chondrocytes. A cyclooxygenase (COX) blocker is frequently used for the treatment of OA. In the present study, we examined in vitro effects of selective blockers of COX on the TNFα-induced activation of ICl,vol in rabbit chondrocytes using the patch-clamp technique. Exposure of isolated chondrocytes to TNFα resulted in an obvious increase in membrane Cl− conductance. The TNFα-evoked Cl− current exhibited electrophysiological and pharmacological properties similar to those of ICl,vol. Pretreatment of cells with selective COX-2 blocker etodolac markedly inhibited ICl,vol activation by TNFα as well as subsequent apoptotic events such as apoptotic cell volume decrease (AVD) and elevation of caspase-3/7 activity. In contrast, a COX-1 blocker had no effect on the decrease in cell volume or the increase in caspase-3/7 activity induced by TNFα. Thus, the COX-2-selective blocker had an inhibitory effect on TNFα-induced apoptotic events, which suggests that this drug would have efficacy for the treatment of OA.

Extracellular and intracellular mechanisms of mechanotransduction in three-dimensionally embedded rat chondrocytes.

Purpose Articular cartilage homeostasis involves modulation of chondrocyte matrix synthesis in response to mechanical stress (MS). We studied extracellular and intracellular mechanotransduction pathways mediating this response. Methods We first confirmed rapid up-regulation of the putative chondro-protective cytokine, interleukin (IL)-4, as an immediate response to MS. We then studied the role of IL-4 by investigating responses to exogenous IL-4 or a specific IL-4 inhibitor, combined with MS. Next we investigated the intracellular second messengers. Since chondrocyte phenotype alters according to the extracellular environment, we characterized the response to mechanotransduction in 3-dimensionally embedded chondrocytes. Results Expression of aggrecan and type II collagen was significantly up-regulated by exogenous IL-4 whereas MS-induced matrix synthesis was inhibited by an IL-4 blocker. Further, MS-induced matrix synthesis was completely blocked by a p38 MAPK inhibitor, while it was only partially blocked by inhibitors of other putative second messengers. Conclusion IL-4 mediates an extracellular pathway of mechanotransduction, perhaps via an autocrine/paracrine loop, while p38 mediates an intracellular pathway prevalent only in a 3-dimensional environment.

FRI0179 Reducion in Complement C3 and C4 Levels Greater with Tocilizumab as Compared to Anti-TNF in Patients with Rheumatoid Arthritis

Background A reduction in complement levels during anti-TNF treatment in patients with rheumatoid arthritis (RA) has been described. However, there are no reports regarding changes in complement levels with the use of Tocilizumab (TCZ). Objectives In this study, we investigated changes in complement levels during TCZ treatment and compared them to those with anti-TNF treatment in patients with RA. Methods We measured C3 and C4 levels in 25 patients treated with TCZ and 44 treated with anti-TNF. In addition, major disease activity indicators were monitored before and 1 year after the initiation of treatment. Results Disease activities (DAS28-ESR) were significantly improved in both groups after 1 year. Furthermore, C3 and C4 concentrations were significantly reduced in both after 6 months and 1 year, though those effects were significantly more pronounced in the TCZ group than the anti-TNF group (graphs A and B, p<0.001). Notably, patients in the TCZ group who showed a reduction in C4 to under the lower limit had significantly lower disease activity than others (graph C). Conclusions TCZ more effectively reduces complement C3 and C4 in patients with RA as compared with anti-TNF. Our results also indicate that C4 may be usable as a biomarker in RA patients undergoing TCZ treatment. References Di Mucio G, Perricone C, Ballanti E, et al. 2011. Complement system and rheumatoid arthritis: relationships with autoantibodies, serological, clinical features, and anti-TNF treatment. Int J Immunopathol Pharmacol; 24(2): 357-66. Disclosure of Interest None declared