Objective. Autophagy, an evolutionarily conserved process for the bulk degradation of cytoplasmic components, serves as a cell survival mechanism. The purpose of this study was to elucidate the role of autophagy in human chondrocytes and pathophysiology of osteoarthritis (OA).Methods. Autophagy in articular cartilage and primary chondrocytes was assessed using antibodies for the autophagy markers light chain 3 and beclin 1. The states of autophagy under catabolic and nutritional stresses were examined. We also examined the effects of inhibition or induction of autophagy under stimulation with interleukin-1␤. Autophagy was inhibited by small interfering RNA targeting ATG5, and autophagy was induced by rapamycin. The effects of inhibition or induction of autophagy were examined by real-time polymerase chain reaction for aggrecan, COL2A1, MMP13, and ADAMTS5 messenger RNA. To further examine the mechanism of autophagy regulation in OA human chondrocytes, we investigated whether autophagy modulates apoptosis and reactive oxygen species (ROS).Results. Autophagy was increased in OA chondrocytes and cartilage. Catabolic and nutritional stresses increased autophagy. In addition, the inhibition of autophagy caused OA-like gene expression changes, while the induction of autophagy prevented them. Furthermore, the inhibition of autophagy increased the amount of cleaved poly(ADP-ribose) polymerase and cleaved caspase 9, while the induction of autophagy inhibited these increases. ROS activity was also decreased by induction of autophagy.Conclusion. These observations suggested that increased autophagy is an adaptive response to protect cells from stresses, and that autophagy regulates OAlike gene expression changes through the modulation of apoptosis and ROS. Further studies about autophagy in chondrocytes will provide novel insights into the pathophysiology of OA.

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IntroductionRecent studies have revealed that rapamycin activates autophagy in human chondrocytes preventing the development of osteoarthritis (OA) like changes in vitro, while the systemic injection of rapamycin reduces the severity of experimental osteoarthritis in a murine model of OA in vivo. Since the systemic use of rapamycin is associated with numerous side effects, the goal of the current study was to examine the beneficial effect of local intra-articular injection of rapamycin in a murine model of OA and to elucidate the mechanism of action of rapamycin on articular cartilage.MethodsDestabilization of the medial meniscus (DMM) was performed on 10-week-old male mice to induce OA. Intra-articular injections of 10 μl of rapamycin (10 μM) were administered twice weekly for 8 weeks. Articular cartilage damage was analyzed by histology using a semi-quantitative scoring system at 8 and 12 weeks after surgery. Mammalian target of rapamycin (mTOR), light chain 3 (LC3), vascular endothelial growth factor (VEGF), collagen, type X alpha 1 (COL10A1), and matrix metallopeptidase 13 (MMP13) expressions were analyzed by immunohistochemistry. VEGF, COL10A1, and MMP13 expressions were further examined via quantitative RT-PCR (qPCR).ResultsIntra-articular injection of rapamycin significantly reduced the severity of articular cartilage degradation at 8 and 12 weeks after DMM surgery. A reduction in mTOR expression and the activation of LC3 (an autophagy marker) in the chondrocytes was observed in the rapamycin treated mice. Rapamycin treatment also reduced VEGF, COL10A1, and MMP13 expressions at 8 and 12 weeks after DMM surgery.ConclusionThese results demonstrate that the intra-articular injection of rapamycin could reduce mTOR expression, leading to a delay in articular cartilage degradation in our OA murine model. Our observations suggest that local intra-articular injection of rapamycin could represent a potential therapeutic approach to prevent OA.

Selective interference of mTORC1/RAPTOR protects against inflammation-induced apoptosis, senescence, and matrix catabolism possibly through Akt and autophagy induction in human disc cells.

Medial transfer of the tibial tuberosity has been commonly used for treatment of recurrent dislocation of the patella and patellofemoral malalignment. In this study, six fresh human cadaveric knees were used. Static intrajoint loads were recorded using Fuji Prescale pressure-sensitive film for contact pressure and contact area determination in a closed kinetic chain knee testing protocol. Peak pressures, average contact pressures, and contact areas of the patellofemoral and tibiofemoral joints were calculated on native intact knee specimens and after tibial tuberosity transfer. All native intact knee specimens had a normal Q angle. Medialization of the tibial tuberosity significantly increased the patellofemoral contact pressure. Medial displacement of the tibial tuberosity also significantly increased the average contact pressure of the medial tibiofemoral compartment and changed the balance of tibiofemoral joint loading. The results of our study suggest that caution should be used when transferring a patellar tendon in the face of a preexisting normal Q angle as this will result in abnormally high peak pressure within the tibiofemoral joint. Overmedialization of the tibial tuberosity should be avoided in the varus knee, the knee after medial meniscectomy, and the knee with preexisting degenerative arthritis of the medial compartment.

1 The present study examined eects of agonist enzymes and receptor-activating peptides for protease-activated receptors (PARs) on duodenal motility in the rat, and also investigated possible mechanisms underlying the evoked responses. 2 Thrombin at 0.03 ± 0.1 mM and the PAR-1-activating peptide SFLLR-NH 2 at 3 ± 100 mM or TFLLR-NH 2 at 10 ± 50 mM produced a dual action, relaxation followed by contraction of the duodenal longitudinal muscle. The PAR-2-activating peptide SLIGRL-NH 2 at 10 ± 100 mM elicited only small contraction. Trypsin at 0.08 mM induced small contraction, or relaxation followed by contraction, depending on preparations. The PAR-4-activating peptide GYPGKF-NH 2 at 1000 mM exhibited no eect. 3 The contractile responses of the duodenal strips to TFLLR-NH 2 and to SLIGRL-NH 2 were partially attenuated by the L-type calcium channel blocker nifedipine (1 mM), the protein kinase C inhibitor GF109203X (1 mM) and the tyrosine kinase inhibitor genistein (15 mM), but were resistant to indomethacin (3 mM) and tetrodotoxin (1 ± 10 mM). 4 The relaxation of the preparations exerted by TFLLR-NH 2 was unaected by indomethacin (3 mM), propranolol (5 mM), N G -nitro-L-arginine methyl ester (100 mM) and tetrodotoxin (1 ± 10 mM). This relaxation was resistant to either GF109203X (1 mM) or genistein (15 mM), but was, remarkably, attenuated by combined application of these two kinase inhibitors. 5 Apamin (0.1 mM), an inhibitor of calcium-activated, small-conductance potassium channels, but not charybdotoxin (0.1 mM), completely abolished the PAR-1-mediated duodenal relaxation, and signi®cantly enhanced the PAR-1-mediated contraction. 6 These ®ndings demonstrate that PAR-1 plays a dual role, suppression and facilitation of smooth muscle motility in the rat duodenum, while PAR-2 plays a minor excitatory role in the muscle, and that PAR-4 is not involved in the duodenal tension modulation. The results also suggest that the contractile responses to PAR-1 and PAR-2 activation are mediated, in part, by activation of L-type calcium channels, protein kinase C and tyrosine kinase, and that the relaxation response to PAR-1 activation occurs via activation of apamin-sensitive, but charybdotoxin-insensitive, potassium channels, in which both protein kinase C and tyrosine kinase might be involved synergistically.

Proteinase‐activated receptor‐2 (PAR‐2) is expressed throughout the gastrointestinal tract including the pancreas, and may be involved in digestive functions. The aim of our study was to evaluate a potential role for PAR‐2 in regulating salivary and pancreatic exocrine secretion in vivo. PAR‐2‐activating peptides (PAR‐2‐APs), but not selective PAR‐1‐APs, administered intravenously, increased salivary secretion in the mouse or rat; this effect of the PAR‐2‐APs was unaffected by atropine, phentolamine, propranolol or indomethacin. Secretion (amylase) by rat parotid gland slices in vitro was also stimulated by PAR‐2‐APs and trypsin, but not by activation of other PARs. PAR‐2‐APs, administered to rats in vivo, caused a prompt effect on pancreatic exocrine secretion. PAR‐2 mRNA, known to be present in pancreatic tissue, was also detected in parotid tissue. Our results indicate that in addition to a potential role in regulating cardiovascular and respiratory functions, PAR‐2 may also play a general role in vivo for the direct regulation of glandular exocrine secretion. British Journal of Pharmacology (2000) 129, 1808–1814; doi:

To develop potent and metabolically stable agonists for protease-activated receptor-2 (PAR-2), we prepared 2-furoylated (2f) derivatives of native PAR-2-activating peptides, 2f-LIGKV-OH, 2f-LIGRL-OH, 2f-LIGKV-NH 2 , and 2f-LIGRL-NH 2 , and systematically evaluated their activity in PAR-2-responsive cell lines and tissues. In both HCT-15 cells and NCTC2544 cells overexpressing PAR-2, all furoylated peptides increased cytosolic Ca 2ϩ levels with a greater potency than the corresponding native peptides, although a similar maximum response was recorded. The absolute potency of each peptide was greater in NCTC2544, possibly due to a higher level of receptor expression. Furthermore, the difference in potency between the 2-furoylated peptides and the native peptides was enhanced when evaluated in the rat superior mesenteric artery and further increased when measuring PAR-2-mediated salivation in ddY mice in vivo. The potency of 2f-LIGRL-NH 2 , the most powerful peptide, relative to SLIGKV-OH, was about 100 in the cultured cell Ca 2ϩ signaling assays, 517 in the vasorelaxation assay, and 1100 in the salivation assay. Amastatin, an aminopeptidase inhibitor, augmented salivation caused by native peptides, but not furoylated peptides. The PAR-2-activating peptides, including the furoylated derivatives, also produced salivation in the wild-type C57BL/6 mice, but not the PAR-2-deficient mice. Our data thus demonstrate that substitution of the N-terminal serine with a furoyl group in native PAR-2-activating peptides dramatically enhances the agonistic activity and decreases degradation by aminopeptidase, leading to development of 2f-LIGRL-NH 2 , the most potent peptide. Furthermore, the data from PAR-2-deficient mice provide ultimate evidence for involvement of PAR-2 in salivation and the selective nature of the 2-furoylated peptides.