sirolimus and Osteoarthritis

Degradation of the articular cartilage is at the centre of the pathogenesis of osteoarthritis (OA), for which age is the major risk factor. Maintaining the chondrocytes in a healthy condition appears to be an important factor for preservation of the entire cartilage and preventing its degeneration. Autophagy, which is an essential cellular homeostatic mechanism for the removal of dysfunctional cellular organelles and macromolecules, is increased by catabolic and nutritional stresses. Autophagy is increased in OA chondrocytes and cartilage, particularly during the initial degenerative phase, to regulate changes in OA-like gene expression through modulation of apoptosis and reactive oxygen species (ROS). In this way, autophagy acts as an adaptive response to protect chondrocytes from various environmental changes, while with gradual cartilage degradation, decreased autophagy is linked with cell death. Rapamycin, which is a specific inhibitor of the mTOR signaling pathway, enhances expression of autophagy regulators and prevents chondrocyte death. In the future, pharmacological activation of autophagy may be an effective therapeutic approach for OA.

Topics: Autophagy; Cartilage, Articular; Chondrocytes; Humans; Immunosuppressive Agents; Inflammation; Osteoarthritis; Sirolimus

Osteoarticular disorders are the major cause of disability in Europe and North America. It is estimated that rheumatoid arthritis affects 1 % of the population and that more than two third of people over age 55 develop osteoarthritis. Because there are no satisfactory treatments, gene therapy offers a new therapeutic approach. The delivery of cDNA encoding anti-arthritic proteins to articular cells has shown therapeutic efficacy in numerous animal models in vivo. Through the development and the experimental progresses that have been made for both rheumatoid arthritis and osteoarthritis, this review discusses the different gene therapy strategies available today and the safety issues with which they may be associated. Among the different vectors available today, adeno-associated virus seems the best candidate for a direct in vivo gene delivery approach for the treatment of joint disorders.

Topics: Aged; Animals; Arthritis, Rheumatoid; Cartilage, Articular; Cytokines; Dependovirus; DNA, Complementary; Dogs; Doxycycline; Etanercept; Gene Expression; Genes, Synthetic; Genetic Therapy; Genetic Vectors; Haplorhini; Horses; Humans; Immunoglobulin G; Injections, Intra-Articular; Mice; Middle Aged; Osteoarthritis; Receptors, Tumor Necrosis Factor; Receptors, Tumor Necrosis Factor, Type II; Sirolimus

Mesenchymal stem cell (MSC) therapy is a promising strategy for treating osteoarthritis (OA). However, the inflammatory microenvironment, apoptosis of transplanted cells, and shear forces during direct injection limit the therapeutic efficacy. This study aimed to explore the role of rapamycin combined with human umbilical-cord-derived mesenchymal stem cells (hUMSCs) in OA rabbits. OA rabbits received an intra-articular injection of a collagenase solution. Gross observations, X-ray examinations, and histological examinations were performed to detect cartilage degradation levels. The fluorescent membrane dye DiR was used to label hUMSCs. In the combination therapy group, rapamycin was injected into the rabbit knee joint one day post the intra-articular injection of hUMSCs. Bioinformatics and transcriptome profiling of the knee meniscus were used to evaluate the potential molecular mechanisms of the combination therapy.. Our study shows that rapamycin combined with hUMSCs significantly ameliorated OA severity. Our study indicates that the combination therapy of rapamycin and hUMSCs may promote cartilage repair in OA rabbits through the mTOR pathway and offers a novel approach for OA therapy.. Our study provides new evidence to support the use of hUMSCs in combination with rapamycin as a potential candidate for OA treatment.

Topics: Animals; Cartilage, Articular; Humans; Mesenchymal Stem Cells; Osteoarthritis; Proto-Oncogene Proteins c-akt; Rabbits; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

Liposomes show promise as biolubricants for damaged cartilage, but their small size results in low joint and cartilage retention. We developed a zinc ion-based liposomal drug delivery system for local osteoarthritis therapy, focusing on sustained release and tribological protection from phospholipid lubrication properties. Our strategy involved inducing aggregation of negatively charged liposomes with zinc ions to extend rapamycin (RAPA) release and improve cartilage lubrication. Liposomal aggregation occurred within 10 min and was irreversible, facilitating excess cation removal. The aggregates extended RAPA release beyond free liposomes and displayed irregular morphology influenced by RAPA. At nearly 100 µm, the aggregates were large enough to exceed the previously reported size threshold for increased joint retention. Tribological assessment on silicon surfaces and ex vivo porcine cartilage revealed the system's excellent protective ability against friction at both nano- and macro-scales. Moreover, RAPA was shown to attenuate the fibrotic response in human OA synovial fibroblasts. Our findings suggest the zinc ion-based liposomal drug delivery system has potential to enhance OA therapy through extended release and cartilage tribological protection, while also illustrating the impact of a hydrophobic drug like RAPA on liposome aggregation and morphology.

Topics: Cartilage, Articular; Friction; Humans; Liposomes; Lubrication; Osteoarthritis; Phospholipids; Sirolimus

Osteoarthritis (OA) is a prevalent joint disease that affects all the tissues within the joint and currently lacks disease-modifying treatments in clinical practice. Despite the potential of rapamycin for OA disease alleviation, its clinical application is hindered by the challenge of achieving therapeutic concentrations, which necessitates multiple injections per week. To address this issue, rapamycin was loaded into poly(lactic-co-glycolic acid) nanoparticles (RNPs), which are nontoxic, have a high encapsulation efficiency and exhibit sustained release properties for OA treatment. The RNPs were found to promote chondrogenic differentiation of ATDC5 cells and prevent senescence caused by oxidative stress in primary mouse articular chondrocytes. Moreover, RNPs were capable to alleviate metabolism homeostatic imbalance of primary mouse articular chondrocytes in both monolayer and 3D cultures under inflammatory or oxidative stress. In the mouse destabilization of the medial meniscus (DMM) model, intra-articular injection of RNPs effectively mitigated joint cartilage destruction, osteophyte formation, chondrocytes hypertrophy, synovial inflammation, and pain. Our study demonstrates the feasibility of using RNPs as a potential clinically translational therapy to prevent the progression of post-traumatic OA.

Topics: Animals; Cartilage, Articular; Disease Models, Animal; Mice; Nanoparticles; Osteoarthritis; Sirolimus

Osteoarthritis (OA) is a common type of degenerative joint diseases that is regulated by a combination of complex intercellular signals and modulators, including non-coding RNAs. Mounting evidence suggests that miR-31-5p is physiologically involved in the regulation of chondrocytes, but the mechanism remains unclear.. Expression levels of miR-31-5p and SOX4 in OA cartilage tissues and in IL-1β-stimulated chondrocytes were examined by quantification polymerase chain reaction (q-PCR) or immunohistochemistry assays. Cell proliferation and apoptosis were detected by Cell Counting Kit-8 (CCK-8) and flow cytometry assays, respectively. Expression of LC3 was detected using immunofluorescence staining. Expressions of autophagy-related proteins and extracellular regulated protein kinase (ERK)/mechanical target of rapamycin kinase (mTORC1) signal-related proteins were measured by Western blot analysis. Molecular interaction was validated by dual luciferase reporter assay.. Downregulation of miR-31-5p and upregulation of SOX4 were observed in both OA patients and OA chondrocytes. Mechanistic experiments revealed that miR-31-5p negatively modulated SOX4 expression by directly targeting its 3'- untranslated region. Moreover, overexpression of miR-31-5p suppressed the activation of mTORC1 in an ERK-dependent manner by inhibiting SOX4. Further functional experiments demonstrated that overexpressing miR-31-5p in OA chondrocytes markedly promoted its proliferation and autophagy while inhibiting apoptosis. However, these effects were abolished by overexpression of SOX4 or treatment with 3BDO, an mTOR activator.. These results demonstrated that miR-31-5p enhanced survival and autophagy of OA chondrocytes through inactivation of mTORC1 via directly targeting SOX4, suggesting that miR-31-5p may play a protective role in OA progression.

Topics: Apoptosis; Autophagy; Chondrocytes; Humans; MicroRNAs; Osteoarthritis; Protein Kinases; Sirolimus; SOXC Transcription Factors

During osteoarthritis (OA) development, chondrocytes progressively decompensate, upregulating proteolytic enzymes and reducing the key growth factors involved in promoting chondrocyte anabolism. A combined therapeutic approach is needed to address this multifactorial pathology, which affects the whole joint. Based on the literature, three promising targets for OA treatment have been selected: MMP3 (matrix metallopeptidase 3), TRPV4 (transient receptor potential cation channel subfamily V member 4) and mTOR (mammalian target of rapamycin). In this study, a novel water-soluble and biocompatible amphiphilic polymer named "sHA-oleylamide" was synthesized and screened from a series of hyaluronic acid derivatives for its anticatabolic activity. This MMP inhibitor showed no cytotoxicity, and in an in vitro model of inflammatory OA, it reversed the inflammatory outcome at a concentration of 0.011 mg/mL. The ability of sHA-oleylamide to form 20-50 nm micelles in water with a critical micelle concentration of 0.27±0.1 mg/mL, was confirmed by TEM images and measured by Nile red staining. RN-1747 and rapamycin molecules were successfully loaded in sHA-oleylamide, previously prepared at 12 mg/mL in PBS; both formulations were stable, sterile and confirmed in vitro to have mTOR inhibition by rapamycin and TRPV4 activation activity by RN-1747. The controlled release of RN-1747 from the micellar formulation with sHA-oleylamide showed that only approximately 60% of the total loaded RN-1747 was released within 7 days. These micellar formulations can potentially increase the bioavailability and pharmaceutical efficacy of the selected active molecules, combining their anti-catabolic and pro-anabolic activities and making them suitable for i.a. administration as OA treatments.

Topics: Drug Delivery Systems; Humans; Hyaluronic Acid; Micelles; Osteoarthritis; Sirolimus; Sulfates; TOR Serine-Threonine Kinases; TRPV Cation Channels; Water

Local inflammation in the joint is considered to contribute to osteoarthritis (OA) progression. Here, we describe an immunomodulating nanoparticle for OA treatment. Intradermal injection of lipid nanoparticles (LNPs) loaded with type II collagen (Col II) and rapamycin (LNP-Col II-R) into OA mice effectively induced Col II-specific anti-inflammatory regulatory T cells, substantially increased anti-inflammatory cytokine expression, and reduced inflammatory immune cells and proinflammatory cytokine expression in the joints. Consequently, LNP-Col II-R injection inhibited chondrocyte apoptosis and cartilage matrix degradation and relieved pain, while injection of LNPs loaded with a control peptide and rapamycin did not induce these events. Adoptive transfer of CD4

Topics: Animals; Anti-Inflammatory Agents; Collagen Type II; Cytokines; Mice; Nanoparticles; Osteoarthritis; Sirolimus; T-Lymphocytes, Regulatory

The objective of this study was to determine if mechanistic target of rapamycin (mTOR) inhibition with or without AMP-activated protein kinase (AMPK) activation can protect against primary, age-related OA.. Dunkin-Hartley guinea pigs develop mild primary OA pathology by 5 months of age that progresses to moderate OA by 8 months of age. At 5 months, guinea pigs served as young control (n = 3) or were fed either a control diet (n = 8), a diet enriched with the mTOR-inhibitor rapamycin (Rap, 14 ppm, n = 8), or Rap with the AMPK-activator metformin (Rap+Met, 1000 ppm, n = 8) for 12 weeks. Knee joints were evaluated by OARSI scoring, micro-computed tomography, and immunohistochemistry. Glenohumeral articular cartilage was collected for western blotting.. Rap- and Rap+Met-treated guinea pigs displayed lower body weight than control. Rap and Rap+Met inhibited articular cartilage mTORC1 but not mTORC2 signaling. Rap+Met, but not Rap alone, stimulated AMPK. Despite lower body weight and articular cartilage mTORC1 inhibition, Rap- and Rap+Met-treated guinea pigs had greater OA severity in the medial tibial plateau due to articular cartilage structural damage and/or proteoglycan loss. Rap and Rap+Met increased plasma glucose compared to control. Plasma glucose concentration was positively correlated with proteoglycan loss, suggesting hyperglycemic stress after Rap treatment was related to worsened OA.. This is the first study to show that Rap induced increase in plasma glucose was associated with greater OA severity. Further, articular cartilage mTORC1 inhibition and bodyweight reduction by dietary Rap and Rap+Met did not appear to protect against primary OA during the prevailing hyperglycemia.

Topics: Animals; Cartilage, Articular; Guinea Pigs; Hyperglycemia; Osteoarthritis; Sirolimus; X-Ray Microtomography

Topics: Animals; Apoptosis; Autophagy; Chondrocytes; Immunosuppressive Agents; Inflammation; Interleukin-18; Male; Osteoarthritis; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Rats; Rats, Sprague-Dawley; Signal Transduction; Sirolimus

Rapamycin has been considered as a potential treatment for osteoarthritis (OA). Drug carriers fabricated from liposomes can prolong the effects of drugs and reduce side effects of drugs. Low-intensity pulsed ultrasound (LIPUS) has been found to possess anti-OA effects.. The anti-osteoarthritic effects of liposome-encapsulated rapamycin (L-rapa) combined with LIPUS were examined by culture of normal and OA chondrocytes in alginate beads and further validated in OA prone Dunkin-Hartley guinea pigs.. L-rapa with LIPUS largely up-regulated aggrecan and type II collagen mRNA in human OA chondrocytes (HOACs). L-rapa with LIPUS caused significant enhancement in proteoglycan and type II collagen production in HOACs. Large decreases in both MMP-13 and IL-6 proteins were found in the HOACs exposed to L-rapa with LIPUS. Intra-articular injection of 40 μL L-rapa at both 5 μM and 50 μM twice a week combined with LIPUS thrice a week for 8 weeks significantly increased GAGs and type II collagen in the cartilage of knee. Results on OARSI score showed that intra-articular injection of 5 μM L-rapa with LIPUS displayed the greatest anti-OA effects. Immunohistochemistry revealed that L-rapa with or without LIPUS predominantly reduced MMP-13 in vivo. The values of complete blood count and serum biochemical examinations remained in the normal ranges after the injections with or without LIPUS. These data indicated that intra-articular injection of L-rapa collaborated with LIPUS is not only effective against OA but a safe OA therapy.. Taken together, L-rapa combined with LIPUS possessed the most consistently and effectively anabolic and anti-catabolic effects in HOACs and the spontaneous OA guinea pigs. This study evidently revealed that liposome-encapsulation collaborated with LIPUS is able to reduce the effective dose and administration frequency of rapamycin and further stably reinforce its therapeutic actions against OA.

Topics: Animals; Body Weight; Cell Proliferation; Cells, Cultured; Chondrocytes; Collagen Type II; Drug Liberation; Guinea Pigs; Humans; Injections, Intra-Articular; Interleukin-6; Liposomes; Male; Matrix Metalloproteinase 13; Middle Aged; Osteoarthritis; Proteoglycans; RNA, Messenger; Sirolimus; Ultrasonic Waves

Osteoarthritis (OA) is a joint disease characterized by progressive damage of articular cartilage and the adjoining subchondral bone. Chondrocytes, the primary cells of the cartilage, have limited regenerative capacity and when they undergo stress due to trauma or with aging, they senesce or become apoptotic. Rapamycin, a potent immunomodulator, has shown promise in OA treatment. It activates autophagy and is known to prevent senescence. However, its clinical translation for OA is hampered due to systemic toxicity as high and frequent doses are required. Here, we have fabricated rapamycin encapsulated poly(lactic-co-glycolic acid) (PLGA) based carriers that induced autophagy and prevented cellular senescence in human chondrocytes. The microparticle (MP) delivery system showed sustained release of the drug for several weeks. Rapamycin microparticles protected in vitro cartilage mimics (micromass cultures) from degradation, allowing sustained production of sGAG, and demonstrated a prolonged senescence preventive effect under oxidative and genomic stress conditions. These microparticles also exhibited a residence time of ∼30 days after intra-articular injections in murine knee joints. Such particulate systems are promising candidates for intra-articular delivery of rapamycin for the treatment of osteoarthritis.

Topics: Animals; Cartilage, Articular; Chondrocytes; Mice; Osteoarthritis; Pharmaceutical Preparations; Sirolimus

Topics: Apoptosis; Autophagy; Chondrocytes; Humans; NF-kappa B; Osteoarthritis; Sirolimus

This study investigated the influence of autophagy on the expression of Collagen type II and light chain 3 (LC-3) in the articular cartilage of osteoarthritis (OA) models. The expression of OA associated biomarkers namely Matrix metalloproteinase (MMP-13), NOD-, LRR- and pyrin domain-containing 3 (NLRP3) induced by destabilizing the medial meniscus operation (DMM) were also investigated. A total of 60 C57BL/6 mice were divided into (1) control; (2) DMM2; (3) DMM8; (4) rapamycin 2 weeks; and (5) rapamycin 8 weeks groups. Saffranin O-Fast green staining, histomorphometry and immunohistochemical methods were used for analysis. In the DMM group, the expression of the OA biomarkers MMP-13, NLRP3 significantly increased, whilst Collagen II and LC-3B levels were significantly lower than other experimental groups. We hypothesized that NLRP3 inhibits autophagy activation and delays disease progression.

Topics: Animals; Autophagy; Cartilage, Articular; Collagen Type II; Immunosuppressive Agents; Male; Matrix Metalloproteinase 13; Menisci, Tibial; Mice; Mice, Inbred C57BL; Mice, Inbred NOD; Microtubule-Associated Proteins; NLR Family, Pyrin Domain-Containing 3 Protein; Osteoarthritis; Sirolimus

Osteoarthritis (OA) is hallmarked by a progressive degradation of articular cartilage. Besides risk factors including trauma, obesity or genetic predisposition, inflammation has a major impact on the development of this chronic disease. During the course of inflammation, cytokines such as tumor necrosis factor-alpha(TNF-α) and interleukin (IL)-1β are secreted by activated chondrocytes as well as synovial cells and stimulate the production of other inflammatory cytokines and matrix degrading enzymes. The mTORC1 inhibitor rapamycin is a clinical approved immunosuppressant and several studies also verified its chondroprotective effects in OA. However, the effect of blocking the mechanistic target of rapamycin complex (mTORC)1 on the inflammatory status within OA is not well studied. Therefore, we aimed to investigate if inhibition of mTORC1 by rapamycin can preserve and sustain chondrocytes in an inflammatory environment. Patient-derived chondrocytes were cultured in media supplemented with or without the mTORC1 inhibitor rapamycin. To establish an inflammatory environment, either TNF-α or IL-1β was added to the media (=OA-model). The chondroprotective and anti-inflammatory effects of rapamycin were evaluated using sulfated glycosaminoglycan (sGAG) release assay, Caspase 3/7 activity assay, lactate dehydrogenase (LDH) assay and quantitative real time polymerase chain reaction (PCR). Blocking mTORC1 by rapamycin reduced the release and therefore degradation of sGAGs, which are components of the extracellular matrix secreted by chondrocytes. Furthermore, blocking mTORC1 in OA chondrocytes resulted in an enhanced expression of the main chondrogenic markers. Rapamycin was able to protect chondrocytes from cell death in an OA-model shown by reduced Caspase 3/7 activity and diminished LDH release. Furthermore, inhibition of mTORC1 preserved the chondrogenic phenotype of OA chondrocytes, but also reduced inflammatory processes within the OA-model. This study highlights that blocking mTORC1 is a new and promising approach for treating OA. Low side effects make rapamycin an attractive implementation to existing therapeutic strategies. We showed that rapamycin's chondroprotective property might be due to an interference with IL-1β triggered inflammatory processes.

Topics: Caspase 3; Caspase 7; Cells, Cultured; Chondrocytes; Collagen Type I; Cytokines; Glycosaminoglycans; Humans; Hydrogel, Polyethylene Glycol Dimethacrylate; Immunohistochemistry; L-Lactate Dehydrogenase; Osteoarthritis; Sirolimus; Tumor Necrosis Factor-alpha

Articular chondrocyte activation, involving aberrant proliferation and prehypertrophic differentiation, is essential for osteoarthritis (OA) initiation and progression. Disruption of mechanistic target of rapamycin complex 1 (mTORC1) promotes chondrocyte autophagy and survival, and decreases the severity of experimental OA. However, the role of cartilage mTORC1 activation in OA initiation is unknown. In this study, we elucidated the specific role of mTORC1 activation in OA initiation, and identify the underlying mechanisms.. Expression of mTORC1 in articular cartilage of OA patients and OA mice was assessed by immunostaining. Cartilage-specific tuberous sclerosis complex 1 (Tsc1, mTORC1 upstream inhibitor) knockout (TSC1CKO) and inducible Tsc1 KO (TSC1CKO. mTORC1 activation stimulates articular chondrocyte proliferation and differentiation to initiate OA, in part by downregulating FGFR3 and PPR.

Topics: Adult; Aged; Animals; Butylamines; Cartilage, Articular; Cell Proliferation; Chondrocytes; Down-Regulation; Female; Humans; Hypertrophy; Immunosuppressive Agents; Knee Joint; Male; Mechanistic Target of Rapamycin Complex 1; Menisci, Tibial; Mice; Mice, Knockout; Middle Aged; Osteoarthritis; Osteoarthritis, Knee; Receptor, Fibroblast Growth Factor, Type 3; Receptor, Parathyroid Hormone, Type 1; Reverse Transcriptase Polymerase Chain Reaction; Sirolimus; TOR Serine-Threonine Kinases; Tuberous Sclerosis Complex 1 Protein; Tumor Suppressor Proteins; Young Adult

Autophagy, a key homeostasis mechanism, is defective in Osteoarthritis (OA) and Type 2 Diabetes (T2D). T2D has been proposed as a risk factor for OA. We hypothesized that diabetes impairs articular cartilage integrity by decreasing autophagy. Our objective was to investigate the effects of high glucose and insulin, characteristics of T2D, on cartilage homeostasis.. Immortalized human chondrocytes (TC28a2) and primary human chondrocytes (HC) were cultured in 25 mM or 0 mM glucose and treated with insulin (10, 100, 500 nM) for 2, 6 or 24 h. Activity of LC3-II, Akt and rpS6 was evaluated by Western blotting (WB). Human cartilage explants were cultivated with 25 mM glucose and insulin (100,1000 nM) for 24 h to evaluate histopathology. MMP-13 and IL-1β expression was determined by immunohistochemistry and WB. Effects of Rapamycin (10 μM) were analyzed by WB. LC3 and rpS6 expression was determined by WB in chondrocytes from Healthy, Non Diabetic-OA and Diabetic-OA patients.. Insulin downregulates autophagy by reducing LC3 II expression and increasing Akt and rpS6 phosphorylation. Loss of proteoglycans and increased MMP-13 and IL-1β expression was observed after insulin treatment. Autophagy activation by rapamycin reversed insulin effects. Importantly, chondrocytes from diabetic-OA patients showed decreased LC3 and increased p-rpS6 expression compared to Healthy and Non-Diabetic OA patients.. These results suggest that decreased autophagy might be a mechanism by which diabetes influences cartilage degradation. Pharmacological activation of autophagy may be an effective therapeutic approach to prevent T2D-induced cartilage damage.

Topics: Aged; Aged, 80 and over; Autophagy; Cartilage, Articular; Cells, Cultured; Chondrocytes; Diabetes Mellitus, Type 2; Dose-Response Relationship, Drug; Glucose; Humans; Insulin; Interleukin-1beta; Matrix Metalloproteinase 13; Middle Aged; Osteoarthritis; Proteoglycans; Proto-Oncogene Proteins c-akt; Sirolimus; Tissue Culture Techniques; TOR Serine-Threonine Kinases; Up-Regulation

Mammalian target of rapamycin (mTOR) (a serine/threonine protein kinase) is a major repressor of autophagy, a cell survival mechanism. The specific in vivo mechanism of mTOR signalling in OA pathophysiology is not fully characterised. We determined the expression of mTOR and known autophagy genes in human OA cartilage as well as mouse and dog models of experimental OA. We created cartilage-specific mTOR knockout (KO) mice to determine the specific role of mTOR in OA pathophysiology and autophagy signalling in vivo.. Inducible cartilage-specific mTOR KO mice were generated and subjected to mouse model of OA. Human OA chondrocytes were treated with rapamycin and transfected with Unc-51-like kinase 1 (ULK1) siRNA to determine mTOR signalling.. mTOR is overexpressed in human OA cartilage as well as mouse and dog experimental OA. Upregulation of mTOR expression co-relates with increased chondrocyte apoptosis and reduced expression of key autophagy genes during OA. Subsequently, we show for the first time that cartilage-specific ablation of mTOR results in increased autophagy signalling and a significant protection from destabilisation of medial meniscus (DMM)-induced OA associated with a significant reduction in the articular cartilage degradation, apoptosis and synovial fibrosis. Furthermore, we show that regulation of ULK1/adenosine monophosphate-activated protein kinase (AMPK) signalling pathway by mTOR may in part be responsible for regulating autophagy signalling and the balance between catabolic and anabolic factors in the articular cartilage.. This study provides a direct evidence of the role of mTOR and its downstream modulation of autophagy in articular cartilage homeostasis.

Topics: Aged; Aged, 80 and over; AMP-Activated Protein Kinases; Animals; Apoptosis; Autophagy; Autophagy-Related Protein-1 Homolog; Cartilage, Articular; Cells, Cultured; Chondrocytes; Disease Models, Animal; Dogs; Gene Silencing; Humans; Immunosuppressive Agents; Intracellular Signaling Peptides and Proteins; Male; Matrix Metalloproteinase 13; Mice; Mice, Inbred C57BL; Mice, Knockout; Middle Aged; Osteoarthritis; Protein Serine-Threonine Kinases; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Up-Regulation

Chondrocyte-derived extracellular organelles known as articular cartilage vesicles (ACVs) participate in non-classical protein secretion, intercellular communication, and pathologic calcification. Factors affecting ACV formation and release remain poorly characterized; although in some cell types, the generation of extracellular vesicles is associated with up-regulation of autophagy. We sought to determine the role of autophagy in ACV production by primary articular chondrocytes. Using an innovative dynamic model with a light scatter nanoparticle counting apparatus, we determined the effects of autophagy modulators on ACV number and content in conditioned medium from normal adult porcine and human osteoarthritic chondrocytes. Healthy articular chondrocytes release ACVs into conditioned medium and show significant levels of ongoing autophagy. Rapamycin, which promotes autophagy, increased ACV numbers in a dose- and time-dependent manner associated with increased levels of autophagy markers and autophagosome formation. These effects were suppressed by pharmacologic autophagy inhibitors and short interfering RNA for ATG5. Caspase-3 inhibition and a Rho/ROCK inhibitor prevented rapamycin-induced increases in ACV number. Osteoarthritic chondrocytes, which are deficient in autophagy, did not increase ACV number in response to rapamycin. SMER28, which induces autophagy via an mTOR-independent mechanism, also increased ACV number. ACVs induced under all conditions had similar ecto-enzyme specific activities and types of RNA, and all ACVs contained LC3, an autophagosome-resident protein. These findings identify autophagy as a critical participant in ACV formation, and augment our understanding of ACVs in cartilage disease and repair.

Topics: Adult; Animals; Apoptosis; Autophagy; Biological Transport; Blotting, Western; Cartilage, Articular; Caspase 3; Cell Proliferation; Cells, Cultured; Chondrocytes; Flow Cytometry; Humans; Immunosuppressive Agents; Middle Aged; Organelles; Osteoarthritis; Phagosomes; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sirolimus; Swine; TOR Serine-Threonine Kinases

Autophagy is a cellular homeostasis mechanism that may have a protective role against osteoarthritis (OA). The present study investigated the therapeutic effect of local administration of rapamycin, a potent activator of autophagy, against OA. To achieve controlled intra-articular administration of rapamycin, gelatin hydrogels incorporating rapamycin-micelles were created and the release profile was evaluated in vitro. The therapeutic effects of gelatin hydrogels incorporating rapamycin-micelles were then tested in a murine OA model. Mice were divided into four groups: Group 1, gelatin hydrogels alone; Group 2, single injection of 1 μg rapamycin; and Groups 3 and 4, gelatin hydrogels incorporating 100 ng or 1 μg rapamycin-micelles, respectively. Immunohistochemical analysis revealed that autophagic marker-positive chondrocytes were increased in the rapamycin-treated mice at 10 weeks after surgery. The histologic score was better in Groups 3 and 4 than in Groups 1 and 2, and Group 2 had a better score than Group 1. Delayed OA progression was maintained even at 16 weeks after surgery in Group 4. Microarray and real-time polymerase chain reaction analysis indicated that OA mediator genes were downregulated in the rapamycin-treated mice. Our novel system for intra-articular administration of rapamycin could be a novel therapeutic approach for treating patients with OA.

Topics: Animals; Autophagy; Chondrocytes; Drug Carriers; Gelatin; Hydrogels; Immunosuppressive Agents; Injections, Intra-Articular; Male; Mice; Mice, Inbred C57BL; Micelles; Osteoarthritis; Sirolimus

Recent 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.. Destabilization 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).. Intra-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.. These 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.

Topics: Animals; Cartilage, Articular; Disease Models, Animal; Immunosuppressive Agents; Injections, Intra-Articular; Male; Mice; Osteoarthritis; Sirolimus

The present study aimed to analyze the responses to autophagy in osteoarthritis (OA) and aging chondrocytes in order to elucidate the role of autophagy in the pathogenesis of OA. We used multiple assays to confirm that autophagic activity was downregulated in chondrocytes of aged articular cartilage. Surprisingly, we found that the expression of autophagy-related proteins was not decreased in the tissues of patients with OA. We also observed that rapamycin-induced autophagy prevented the accumulation of subdiploid cells in young chondrocytes, while it induced cell death by autophagy in OA chondrocytes. Our results demonstrate that autophagic activity decreases with aging, and may be responsible for the cytoprotective effects in young cartilage. However, we found that autophagic activity in patients with OA was higher than in the aging group, and reported autophagic cell death in OA chondrocytes. These results suggest that autophagy plays both a cytoprotective and death-promoting role in the pathogenesis of OA.

Topics: Adenine; Aged; Apoptosis Regulatory Proteins; Autophagy; Beclin-1; Cartilage, Articular; Cell Cycle; Cell Hypoxia; Cell Survival; Cells, Cultured; Chondrocytes; Female; Green Fluorescent Proteins; Humans; Male; Membrane Proteins; Microtubule-Associated Proteins; Middle Aged; Osteoarthritis; Sirolimus; Young Adult

Osteoarthritis is characterized by degenerative alterations of articular cartilage including both the degradation of extracellular matrix and the death of chondrocytes. The PI3K/Akt pathway has been demonstrated to involve in both processes. Inhibition of its downstream target NF-kB reduces the degradation of extracellular matrix via decreased production of matrix metalloproteinases while inhibition of mTOR increased autophagy to reduce chondrocyte death. However, mTOR feedback inhibits the activity of the PI3K/Akt pathway and inhibition of mTOR could result in increased activity of the PI3K/Akt/NF-kB pathway. We proposed that the use of dual inhibitors of PI3K and mTOR could be a promising approach to more efficiently inhibit the PI3K/Akt pathway than rapamycin or PI3K inhibitor alone and produce better treatment outcome.

Topics: Autophagy; Cell Proliferation; Extracellular Matrix; Humans; Molecular Targeted Therapy; NF-kappa B; Oncogene Protein v-akt; Osteoarthritis; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

To investigate apoptosis of osteoarthritic (OA) chondrocytes stimulated with different inhibitors targeting tumor necrosis factor-alpha (TNFα) pathway, we isolated first passage chondrocytes from OA patients and then treated them with the inhibitors in combination with TNFα, and then collected the stimulated chondrocytes for Western blotting. Chondrocytes from OA patients expressed cleaved caspase-3 and PARP, suggesting apoptotic background. We here, validated that 10 ng/ml of TNFα couldn't induce more chondrocytes apoptosis. PI3K inhibitor LY294002 or NF-κB inhibitor CAPE, but not mTOR inhibitor rapamycin and MEK1/2 inhibitor U0126 in combination with TNFα could facilitate apoptosis. CAPE-induced more apoptosis could be explained by c-FLIP downregulation more than cIAP1 upregulation. And, we showed the first time that PI3K-NF-κB pathway, but not mTOR pathway could prevent chondrocytes apoptosis induced by a pro-apoptotic factor TNFα and call for attention while trying to inhibit NF-κB as a therapeutic target.

Topics: Aged; Apoptosis; Butadienes; Caffeic Acids; Caspase 3; Cells, Cultured; Chondrocytes; Chromones; Female; Humans; Middle Aged; Morpholines; NF-kappa B; Nitriles; Osteoarthritis; Phenylethyl Alcohol; Phosphoinositide-3 Kinase Inhibitors; Poly(ADP-ribose) Polymerases; Sirolimus; TOR Serine-Threonine Kinases; Tumor Necrosis Factor-alpha

Osteoarthritis is associated with cell death and extracellular matrix degradation in articular cartilage. Autophagy is an essential cellular homeostasis mechanism that was found to be deficient in ageing and osteoarthritic cartilage. This study determined whether pharmacological inhibition of the mammalian target of rapamycin (mTOR), a key inhibitor of autophagy, has disease-modifying activity in experimental osteoarthritis.. Experimental osteoarthritis was induced by transection of the medial meniscotibial ligament and the medial collateral ligament in 2-month-old C57Bl/6 mice (n=36). Rapamycin (1 mg/kg weight/day) (n=18 mice) or dimethyl sulphoxide vehicle control (n=18 mice) was administered intraperitoneally for 10 weeks. Histopathological changes in articular cartilage and synovium were examined by using semiquantitative scoring systems. Rapamycin effects on mTOR signalling, autophagy, cartilage homeostasis and inflammation were analysed by immunohistochemistry and immunofluorescence staining.. Rapamycin affected the mTOR signalling pathway in mouse knee joints as indicated by the inhibition of ribosomal protein S6 phosphorylation, a target of mTOR and activation of LC3, a main marker of autophagy. The severity of cartilage degradation was significantly (p<0.01) reduced in the rapamycin-treated group compared with the control group and this was associated with a significant (p<0.05) decrease in synovitis. Rapamycin treatment also maintained cartilage cellularity and decreased ADAMTS-5 and interleukin-1β expression in articular cartilage.. These results suggest that rapamycin, at least in part by autophagy activation, reduces the severity of experimental osteoarthritis. Pharmacological activation of autophagy may be an effective therapeutic approach for osteoarthritis.

Topics: ADAM Proteins; ADAMTS5 Protein; Animals; Anti-Inflammatory Agents; Arthritis, Experimental; Autophagy; Cartilage, Articular; Chondrocytes; Drug Evaluation, Preclinical; Immunosuppressive Agents; Male; Mice; Mice, Inbred C57BL; Osteoarthritis; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Treatment Outcome

Topics: Animals; Autophagy; Disease Models, Animal; Humans; Immunosuppressive Agents; Osteoarthritis; Sirolimus; TOR Serine-Threonine Kinases