New approach to testing treatments for osteoarthritis: FastOA.

Animal models of post traumatic osteoarthritis have shown many promising treatments for disease, but human trials have mostly failed to identify effective treatments. This viewpoint suggests that the frequent failure of drug and treatment development in osteoarthritis is due, in part, to the advanced stage of disease of patients in trials and suggests that mirroring the animal model approach might be more successful. It suggests a path forward by enriching trial enrollees with those likely to develop post traumatic OA quickly.

Human-specific duplicate CHRFAM7A gene is associated with more severe osteoarthritis and amplifies pain behaviours.

OBJECTIVES: CHRFAM7A is a uniquely human fusion gene that functions as a dominant negative regulator of alpha 7 acetylcholine nicotinic receptor (α7nAChR) in vitro. This study determined the impact of CHRFAM7A on α7nAChR agonist responses, osteoarthritis (OA) severity and pain behaviours and investigated mechanisms. METHODS: Transgenic CHRFAM7A (TgCHRFAM7A) mice were used to determine the impact of CHRFAM7A on knee OA histology, pain severity in OA and other pain models, response to nAchR agonist and IL-1ß. Mouse and human cells were used for mechanistic studies. RESULTS: Transgenic (Tg) TgCHRFAM7A mice developed more severe structural damage and increased mechanical allodynia than wild type (WT) mice in the destabilisation of medial meniscus model of OA. This was associated with a decreased suppression of inflammation by α7nAchR agonist. TgCHRFAM7A mice displayed a higher basal sensitivity to pain stimuli and increased pain behaviour in the monoiodoacetate and formalin models. Dorsal root ganglia of TgCHRFAM7A mice showed increased macrophage infiltration and expression of the chemokine fractalkine and also had a compromised antinociceptive response to the α7nAchR agonist nicotine. Both native CHRNA7 and CHRFAM7A subunits were expressed in human joint tissues and the CHRFAM7A/CHRNA7 ratio was increased in OA cartilage. Human chondrocytes with two copies of CHRFAM7A had reduced anti-inflammatory responses to nicotine. CONCLUSION: CHRFAM7A is an aggravating factor for OA-associated inflammation and tissue damage and a novel genetic risk factor and therapeutic target for pain.

Senescent cell population with ZEB1 transcription factor as its main regulator promotes osteoarthritis in cartilage and meniscus.

OBJECTIVES: Single-cell level analysis of articular cartilage and meniscus tissues from human healthy and osteoarthritis (OA) knees. METHODS: Single-cell RNA sequencing (scRNA-seq) analyses were performed on articular cartilage and meniscus tissues from healthy (n=6, n=7) and OA (n=6, n=6) knees. Expression of genes of interest was validated using immunohistochemistry and RNA-seq and function was analysed by gene overexpression and depletion. RESULTS: scRNA-seq analyses of human knee articular cartilage (70 972 cells) and meniscus (78 017 cells) identified a pathogenic subset that is shared between both tissues. This cell population is expanded in OA and has strong OA and senescence gene signatures. Further, this subset has critical roles in extracellular matrix (ECM) and tenascin signalling and is the dominant sender of signals to all other cartilage and meniscus clusters and a receiver of TGFß signalling. Fibroblast activating protein (FAP) is also a dysregulated gene in this cluster and promotes ECM degradation. Regulons that are controlled by transcription factor ZEB1 are shared between the pathogenic subset in articular cartilage and meniscus. In meniscus and cartilage cells, FAP and ZEB1 promote expression of genes that contribute to OA pathogenesis, including senescence. CONCLUSIONS: These single-cell studies identified a senescent pathogenic cell cluster that is present in cartilage and meniscus and has FAP and ZEB1 as main regulators which are novel and promising therapeutic targets for OA-associated pathways in both tissues.

Targeting FoxO transcription factors with HDAC inhibitors for the treatment of osteoarthritis.

OBJECTIVES: Osteoarthritis (OA) features ageing-related defects in cellular homeostasis mechanisms in articular cartilage. These defects are associated with suppression of forkhead box O (FoxO) transcription factors. FoxO1 or FoxO3 deficient mice show early onset OA while FoxO1 protects against oxidative stress in chondrocytes and promotes expression of autophagy genes and the essential joint lubricant proteoglycan 4 (PRG4). The objective of this study was to identify small molecules that can increase FoxO1 expression. METHODS: We constructed a reporter cell line with FoxO1 promoter sequences and performed high-throughput screening (HTS) of the Repurposing, Focused Rescue and Accelerated Medchem (ReFRAME) library . Hits from the HTS were validated and function was assessed in human chondrocytes, meniscus cells and synoviocytes and following administration to mice. The most promising hit, the histone deacetylase inhibitor (HDACI) panobinostat was tested in a murine OA model. RESULTS: Among the top hits were HDACI and testing in human chondrocytes, meniscus cells and synoviocytes showed that panobinostat was the most promising compound as it increased the expression of autophagy genes and PRG4 while suppressing the basal and IL-1ß induced expression of inflammatory mediators and extracellular matrix degrading enzymes. Intraperitoneal administration of panobinostat also suppressed the expression of mediators of OA pathogenesis induced by intra-articular injection of IL-1ß. In a murine OA model, panobinostat reduced the severity of histological changes in cartilage, synovium and subchondral bone and improved pain behaviours. CONCLUSION: Panobinostat has a clinically relevant activity profile and is a candidate for OA symptom and structure modification.

FOXO1 and FOXO3 transcription factors have unique functions in meniscus development and homeostasis during aging and osteoarthritis.

The objective of this study was to examine FoxO expression and FoxO function in meniscus. In menisci from human knee joints with osteoarthritis (OA), FoxO1 and 3 expression were significantly reduced compared with normal menisci from young and old normal donors. The expression of FoxO1 and 3 was also significantly reduced in mouse menisci during aging and OA induced by surgical meniscus destabilization or mechanical overuse. Deletion of FoxO1 and combined FoxO1, 3, and 4 deletions induced abnormal postnatal meniscus development in mice and these mutant mice spontaneously displayed meniscus pathology at 6 mo. Mice with Col2Cre-mediated deletion of FoxO3 or FoxO4 had normal meniscus development but had more severe aging-related damage. In mature AcanCreERT2 mice, the deletion of FoxO1, 3, and 4 aggravated meniscus lesions in all experimental OA models. FoxO deletion suppressed autophagy and antioxidant defense genes and altered several meniscus-specific genes. Expression of these genes was modulated by adenoviral FoxO1 in cultured human meniscus cells. These results suggest that FoxO1 plays a key role in meniscus development and maturation, and both FoxO1 and 3 support homeostasis and protect against meniscus damage in response to mechanical overuse and during aging and OA.

Krüppel-like factor-4 and Krüppel-like factor-2 are important regulators of joint tissue cells and protect against tissue destruction and inflammation in osteoarthritis.

OBJECTIVES: Analysing expression patterns of Krüppel-like factor (KLF) transcription factors in normal and osteoarthritis (OA) human cartilage, and determining functions and mechanisms of KLF4 and KLF2 in joint homoeostasis and OA pathogenesis. METHODS: Experimental approaches included human joint tissues cells, transgenic mice and mouse OA model with viral KLF4 gene delivery to demonstrate therapeutic benefit in structure and pain improvement. Mechanistic studies applied global gene expression analysis and chromatin immunoprecipitation sequencing (ChIP-seq). RESULTS: Several KLF genes were significantly decreased in OA cartilage. Among them, KLF4 and KLF2 were strong inducers of cartilage collagen genes and Proteoglycan-4. Cartilage-specific deletion of Klf2 in mature mice aggravated severity of experimental OA. Transduction of human chondrocytes with Adenovirus (Ad) expressing KLF4 or KLF2 enhanced expression of major cartilage extracellular matrix (ECM) genes and SRY-box transcription factor-9, and suppressed mediators of inflammation and ECM-degrading enzymes. Ad-KLF4 and Ad-KLF2 enhanced similar protective functions in meniscus cells and synoviocytes, and promoted chondrocytic differentiation of human mesenchymal stem cells. Viral KLF4 delivery into mouse knees reduced severity of OA-associated changes in cartilage, meniscus and synovium, and improved pain behaviours. ChIP-seq analysis suggested that KLF4 directly bound cartilage signature genes. Ras-related protein-1 signalling was the most enriched pathway in KLF4-transduced cells, and its signalling axis was involved in upregulating cartilage ECM genes by KLF4 and KLF2. CONCLUSIONS: KLF4 and KLF2 may be central transcription factors that increase protective and regenerative functions in joint tissue cells, suggesting that KLF gene transfer or molecules upregulating KLFs are therapeutic candidates for OA.

FOXO1 transcription factor regulates chondrogenic differentiation through transforming growth factor ß1 signaling.

The forkhead box O (FOXO) proteins are transcription factors involved in the differentiation of many cell types. Type II collagen (Col2) Cre-Foxo1-knockout and Col2-Cre-Foxo1,3,4 triple-knockout mice exhibit growth plate malformation. Moreover, recent studies have reported that in some cells, the expressions and activities of FOXOs are promoted by transforming growth factor ß1 (TGFß1), a growth factor playing a key role in chondrogenic differentiation. Here, using a murine chondrogenic cell line (ATDC5), mouse embryos, and human mesenchymal stem cells, we report the mechanisms by which FOXOs affect chondrogenic differentiation. FOXO1 expression increased along with chondrogenic differentiation, and FOXO1 inhibition suppressed chondrogenic differentiation. TGFß1/SMAD signaling promoted expression and activity of FOXO1. In ATDC5, FOXO1 knockdown suppressed expression of sex-determining region Y box 9 (Sox9), a master regulator of chondrogenic differentiation, resulting in decreased collagen type II α1 (Col2a1) and aggrecan (Acan) expression after TGFß1 treatment. On the other hand, chemical FOXO1 inhibition suppressed Col2a1 and Acan expression without suppressing Sox9 To investigate the effects of FOXO1 on chondrogenic differentiation independently of SOX9, we examined FOXO1's effects on the cell cycle. FOXO1 inhibition suppressed expression of p21 and cell-cycle arrest in G0/G1 phase. Conversely, FOXO1 overexpression promoted expression of p21 and cell-cycle arrest. FOXO1 inhibition suppressed expression of nascent p21 RNA by TGFß1, and FOXO1 bound the p21 promoter. p21 inhibition suppressed expression of Col2a1 and Acan during chondrogenic differentiation. These results suggest that FOXO1 is necessary for not only SOX9 expression, but also cell-cycle arrest during chondrogenic differentiation via TGFß1 signaling.

Bioactive proteins delivery through core-shell nanofibers for meniscal tissue regeneration.

Mimicking the ultrastructural morphology of the meniscus with nanofiber scaffolds, coupled with controlled growth-factor delivery to the appropriate cells, can help engineer tissue with the potential to grow, mature, and regenerate after in vivo implantation. We electrospun nanofibers encapsulating platelet-derived growth factor (PDGF-BB), which is a potent mitogen and chemoattractant in a core of serum albumin contained within a shell of polylactic acid. We controlled the local PDGF-BB release by adding water-soluble polyethylene glycol to the polylactic acid shell to serve as a porogen. The novel core-shell nanofibers generated 3D scaffolds with an interconnected macroporous structure, with appropriate mechanical properties and with high cell compatibility. Incorporating PDGF-BB increased cell viability, proliferation, and infiltration, and upregulated key genes involved in meniscal extracellular matrix synthesis in human meniscal and synovial cells. Our results support proof of concept that these core-shell nanofibers can create a cell-favorable nanoenvironment and can serve as a system for sustained release of bioactive factors.

Modulation of matrix metabolism by ATP-citrate lyase in articular chondrocytes.

Certain dysregulated chondrocyte metabolic adaptive responses such as decreased activity of the master regulator of energy metabolism AMP-activated protein kinase (AMPK) promote osteoarthritis (OA). Metabolism intersects with epigenetic and transcriptional responses. Hence, we studied chondrocyte ATP-citrate lyase (ACLY), which generates acetyl-CoA from mitochondrial-derived citrate, and modulates acetylation of histones and transcription factors. We assessed ACLY in normal and OA human knee chondrocytes and cartilages by Western blotting and immunohistochemistry, and quantified acetyl-CoA fluorometrically. We examined histone and transcription factor lysine acetylation by Western blotting, and assessed histone H3K9 and H3K27 occupancy of iNOS, MMP3, and MMP13 promoters by chromatin immunoprecipitation (ChIP) and quantitative PCR (qPCR). We analyzed iNOS, MMP3, MMP13, aggrecan (ACAN), and Col2a1 gene expression by RT-qPCR. Glucose availability regulated ACLY expression and function, nucleocytosolic acetyl-CoA, and histone acetylation. Human knee OA chondrocytes exhibited increased ACLY activation (assessed by Ser-455 phosphorylation), associated with increased H3K9 and H3K27 acetylation. Inhibition of ACLY attenuated IL-1ß-induced transcription of iNOS, MMP3, and MMP13 by suppressing acetylation of p65 NF-κB, H3K9, and H3K27, blunted release of NO, MMP3, and MMP13, and also reduced SOX9 acetylation that promoted SOX9 nuclear translocation, leading to increased aggrecan and Col2a1 mRNA expression. ACLY is a novel player involved in regulation of cartilage matrix metabolism. Increased ACLY activity in OA chondrocytes increased nucleocytosolic acetyl-CoA, leading to increased matrix catabolism via dysregulated histone and transcription factor acetylation. Pharmacologic ACLY inhibition in OA chondrocytes globally reverses these changes and stimulates matrix gene expression and AMPK activation, supporting translational investigation in OA.

Gene targeting of the transcription factor Mohawk in rats causes heterotopic ossification of Achilles tendon via failed tenogenesis.

Cell-based or pharmacological approaches for promoting tendon repair are currently not available because the molecular mechanisms of tendon development and healing are not well understood. Although analysis of knockout mice provides many critical insights, small animals such as mice have some limitations. In particular, precise physiological examination for mechanical load and the ability to obtain a sufficient number of primary tendon cells for molecular biology studies are challenging using mice. Here, we generated Mohawk (Mkx)(-/-) rats by using CRISPR/Cas9, which showed not only systemic hypoplasia of tendons similar to Mkx(-/-) mice, but also earlier heterotopic ossification of the Achilles tendon compared with Mkx(-/-) mice. Analysis of tendon-derived cells (TDCs) revealed that Mkx deficiency accelerated chondrogenic and osteogenic differentiation, whereas Mkx overexpression suppressed chondrogenic, osteogenic, and adipogenic differentiation. Furthermore, mechanical stretch stimulation of Mkx(-/-) TDCs led to chondrogenic differentiation, whereas the same stimulation in Mkx(+/+) TDCs led to formation of tenocytes. ChIP-seq of Mkx overexpressing TDCs revealed significant peaks in tenogenic-related genes, such as collagen type (Col)1a1 and Col3a1, and chondrogenic differentiation-related genes, such as SRY-box (Sox)5, Sox6, and Sox9 Our results demonstrate that Mkx has a dual role, including accelerating tendon differentiation and preventing chondrogenic/osteogenic differentiation. This molecular network of Mkx provides a basis for tendon physiology and tissue engineering.

Guided apicoectomy using a CAD/CAM drilling template.

AIM: Modern microsurgical techniques have increased the success rate of apicoectomy relative to that of traditional approaches. This case report introduces a novel workaround for guided apicoectomy using a patient-specific computer-aided design/computer-aided manufacturing (CAD/CAM) three-dimensional (3D)-printed template. MATERIALS AND METHODS: Apicoectomy was performed on the mesial root of tooth 36 using template-guided trephine drilling, followed by retrograde filling with mineral trioxide aggregate (MTA). Initially, a cone beam computed tomography (CBCT) scan and an intraoral surface scan were imported into the planning software. After superimposition, virtual planning was performed to determine the exact localization for root resection. Subsequently, a tooth-supported drilling template was designed and 3D printed. Endodontic microsurgical approaches, including root-end cavity preparation and root-end filling, completed the surgical treatment. RESULT: The apical resection was easily feasible. There were no postoperative complications. Radiological assessment after a 6-month period showed signs of reossification. CONCLUSION: Guided apicoectomy allowed precise root resection, suggesting that this technique may be advantageous in complex anatomical situations.

Extracellular vesicles in cartilage homeostasis and osteoarthritis.

PURPOSE OF REVIEW: Extracellular vesicles carry bioactive molecules that can be transferred between cells and tissues. The purpose of this review is to describe how extracellular vesicles regulate functions of cells in cartilage and other joint tissues. The potential application of extracellular vesicles in the treatment of osteoarthritis and as biomarkers will also be discussed. RECENT FINDINGS: Extracellular vesicles are found in synovial fluid, in articular cartilage and in the supernatants of synoviocytes and chondrocytes. Extracellular vesicles in cartilage have been proposed to be involved in cross talk between cells in joint tissues and to affect extracellular matrix turnover and inflammation. Extracellular vesicles from arthritic joints can promote abnormal gene expression and changes in cartilage extracellular matrix, including abnormal mineralization. Promising results were obtained in the therapeutic application of mesenchymal stem cell-derived extracellular vesicles for cartilage repair and experimental osteoarthritis. SUMMARY: Extracellular vesicles have emerged as vehicles for the exchange of bioactive signaling molecules within cartilage and between joint tissues to promote joint homeostasis and arthritis pathogenesis. As the molecular content of extracellular vesicles can be customized, they offer utility in therapeutic applications.

Molecular mechanisms of autophagic memory in pathogenic T cells in human arthritis.

T-cell resilience is critical to the immune pathogenesis of human autoimmune arthritis. Autophagy is essential for memory T cell generation and associated with pathogenesis in rheumatoid arthritis (RA). Our aim here was to delineate the role and molecular mechanism of autophagy in resilience and persistence of pathogenic T cells from autoimmune arthritis. We demonstrated "Autophagic memory" as elevated autophagy levels in CD4+ memory T cells compared to CD4+ naive T cells and in Jurkat Human T cell line trained with starvation stress. We then showed increased levels of autophagy in pathogenic CD4+ T cells subsets from autoimmune arthritis patients. Using RNA-sequencing, transcription factor gene regulatory network and methylation analyses we identified MYC as a key regulator of autophagic memory. We validated MYC levels using qPCR and further demonstrated that inhibiting MYC increased autophagy. The present study proposes the novel concept of autophagic memory and suggests that autophagic memory confers metabolic advantage to pathogenic T cells from arthritis and supports its resilience and long term survival. Particularly, suppression of MYC imparted the heightened autophagy levels in pathogenic T cells. These studies have a direct translational valency as they identify autophagy and its metabolic controllers as a novel therapeutic target.

MicroRNA-140 plays dual roles in both cartilage development and homeostasis.

Osteoarthritis (OA), the most prevalent aging-related joint disease, is characterized by insufficient extracellular matrix synthesis and articular cartilage degradation, mediated by several proteinases, including Adamts-5. miR-140 is one of a very limited number of noncoding microRNAs (miRNAs) specifically expressed in cartilage; however, its role in development and/or tissue maintenance is largely uncharacterized. To examine miR-140 function in tissue development and homeostasis, we generated a mouse line through a targeted deletion of miR-140. miR-140(-/-) mice manifested a mild skeletal phenotype with a short stature, although the structure of the articular joint cartilage appeared grossly normal in 1-mo-old miR-140(-/-) mice. Interestingly, miR-140(-/-) mice showed age-related OA-like changes characterized by proteoglycan loss and fibrillation of articular cartilage. Conversely, transgenic (TG) mice overexpressing miR-140 in cartilage were resistant to antigen-induced arthritis. OA-like changes in miR-140-deficient mice can be attributed, in part, to elevated Adamts-5 expression, regulated directly by miR-140. We show that miR-140 regulates cartilage development and homeostasis, and its loss contributes to the development of age-related OA-like changes.

Increased autophagy in CD4+ T cells of rheumatoid arthritis patients results in T-cell hyperactivation and apoptosis resistance.

Rheumatoid arthritis (RA) is an autoimmune disease hallmarked by aberrant cellular homeostasis, resulting in hyperactive CD4+ T cells that are more resistant to apoptosis. Both hyperactivation and resistance to apoptosis may contribute to the pathogenicity of CD4+ T cells in the autoimmune process. A better knowledge of the mechanisms determining such impaired homeostasis could contribute significantly to both the understanding and the treatment of the disease. Here we investigated whether autophagy, is dysregulated in CD4+ T cells of RA patients, resulting in disturbed T-cell homeostasis. We demonstrate that the rate of autophagy is significantly increased in CD4+ T cells from RA patients, and that increased autophagy is also a feature of in vitro activated CD4+ T cells. The increased apoptosis resistance observed in CD4+ T cells from RA patients was significantly reversed upon autophagy inhibition. These mechanisms may contribute to RA pathogenesis, as autophagy inhibition reduced both arthritis incidence and disease severity in a mouse collagen induced arthritis mouse model. Conversely, in Atg5flox/flox -CD4-Cre+ mice, in which all T cells are autophagy deficient, T cells showed impaired activation and proliferation. These data provide novel insight into the pathogenesis of RA and underscore the relevance of autophagy as a promising therapeutic target.

Increased autophagy contributes to the inflammatory phenotype of juvenile idiopathic arthritis synovial fluid T cells.

Objectives: JIA is an autoimmune disease involving disturbed T-cell homeostasis, marked by highly activated effector T cells. Autophagy, a lysosomal degradation pathway, is crucial for maintaining cellular homeostasis by regulating the survival, differentiation and function of a large variety of cells, including T cells. The aim of this study was to examine the rate of autophagy in JIA T cells and to investigate the effect of inhibition of autophagy on the inflammatory phenotype of JIA T cells. Methods: Autophagy-related gene expression was analysed in CD4+ T cells from the SF of JIA patients and healthy controls using RNA sequencing. Autophagy was measured by flow cytometry and western blot. The effect of inhibition of autophagy, using HCQ, on the cellular activation status was analysed using flow cytometry and multiplex immunoassay. Results: Autophagy was increased in T cells derived from the site of inflammation compared with cells from the peripheral blood of patients and healthy controls. This increase in autophagy was not induced by JIA SF, but is more likely to be the result of increased cellular activation. Inhibition of autophagy reduced proliferation, cytokine production and activation marker expression of JIA SF-derived CD4+ T cells. Conclusion: These data indicate that autophagy is increased in JIA SF-derived T cells and that targeting autophagy could be a promising therapeutic strategy to restore the disrupted T-cell homeostasis in JIA.

Relevance of meniscal cell regional phenotype to tissue engineering.

PURPOSE: Meniscus contains heterogeneous populations of cells that have not been fully characterized. Cell phenotype is often lost during culture; however, culture expansion is typically required for tissue engineering. We examined and compared cell-surface molecule expression levels on human meniscus cells from the vascular and avascular regions and articular chondrocytes while documenting changes during culture-induced dedifferentiation. MATERIALS AND METHODS: Expressions of 16 different surface molecules were examined by flow cytometry after monolayer culture for 24 h, 1 week, and 2 weeks. Menisci were also immunostained to document the spatial distributions of selected surface molecules. RESULTS: Meniscus cells and chondrocytes exhibited several similarities in surface molecule profiles with dynamic changes during culture. A greater percentage of meniscal cells were positive for CD14, CD26, CD49c, and CD49f compared to articular chondrocytes. Initially, more meniscal cells from the vascular region were positive for CD90 compared to cells from the avascular region or chondrocytes. Cells from the vascular region also expressed higher levels of CD166 and CD271 compared to cells from the avascular region. CD90, CD166, and CD271-positive cells were predominately perivascular in location. However, CD166-positive cells were also located in the superficial layer and in the adjacent synovial and adipose tissue. CONCLUSIONS: These surface marker profiles provide a target phenotype for differentiation of progenitors in tissue engineering. The spatial location of progenitor cells in meniscus is consistent with higher regenerative capacity of the vascular region, while the surface progenitor subpopulations have potential to be utilized in tears created in the avascular region.

Glucosamine activates autophagy in vitro and in vivo.

OBJECTIVE: Aging-associated changes in articular cartilage represent a main risk factor for osteoarthritis (OA). Autophagy is an essential cellular homeostasis mechanism. Aging-associated or experimentally induced defects in autophagy contribute to organismal- and tissue-specific aging, while enhancement of autophagy may protect against certain aging-related pathologies such as OA. The objective of this study was to determine whether glucosamine can activate autophagy. METHODS: Chondrocytes from normal human articular cartilage were treated with glucosamine (0.1- 10 mM). Autophagy activation and phosphorylation levels of Akt, FoxO3, and ribosomal protein S6 were determined by Western blotting. Autophagosome formation was analyzed by confocal microscopy. Reporter mice systemically expressing green fluorescent protein (GFP) fused to light chain 3 (LC3) (GFP-LC3-transgenic mice) were used to assess changes in autophagy in response to starvation and glucosamine treatment. RESULTS: Glucosamine treatment of chondrocytes activated autophagy, as indicated by increased LC3-II levels, formation of LC3 puncta, and increased LC3 turnover. This was associated with glucosamine-mediated inhibition of the Akt/FoxO3/mammalian target of rapamycin pathway. Administration of glucosamine to GFP-LC3-transgenic mice markedly activated autophagy in articular cartilage. CONCLUSION: Glucosamine modulates molecular targets of the autophagy pathway in vitro and in vivo, and the enhancement of autophagy is mainly dependent on the Akt/FoxO/mTOR pathway. These findings suggest that glucosamine is an effective autophagy activator and should motivate future studies on the efficacy of glucosamine in modifying aging-related cellular changes and supporting joint health.

Zone-specific gene expression patterns in articular cartilage.

OBJECTIVE: To identify novel genes and pathways specific to the superficial zone (SZ), middle zone (MZ), and deep zone (DZ) of normal articular cartilage. METHODS: Articular cartilage was obtained from the knees of 4 normal human donors. The cartilage zones were dissected on a microtome. RNA was analyzed on human genome arrays. The zone-specific DNA array data obtained from human tissue were compared to array data obtained from bovine cartilage. Genes differentially expressed between zones were evaluated using direct annotation for structural or functional features, and by enrichment analysis for integrated pathways or functions. RESULTS: The greatest differences in genome-wide RNA expression data were between the SZ and DZ in both human and bovine cartilage. The MZ, being a transitional zone between the SZ and DZ, thereby shared some of the same pathways as well as structural/functional features of the adjacent zones. Cellular functions and biologic processes that were enriched in the SZ relative to the DZ included, most prominently, extracellular matrix-receptor interactions, cell adhesion molecule functions, regulation of actin cytoskeleton, ribosome-related functions, and signaling aspects such as the IFN, IL4, Cdc42/Rac, and JAK/STAT signaling pathways. Two pathways were enriched in the DZ relative to the SZ, including PPARG and EGFR/SMRTE. CONCLUSION: These differences in cartilage zonal gene expression identify new markers and pathways that govern the unique differentiation status of chondrocyte subpopulations.

Transcription factor Mohawk and the pathogenesis of human anterior cruciate ligament degradation.

OBJECTIVE: To investigate the expression and function of Mohawk (MKX) in human adult anterior cruciate ligament (ACL) tissue and ligament cells from normal and osteoarthritis (OA)-affected knees. METHODS: Knee joints were obtained at autopsy (within 24-48 hours postmortem) from 13 donors with normal knees (mean ± SD age 36.9 ± 11.0 years), 16 donors with knee OA (age 79.7 ± 11.4 years), and 8 aging donors without knee OA (age 76.9 ± 12.9 years). All cartilage surfaces were graded macroscopically. MKX expression was analyzed by immunohistochemistry and quantitative polymerase chain reaction. ACL-derived cells were used to study regulation of MKX expression by interleukin-1ß (IL-1ß). MKX was knocked down with small interfering RNA (siRNA) to analyze the function of MKX in extracellular matrix (ECM) production and differentiation in ACL-derived cells. RESULTS: The expression of MKX was significantly decreased in ACL-derived cells from OA knees compared with normal knees. Consistent with this finding, immunohistochemistry analysis showed that MKX-positive cells were significantly reduced in ACL tissue from OA donors, in particular in cells located in disorientated fibers. In ACL-derived cells, IL-1ß strongly suppressed MKX expression and reduced expression of the ligament ECM genes COL1A1 and TNXB. In contrast, SOX9, a chondrocyte master transcription factor, was up-regulated by IL-1ß treatment. Importantly, knockdown of MKX expression with siRNA up-regulated SOX9 expression in ACL-derived cells, whereas the expression of COL1A1 and TNXB was reduced. CONCLUSION: Reduced expression of MKX is a feature of degenerated ACL in OA-affected joints, and this may be mediated in part by IL-1ß. MKX appears necessary to maintain the tissue-specific cellular differentiation status and ECM production in adult human tendons and ligaments.