Mocetinostat activates Krüppel-like factor 4 and protects against tissue destruction and inflammation in osteoarthritis.

Osteoarthritis (OA) is the most common joint disorder, and disease-modifying OA drugs (DMOADs) represent a major need in OA management. Krüppel-like factor 4 (KLF4) is a central transcription factor upregulating regenerative and protective functions in joint tissues. This study was aimed to identify small molecules activating KLF4 expression and to determine functions and mechanisms of the hit compounds. High-throughput screening (HTS) with 11,948 clinical-stage compounds was performed using a reporter cell line detecting endogenous KLF4 activation. Eighteen compounds were identified through the HTS and confirmed in a secondary screen. After testing in SW1353 chondrosarcoma cells and human chondrocytes, mocetinostat - a class I selective histone deacetylase (HDAC) inhibitor - had the best profile of biological activities. Mocetinostat upregulated cartilage signature genes in human chondrocytes, meniscal cells, and BM-derived mesenchymal stem cells, and it downregulated hypertrophic, inflammatory, and catabolic genes in those cells and synoviocytes. I.p. administration of mocetinostat into mice reduced severity of OA-associated changes and improved pain behaviors. Global gene expression and proteomics analyses revealed that regenerative and protective effects of mocetinostat were dependent on peroxisome proliferator-activated receptor γ coactivator 1-α. These findings show therapeutic and protective activities of mocetinostat against OA, qualifying it as a candidate to be used as a DMOAD.

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.

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.

Mohawk is a transcription factor that promotes meniscus cell phenotype and tissue repair and reduces osteoarthritis severity.

Meniscus tears are common knee injuries and a major osteoarthritis (OA) risk factor. Knowledge gaps that limit the development of therapies for meniscus injury and degeneration concern transcription factors that control the meniscus cell phenotype. Analysis of RNA sequencing data from 37 human tissues in the Genotype-Tissue Expression database and RNA sequencing data from meniscus and articular cartilage showed that transcription factor Mohawk (MKX) is highly enriched in meniscus. In human meniscus cells, MKX regulates the expression of meniscus marker genes, OA-related genes, and other transcription factors, including Scleraxis (SCX), SRY Box 5 (SOX5), and Runt domain-related transcription factor 2 (RUNX2). In mesenchymal stem cells (MSCs), the combination of adenoviral MKX (Ad-MKX) and transforming growth factor-ß3 (TGF-ß3) induced a meniscus cell phenotype. When Ad-MKX-transduced MSCs were seeded on TGF-ß3-conjugated decellularized meniscus scaffold (DMS) and inserted into experimental tears in meniscus explants, they increased glycosaminoglycan content, extracellular matrix interconnectivity, cell infiltration into the DMS, and improved biomechanical properties. Ad-MKX injection into mouse knee joints with experimental OA induced by surgical destabilization of the meniscus suppressed meniscus and cartilage damage, reducing OA severity. Ad-MKX injection into human OA meniscus tissue explants corrected pathogenic gene expression. These results identify MKX as a previously unidentified key transcription factor that regulates the meniscus cell phenotype. The combination of Ad-MKX with TGF-ß3 is effective for differentiation of MSCs to a meniscus cell phenotype and useful for meniscus repair. MKX is a promising therapeutic target for meniscus tissue engineering, repair, and prevention of OA.

Genome-Wide Occupancy Profiling Reveals Critical Roles of FoxO1 in Regulating Extracellular Matrix and Circadian Rhythm Genes in Human Chondrocytes.

OBJECTIVE: Osteoarthritis (OA) is the most common age-related joint disease. With aging and in OA, the expression of FoxO transcription factors is reduced, diminishing their chondroprotective actions. In order to elucidate the molecular mechanisms by which FoxO1 protects chondrocytes, we sought to identify the genome-wide occupancy profile of FoxO1. METHODS: We performed FoxO1 chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-Seq) on human primary chondrocytes. ChIP-Seq data were integrated with RNA sequencing (RNA-Seq) data sets. Bioinformatics results were confirmed in primary chondrocytes that were treated with a FoxO1 inhibitor. RESULTS: Analysis of FoxO1 ChIP-Seq on human primary chondrocytes showed that pathways implicated in OA pathogenesis are mainly regulated by FoxO1 binding to tissue-specific enhancers with suboptimal binding sites (20% of the peaks), while more ubiquitous FoxO1 pathways are regulated at the promoter level through interaction with its canonical binding motif (7% of the peaks). Integrating FoxO1 occupancy data with RNA-Seq data comparing OA and healthy human cartilage revealed 428 putative FoxO1 target genes that are dysregulated in OA. Pathway analysis showed enrichment for genes belonging to the senescence pathway (logP = -6.73), extracellular matrix (ECM) pathway (logP = -12.97), and circadian clock pathway (logP = -6.30), which suggests that FoxO1 dysregulation plays an important role in their abnormal expression in OA. Using an inhibitor of FoxO1, we confirmed that FoxO1 regulates these pathways in cultured human chondrocytes. CONCLUSION: FoxO1 regulates ubiquitous and cartilage-specific genes in chondrocytes by using different mechanisms. The FoxO1 transcriptional network is a key player in regulating homeostasis, ECM, and circadian clock genes and plays an important role in the abnormal expression of these pathways observed in OA pathogenesis.

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.

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.

FOXO are required for intervertebral disk homeostasis during aging and their deficiency promotes disk degeneration.

Intervertebral disk (IVD) degeneration is a prevalent age-associated musculoskeletal disorder and a major cause of chronic low back pain. Aging is the main risk factor for the disease, but the molecular mechanisms regulating IVD homeostasis during aging are unknown. The aim of this study was to investigate the function of FOXO, a family of transcription factors linked to aging and longevity, in IVD aging and age-related degeneration. Conditional deletion of all FOXO isoforms (FOXO1, 3, and 4) in IVD using the Col2a1Cre and AcanCreER mouse resulted in spontaneous development of IVD degeneration that was driven by severe cell loss in the nucleus pulposus (NP) and cartilaginous endplates (EP). Conditional deletion of individual FOXO in mature mice showed that FOXO1 and FOXO3 are the dominant isoforms and have redundant functions in promoting IVD homeostasis. Gene expression analyses indicated impaired autophagy and reduced antioxidant defenses in the NP of FOXO-deficient IVD. In primary human NP cells, FOXO directly regulated autophagy and adaptation to hypoxia and promoted resistance to oxidative and inflammatory stress. Our findings demonstrate that FOXO are critical regulators of IVD homeostasis during aging and suggest that maintaining or restoring FOXO expression can be a therapeutic strategy to promote healthy IVD aging and delay the onset of IVD degeneration.

Age-related reduction in the expression of FOXO transcription factors and correlations with intervertebral disc degeneration.

Aging is a main risk factor for intervertebral disc (IVD) degeneration, the main cause of low back pain. FOXO transcription factors are important regulators of tissue homeostasis and longevity. Here, we determined the expression pattern of FOXO in healthy and degenerated human IVD and the associations with IVD degeneration during mouse aging. FOXO expression was assessed by immunohistochemistry in normal and degenerated human IVD samples and in cervical and lumbar IVD from 6-, 12-, 24-, and 36-month-old C57BL/6J mice. Mouse spines were graded for key histological features of disc degeneration in all the time points and expression of two key FOXO downstream targets, sestrin 3 (SESN3) and superoxide dismutase (SOD2), was assessed by immunohistochemistry. Histological analysis revealed that FOXO proteins are expressed in all compartments of human and mouse IVD. Expression of FOXO1 and FOXO3, but not FOXO4, was significantly deceased in human degenerated discs. In mice, degenerative changes in the lumbar spine were seen at 24 and 36 months of age whereas cervical IVD showed increased histopathological scores at 36 months. FOXO expression was significantly reduced in lumbar IVD at 12-, 24-, and 36-month-old mice and in cervical IVD at 36-month-old mice when compared with the 6-month-old group. The reduction of FOXO expression in lumbar IVD was concomitant with a decrease in the expression of SESN3 and SOD2. These findings suggest that reduced FOXO expression occurs in lumbar IVD during aging and precedes the major histopathological changes associated with lumbar IVD degeneration. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:2682-2691, 2017.

TWIST1 induces MMP3 expression through up-regulating DNA hydroxymethylation and promotes catabolic responses in human chondrocytes.

The objective was to investigate the levels of TWIST1 in normal and OA cartilage and examine its role in regulating gene expression in chondrocytes. Human cartilage tissues and chondrocytes were obtained at autopsy from normal knee joints and from OA-affected joints at the time of total knee arthroplasty. TWIST1 expression was increased in human OA knee cartilage compared to normal knee cartilage. TWIST1 induced matrix metalloproteinase 3 (MMP3) expression without direct binding to MMP3 promoter and increased the 5-hydroxymethylcytosine (5hmC) level at the MMP3 promoter. The effect of TWIST1 on expression of TET family (TET1, 2 and 3) was measured in stable TWIST1 transfected TC28 cells, and TET1 expression was up-regulated. TWIST1 dependent upregulation of Mmp3 expression was suppressed in Tet triple KO fibroblast derived from mouse ES cells. Increased TWIST1 expression is a feature of OA-affected cartilage. We identified a novel mechanism of catabolic reaction where TWIST1 up-regulates MMP3 expression by enriching 5hmC levels at the MMP3 promoter via TET1 induction. These findings implicate TWIST1 as an important factor regulating OA related gene expression. Clarifying epigenetic mechanisms of 5hmC induced by TWIST1 is a critical molecule to understanding OA pathogenesis.

Mohawk promotes the maintenance and regeneration of the outer annulus fibrosus of intervertebral discs.

The main pathogenesis of intervertebral disc (IVD) herniation involves disruption of the annulus fibrosus (AF) caused by ageing or excessive mechanical stress and the resulting prolapse of the nucleus pulposus. Owing to the avascular nature of the IVD and lack of understanding the mechanisms that maintain the IVD, current therapies do not lead to tissue regeneration. Here we show that homeobox protein Mohawk (Mkx) is a key transcription factor that regulates AF development, maintenance and regeneration. Mkx is mainly expressed in the outer AF (OAF) of humans and mice. In Mkx(-/-) mice, the OAF displays a deficiency of multiple tendon/ligament-related genes, a smaller OAF collagen fibril diameter and a more rapid progression of IVD degeneration compared with the wild type. Mesenchymal stem cells overexpressing Mkx promote functional AF regeneration in a mouse AF defect model, with abundant collagen fibril formation. Our results indicate a therapeutic strategy for AF regeneration.

Hyaluronan concentration and size distribution in human knee synovial fluid: variations with age and cartilage degeneration.

BACKGROUND: One potential mechanism for early superficial cartilage wear in normal joints is alteration of the lubricant content and quality of synovial fluid. The purpose of this study was to determine if the concentration and quality of the lubricant, hyaluronan, in synovial fluid: (1) was similar in left and right knees; (2) exhibited similar age-associated trends, whether collected postmortem or antemortem; and (3) varied with age and grade of joint degeneration. METHODS: Human synovial fluid of donors (23-91 years) without osteoarthritis was analyzed for the concentrations of protein, hyaluronan, and hyaluronan in the molecular weight ranges of 2.5-7 MDa, 1-2.5 MDa, 0.5-1 MDa, and 0.03-0.5 MDa. Similarity of data between left and right knees was assessed by reduced major axis regression, paired t-test, and Bland-Altman analysis. The effect of antemortem versus postmortem collection on biochemical properties was assessed for age-matched samples by unpaired t-test. The relationships between age, joint grade, and each biochemical component were assessed by regression analysis. RESULTS: Joint grade and the concentrations of protein, hyaluronan, and hyaluronan in the molecular weight ranges of 2.5-7 MDa, 1-2.5 MDa, and 0.5-1 MDa in human synovial fluid showed good agreement between left and right knees and were similar between age-matched patient and cadaver knee joints. There was an age-associated decrease in overall joint grade (-15 %/decade) and concentrations of hyaluronan (-10.5 %/decade), and hyaluronan in the molecular weight ranges of 2.5-7 MDa (-9.4 %/decade), 1-2.5 MDa (-11.3 %/decade), 0.5-1 MDa (-12.5 %/decade), and 0.03-0.5 MDa (-13.0 %/decade). Hyaluronan concentration and quality was more strongly associated with age than with joint grade. CONCLUSIONS: The age-related increase in cartilage wear in non-osteoarthritic joints may be related to the altered hyaluronan content and quality of synovial fluid.

Transthyretin deposition in articular cartilage: a novel mechanism in the pathogenesis of osteoarthritis.

OBJECTIVE: Amyloid deposits are prevalent in osteoarthritic (OA) joints. We undertook this study to define the dominant precursor and to determine whether the deposits affect chondrocyte functions. METHODS: Amyloid deposition in human normal and OA knee cartilage was determined by Congo red staining. Transthyretin (TTR) in cartilage and synovial fluid was analyzed by immunohistochemistry and Western blotting. The effects of recombinant amyloidogenic and nonamyloidogenic TTR variants were tested in human chondrocyte cultures. RESULTS: Normal cartilage from young donors did not contain detectable amyloid deposits, but 7 of 12 aged normal cartilage samples (58%) and 12 of 12 OA cartilage samples (100%) had Congo red staining with green birefringence under polarized light. TTR, which is located predominantly at the cartilage surfaces, was detected in all OA cartilage samples and in a majority of aged normal cartilage samples, but not in normal cartilage samples from young donors. Chondrocytes and synoviocytes did not contain significant amounts of TTR messenger RNA. Synovial fluid TTR levels were similar in normal and OA knees. In cultured chondrocytes, only an amyloidogenic TTR variant induced cell death as well as the expression of proinflammatory cytokines and extracellular matrix-degrading enzymes. The effects of amyloidogenic TTR on gene expression were mediated in part by Toll-like receptor 4, receptor for advanced glycation end products, and p38 MAPK. TTR-induced cytotoxicity was inhibited by resveratrol, a plant polyphenol that stabilizes the native tetrameric structure of TTR. CONCLUSION: These findings are the first to suggest that TTR amyloid deposition contributes to cell and extracellular matrix damage in articular cartilage in human OA and that therapies designed to reduce TTR amyloid formation might be useful.

Chondrocyte clusters adjacent to sites of cartilage degeneration have characteristics of progenitor cells.

The purpose of this study was to investigate the site-specific characteristics and roles of chondrocyte clusters in human knee osteoarthritis. Cartilage explants were obtained from 45 knees undergoing total knee replacement surgery. The explants were taken from 4 locations in the knee: the medial femoral condyle, the medial posterior femoral condyle (MPC), the lateral femoral condyle, and the lateral posterior femoral condyle (LPC). Cartilage degeneration, cell density, and cell arrangement were compared histologically. A live/dead cell viability assay and immunohistochemical analyses using antibodies against STRO-1, FGF2, and Ki-67 were performed. Cell proliferation and cartilaginous nodule production in MPC and LPC explants in monolayer culture were compared. Finally, MPC cartilage explants were cultured to observe histological changes. The cell density of the MPC explants was higher than that of the LPC because of clustering. MPC explants contained more live cells than the LPC did, and the expression of IHC markers in MPC explants was higher than that in LPC. Chondrocytes from MPC proliferated faster and produced more nodules in monolayer culture than those from the LPC and MPC explants were repaired during organ culture. In conclusion, chondrocyte clusters adjacent to severe cartilage degeneration have specific characteristics, with progenitor and proliferative potential.

FoxO transcription factors support oxidative stress resistance in human chondrocytes.

OBJECTIVE: A major signaling pathway that regulates cellular aging is the insulin/insulin-like growth factor 1 (IGF-1)/phosphatidylinositol 3-kinase (PI3K)/Akt/FoxO transcription factor axis. We previously observed that FoxO transcription factors are dysregulated in aged and OA cartilage. The objective of this study was to investigate the impact of down-regulated FoxO transcription factors on chondrocytes. METHODS: Small interfering RNAs (siRNAs) targeting FOXO1 (siFOXO1) and FOXO3 (siFOXO3) were transfected into human articular chondrocytes. Cell viability following treatment with the oxidant tert-butyl-hydroperoxide (tBHP) was measured by MTT assay. Caspase 3/7 activation and apoptotic cells were examined. Gene and protein expression of antioxidant proteins and autophagy-related proteins and changes in inflammatory mediators following treatment with interleukin-1ß were assessed. Cells transfected with FOXO plasmids were also analyzed. RESULTS: Cell viability was significantly reduced by siFOXO after treatment with tBHP. Apoptosis accompanied by caspase activation was significantly increased in siFOXO-transfected chondrocytes. Knockdown of FOXO1 and FOXO1+3 resulted in significant reductions in levels of glutathione peroxidase 1 (GPX-1), catalase, light chain 3 (LC3), Beclin1, and sirtuin 1 (SIRT-1) proteins following treatment with tBHP. In contrast, the constitutive active form of FOXO3 increased cell viability while inducing GPX-1, Beclin1, and LC3 in response to tBHP. Expression and production of ADAMTS-4 and chemerin were significantly increased in siFOXO-transfected chondrocytes. CONCLUSION: Reduced expression of FoxO transcription factors in chondrocytes increased susceptibility to cell death induced by oxidative stress. This was associated with reduced levels of antioxidant proteins and autophagy-related proteins. Our data provide evidence for a key role of FoxO transcription factors as regulators of chondrocyte oxidative stress resistance and tissue homeostasis.

Palmitate has proapoptotic and proinflammatory effects on articular cartilage and synergizes with interleukin-1.

OBJECTIVE: Obesity is a major risk factor for the development of osteoarthritis (OA) that is associated with a state of low-grade inflammation and increased circulating levels of adipokines and free fatty acids (FFAs). The aim of this study was to analyze the effects of saturated (palmitate) and monounsaturated (oleate) FFAs on articular chondrocytes, synoviocytes, and cartilage. METHODS: Human articular chondrocytes and fibroblast-like synoviocytes obtained from young healthy donors and OA chondrocytes from patients undergoing total knee replacement surgery were treated with palmitate or oleate alone or in combination with interleukin-1ß (IL-1ß). Cell viability, caspase activation, and gene expression of proinflammatory factors, extracellular matrix (ECM) proteins, and proteases were studied. In addition, chondrocyte viability, IL-6 production, and matrix damage were assessed in bovine and human articular cartilage explants cultured with FFAs in the presence or absence of IL-1ß. RESULTS: Palmitate, but not oleate, induced caspase activation and cell death in IL-1ß-stimulated normal chondrocytes, and up-regulated the expression of IL-6 and cyclooxygenase 2 in chondrocytes and fibroblast-like synoviocytes through Toll-like receptor 4 (TLR-4) signaling. In cartilage explants, palmitate induced chondrocyte death, IL-6 release, and ECM degradation. Palmitate synergized with IL-1ß in stimulating proapoptotic and proinflammatory cellular responses. Pharmacologic inhibition of caspases or TLR-4 signaling reduced palmitate and IL-1ß induced cartilage damage. CONCLUSION: Palmitate acts as a proinflammatory and catabolic factor that, in synergy with IL-1ß, induces chondrocyte apoptosis and articular cartilage breakdown. Collectively, our data suggest that elevated levels of saturated FFAs that are often found in patients who are obese may contribute to the pathogenesis of OA.

Repair of cartilage defects in arthritic tissue with differentiated human embryonic stem cells.

Chondrocytes have been generated in vitro from a range of progenitor cell types and by a number of strategies. However, achieving reconstitution of actual physiologically relevant, appropriately-laminated cartilage in situ that would be applicable to conditions, such as arthritis and cartilage degeneration remains elusive. This lack of success is multifactorial and includes limited cell source, decreased proliferation rate of mature chondrocytes, lack of maintenance of phenotype, reduced matrix synthesis, and poor integration with host tissue. We report an efficient approach for deriving mesenchymal chondroprogenitor cells from human embryonic stem cells. These cells generated tissue containing cartilage-specific matrix proteins that integrated in situ in a partial-thickness defect in ex vivo articular cartilage harvested from human arthritic joints. Given that stem cells provide a virtually inexhaustible supply of starting material and that our technique is easily scalable, cartilaginous tissue primed and grafted in this manner could be suitable for clinical translation.

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.

Cellular and extracellular matrix changes in anterior cruciate ligaments during human knee aging and osteoarthritis.

INTRODUCTION: Anterior cruciate ligament (ACL) degeneration is observed in most osteoarthritis (OA)-affected knee joints. However, the specific spatial and temporal relations of these changes and their association with extracellular matrix (ECM) degeneration are not well understood. The objective of this study was to characterize the patterns and relations of aging-related and OA-associated changes in ACL cells and the ECM. METHODS: Human knee joints from 80 donors (age 23 through 94) were obtained at autopsy. ACL degeneration was assessed histologically by using a quantitative scoring system. Tissue sections were analyzed for cell density, cell organization, ECM components, ECM-degrading enzymes and markers of differentiation, proliferation, and stem cells. RESULTS: Total cell number in normal ACL decreased with aging but increased in degenerated ACL, because of the formation of perivascular cell aggregates and islands of chondrocyte-like cells. Matrix metalloproteinase (MMP)-1, -3, and -13 expression was reduced in aging ACL but increased in degenerated ACL, mainly in the chondrocyte-like cells. Collagen I was expressed throughout normal and degenerated ACL. Collagen II and X were detected only in the areas with chondroid metaplasia, which also expressed collagen III. Sox9, Runt-related transcription factor 2 (Runx2), and scleraxis expression was increased in the chondrocyte-like cells in degenerated ACL. Alpha-smooth muscle actin (α-SMA), a marker of myofibroblasts and the progenitor cell marker STRO-1, decreased with aging in normal ACL. In degenerated ACL, the new cell aggregates were positive for α-SMA and STRO-1. CONCLUSIONS: ACL aging is characterized by reduced cell density and activation. In contrast, ACL degeneration is associated with cell recruitment or proliferation, including progenitor cells or myofibroblasts. Abnormally differentiated chondrocyte-like cell aggregates in degenerated ACL produce abnormal ECM and may predispose to mechanical failure.

Histopathological changes in the human posterior cruciate ligament during aging and osteoarthritis: correlations with anterior cruciate ligament and cartilage changes.

OBJECTIVES: To determine the histological patterns of posterior cruciate ligament (PCL) degeneration during aging and in relation to changes in articular cartilage and anterior cruciate ligament (ACL) across the entire adult age spectrum. METHODS: Human knee joints (n=120 from 65 donors) were processed within 72 h of postmortem. Articular cartilage surfaces were graded macroscopically. Each PCL was histologically evaluated for inflammation, mucinous changes, chondroid metaplasia, cystic changes and orientation of collagen fibres. The severity of PCL degeneration was classified as normal, mild, moderate or severe. PCL scores were compared to ACL and cartilage scores from the same knees. RESULTS: All knees had intact PCL. Histologically, 6% were normal, 76% showed mild, 12% moderate and 9% severe degeneration. Fibre disorientation was the most prevalent and severe change. Histological grades of PCL and ACL correlated, but significantly fewer PCL than ACL showed severe changes. There was a weaker correlation between aging and total histological PCL scores (R=0.26) compared to aging and ACL scores (R=0.42). ACL scores correlated with cartilage scores (R=0.54) while PCL scores increased with the severity of osteoarthritis from grades 0 to III but not between osteoarthritis grades III-IV (R=0.32). In knees with ruptured ACL, the PCL scores correlated with cartilage scores of the lateral compartment. CONCLUSIONS: PCL histopathological changes were less severe than in the ACL. PCL degeneration was associated with ACL and cartilage damage. The lack of correlation with age indicates independent pathways for PCL versus ACL degeneration.