Clinical and biobehavioral phenotypic assessments and data harmonization for the RE-JOIN research consortium: Recommendations for common data element selection.

Background: The Restoring Joint Health and Function to Reduce Pain (RE-JOIN) Consortium is part of the Helping to End Addiction Long-term® (HEAL) Initiative. HEAL is an ambitious, NIH-wide initiative to speed scientific solutions to stem the national opioid public health crisis. The RE-JOIN consortium's over-arching goal is to define how chronic joint pain-mediating neurons innervate different articular and peri-articular tissues, with a focus on the knee and temporomandibular joints (TMJ) across species employing the latest neuroscience approaches. The aim of this manuscript is to elucidate the human data gathered by the RE-JOIN consortium, as well as to expound upon its underlying rationale and the methodologies and protocols for harmonization and standardization that have been instituted by the RE-JOIN Consortium. Methods: The consortium-wide human models working subgroup established the RE-JOIN minimal harmonized data elements that will be collected across all human studies and set the stage to develop parallel pre-clinical data collection standards. Data harmonization considerations included requirements from the HEAL program and recommendations from the consortium's researchers and experts on informatics, knowledge management, and data curation. Results: Multidisciplinary experts - including preclinical and clinical researchers, with both clinician-scientists- developed the RE-JOIN's Minimal Human Data Standard with required domains and outcome measures to be collected across projects and institutions. The RE-JOIN minimal data standard will include HEAL Common Data Elements (CDEs) (e.g., standardized demographics, general pain, psychosocial and functional measures), and RE-JOIN common data elements (R-CDE) (i.e., both general and joint-specific standardized and clinically important self-reported pain and function measures, as well as pressure pain thresholds part of quantitative sensory testing). In addition, discretionary, site-specific measures will be collected by individual institutions (e.g., expanded quantitative sensory testing and gait biomechanical assessments), specific to the knee or TMJ. Research teams will submit datasets of standardized metadata to the RE-JOIN Data Coordinating Center (DCG) via a secure cloud-based central data repository and computing infrastructure for researchers to share and conduct analyses on data collected by or acquired for RE-JOIN. RE-JOIN datasets will have protected health information (PHI) removed and be publicly available on the SPARC portal and accessible through the HEAL Data Ecosystem. Conclusion: Data Harmonization efforts provide the multidisciplinary consortium with an opportunity to effectively collaborate across decentralized research teams, and data standardization sets the framework for efficient future analyses of RE-JOIN data collected by the consortium. The harmonized phenotypic information obtained will significantly enhance our understanding of the neurobiology of the pain-pathology relationships in humans, providing valuable insights for comparison with pre-clinical models.

Shared and Compartment-Specific Processes in Nucleus Pulposus and Annulus Fibrosus During Intervertebral Disc Degeneration.

Elucidating how cell populations promote onset and progression of intervertebral disc degeneration (IDD) has the potential to enable more precise therapeutic targeting of cells and mechanisms. Single-cell RNA-sequencing (scRNA-seq) is performed on surgically separated annulus fibrosus (AF) (19,978; 26,983 cells) and nucleus pulposus (NP) (20,884; 24,489 cells) from healthy and diseased human intervertebral discs (IVD). In both tissue types, depletion of cell subsets involved in maintenance of healthy IVD is observed, specifically the immature cell subsets - fibroblast progenitors and stem cells - indicative of an impairment of normal tissue self-renewal. Tissue-specific changes are also identified. In NP, several fibrotic populations are increased in degenerated IVD, indicating tissue-remodeling. In degenerated AF, a novel disease-associated subset is identified, which expresses disease-promoting genes. It is associated with pathogenic biological processes and the main gene regulatory networks include thrombospondin signaling and FOXO1 transcription factor. In NP and AF cells thrombospondin protein promoted expression of genes associated with TGFß/fibrosis signaling, angiogenesis, and nervous system development. The data reveal new insights of both shared and tissue-specific changes in specific cell populations in AF and NP during IVD degeneration. These identified mechanisms and molecules are novel and more precise targets for IDD prevention and treatment.

vSPACE: Exploring Virtual Spatial Representation of Articular Chondrocytes at the Single-Cell Level.

Single cell RNA sequencing technology has been dramatically changing how gene expression studies are performed. However, its use has been limited to identifying subtypes of cells by comparing cells' gene expression levels in an unbiased manner to produce a 2D plot (e.g., UMAP/tSNE). We developed a new method of placing cells in 2D space. This system, called vSPACE, shows a virtual spatial representation of scRNAseq data obtained from human articular cartilage by emulating the concept of spatial transcriptomics technology, but virtually. This virtual 2D plot presentation of human articular cartage cells generates several zonal distribution patterns, in one or multiple genes at a time, reveling patterns that scientists can appreciate as imputed spatial distribution patterns along the zonal axis. The discovered patterns are explainable and remarkably consistent across all six healthy doners despite their respectively different clinical variables (age and sex), suggesting the confidence of the discovered patterns.

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.

RNA-binding proteins that are highly expressed and enriched in healthy cartilage but suppressed in osteoarthritis.

Objectives: RNA-binding proteins (RBPs) have diverse and essential biological functions, but their role in cartilage health and disease is largely unknown. The objectives of this study were (i) map the global landscape of RBPs expressed and enriched in healthy cartilage and dysregulated in osteoarthritis (OA); (ii) prioritize RBPs for their potential role in cartilage and in OA pathogenesis and as therapeutic targets. Methods: Our published bulk RNA-sequencing (RNA-seq) data of healthy and OA human cartilage, and a census of 1,542 RBPs were utilized to identify RBPs that are expressed in healthy cartilage and differentially expressed (DE) in OA. Next, our comparison of healthy cartilage RNA-seq data to 37 transcriptomes in the Genotype-Tissue Expression (GTEx) database was used to determine RBPs that are enriched in cartilage. Finally, expression of RBPs was analyzed in our single cell RNA-sequencing (scRNA-seq) data from healthy and OA human cartilage. Results: Expression of RBPs was higher than nonRBPs in healthy cartilage. In OA cartilage, 188 RBPs were differentially expressed, with a greater proportion downregulated. Ribosome biogenesis was enriched in the upregulated RBPs, while splicing and transport were enriched in the downregulated. To further prioritize RBPs, we selected the top 10% expressed RBPs in healthy cartilage and those that were cartilage-enriched according to GTEx. Intersecting these criteria, we identified Tetrachlorodibenzodioxin (TCDD) Inducible Poly (ADP-Ribose) Polymerase (TIPARP) as a candidate RBP. TIPARP was downregulated in OA. scRNA-seq data revealed TIPARP was most significantly downregulated in the "pathogenic cluster". Conclusion: Our global analyses reveal expression patterns of RBPs in healthy and OA cartilage. We also identified TIPARP and other RBPs as novel mediators in OA pathogenesis and as potential therapeutic targets.

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.

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.

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.

Suppression of Autoimmune Rheumatoid Arthritis with Hybrid Nanoparticles That Induce B and T Cell Tolerance to Self-Antigen.

Autoimmune diseases affect over 4% of the world's population. Treatments are generally palliative or use broad spectrum immunosuppressants to reduce symptoms and disease progression. In some diseases, antibodies generated to a single autoantigen are the major cause of pathogenic inflammation, suggesting that treatments to induce tolerance to the autoantigen could be therapeutic. Here we report the development of hybrid nanoparticles (NPs) that induce tolerance in both T cells and B cells. The NPs comprise a lipid monolayer encapsulating a PLGA core loaded with rapamycin that promotes development of regulatory T cells (Tregs). The lipid monolayer displays the protein antigen and a ligand of the B cell inhibitory co-receptor CD22 (CD22L) that act together to suppress activation of B cells recognizing the antigen. We demonstrate that the hybrid NPs decorated with ovalbumin (OVA) elicit tolerance to OVA in naïve mice, as judged by low OVA-specific antibody titers after the challenge. In the K/BxN mouse model of rheumatoid arthritis caused by B and T cell-dependent responses to the self-antigen glucose-6-phosphate-isomerase (GPI), we show that GPI hybrid NPs delay development of disease, with some treated mice remaining arthritis-free for 300 days. We provide evidence that the mechanism of rheumatoid arthritis suppression involves induction of B cell tolerance, as measured by low anti-GPI antibodies and decreased plasma cell populations, and T cell tolerance, as measured by increased Tregs. The results show the potential of this versatile NP platform for inducing immune tolerance to a self-antigen and suppressing autoimmune disease.

Urolithin A improves mitochondrial health, reduces cartilage degeneration, and alleviates pain in osteoarthritis.

Osteoarthritis (OA) is the most common age-related joint disorder with no effective therapy. According to the World Health Organization, OA affects over 500 million people and is characterized by degradation of cartilage and other joint tissues, severe pain, and impaired mobility. Mitochondrial dysfunction contributes to OA pathology. However, interventions to rescue mitochondrial defects in human OA are not available. Urolithin A (Mitopure) is a natural postbiotic compound that promotes mitophagy and mitochondrial function and beneficially impacts muscle health in preclinical models of aging and in elderly and middle-aged humans. Here, we showed that Urolithin A improved mitophagy and mitochondrial respiration in primary chondrocytes from joints of both healthy donors and OA patients. Furthermore, Urolithin A reduced disease progression in a mouse model of OA, decreasing cartilage degeneration, synovial inflammation, and pain. These improvements were associated with increased mitophagy and mitochondrial content, in joints of OA mice. These findings indicate that UA promotes joint mitochondrial health, alleviates OA pathology, and supports Urolithin A's potential to improve mobility with beneficial effects on structural damage in joints.

The mechanosensitive ion channel PIEZO1 is expressed in tendons and regulates physical performance.

How mechanical stress affects physical performance via tendons is not fully understood. Piezo1 is a mechanosensitive ion channel, and E756del PIEZO1 was recently found as a gain-of-function variant that is common in individuals of African descent. We generated tendon-specific knock-in mice using R2482H Piezo1, a mouse gain-of-function variant, and found that they had higher jumping abilities and faster running speeds than wild-type or muscle-specific knock-in mice. These phenotypes were associated with enhanced tendon anabolism via an increase in tendon-specific transcription factors, Mohawk and Scleraxis, but there was no evidence of changes in muscle. Biomechanical analysis showed that the tendons of R2482H Piezo1 mice were more compliant and stored more elastic energy, consistent with the enhancement of jumping ability. These phenotypes were replicated in mice with tendon-specific R2482H Piezo1 replacement after tendon maturation, indicating that PIEZO1 could be a target for promoting physical performance by enhancing function in mature tendon. The frequency of E756del PIEZO1 was higher in sprinters than in population-matched nonathletic controls in a small Jamaican cohort, suggesting a similar function in humans. Together, this human and mouse genetic and physiological evidence revealed a critical function of tendons in physical performance, which is tightly and robustly regulated by PIEZO1 in tenocytes.

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.

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.

TAFI deficiency causes maladaptive vascular remodeling after hemophilic joint bleeding.

Excessive vascular remodeling is characteristic of hemophilic arthropathy (HA) and may contribute to joint bleeding and the progression of HA. Mechanisms for pathological vascular remodeling after hemophilic joint bleeding are unknown. In hemophilia, activation of thrombin-activatable fibrinolysis inhibitor (TAFI) is impaired, which contributes to joint bleeding and may also underlie the aberrant vascular remodeling. Here, hemophilia A (factor VIII-deficient; FVIII-deficient) mice or TAFI-deficient mice with transient (antibody-induced) hemophilia A were used to determine the role of FVIII and TAFI in vascular remodeling after joint bleeding. Excessive vascular remodeling and vessel enlargement persisted in FVIII-deficient and TAFI-deficient mice, but not in transient hemophilia WT mice, after similar joint bleeding. TAFI-overexpression in FVIII-deficient mice prevented abnormal vessel enlargement and vascular leakage. Age-related vascular changes were observed with FVIII or TAFI deficiency and correlated positively with bleeding severity after injury, supporting increased vascularity as a major contributor to joint bleeding. Antibody-mediated inhibition of uPA also prevented abnormal vascular remodeling, suggesting that TAFI's protective effects include inhibition of uPA-mediated plasminogen activation. In conclusion, the functional TAFI deficiency in hemophilia drives maladaptive vascular remodeling in the joints after bleeding. These mechanistic insights allow targeted development of potentially new strategies to normalize vascularity and control rebleeding in HA.

Wwp2 maintains cartilage homeostasis through regulation of Adamts5.

The WW domain-containing protein 2 (Wwp2) gene, the host gene of miR-140, codes for the Wwp2 protein, which is an HECT-type E3 ubiquitin ligases abundantly expressed in articular cartilage. However, its function remains unclear. Here, we show that mice lacking Wwp2 and mice in which the Wwp2 E3 enzyme is inactivated (Wwp2-C838A) exhibit aggravated spontaneous and surgically induced osteoarthritis (OA). Consistent with this phenotype, WWP2 expression level is downregulated in human OA cartilage. We also identify Runx2 as a Wwp2 substrate and Adamts5 as a target gene, as similar as miR-140. Analysis of Wwp2-C838A mice shows that loss of Wwp2 E3 ligase activity results in upregulation of Runx2-Adamts5 signaling in articular cartilage. Furthermore, in vitro transcribed Wwp2 mRNA injection into mouse joints reduces the severity of experimental OA. We propose that Wwp2 has a role in protecting cartilage from OA by suppressing Runx2-induced Adamts5 via Runx2 poly-ubiquitination and degradation.

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.

Platelet-derived growth factor-coated decellularized meniscus scaffold for integrative healing of meniscus tears.

The aim of this study was to examine the potential of platelet-derived growth factor (PDGF)-coated decellularized meniscus scaffold in mediating integrative healing of meniscus tears by inducing endogenous cell migration. Fresh bovine meniscus was chemically decellularized and covalently conjugated with heparin and PDGF-BB. In vitro PDGF release kinetics was measured. The scaffold was transplanted into experimental tears in avascular bovine meniscus explants and cultured for 2 and 4 weeks. The number migrating and proliferating cells at the borderline between the scaffold and injured explant and PDGF receptor-ß (PDGFRß) expressing cells were counted. The alignment of the newly produced ECM and collagen was analyzed by Safranin-O, picrosirius red staining, and differential interference contrast (DIC). Tensile testing of the explants was performed after culture for 2 and 4 weeks. Heparin conjugated scaffold showed immobilization of high levels of PDGF-BB, with sustained release over 2 weeks. Insertion of the PDGF-BB treated scaffold in defects in avascular meniscus led to increased PDGFRß expression, cell migration and proliferation into the defect zone. Safranin-O, picrosirius red staining and DIC showed tissue integration between the scaffold and injured explants. Tensile properties of injured explants treated with PDGF-BB coated scaffold were significantly higher than in the scaffold without PDGF. In conclusion, PDGF-BB-coated scaffold increased PDGFRß expression and promoted migration of endogenous meniscus cells to the defect area. New matrix was formed that bridged the space between the native meniscus and the scaffold and this was associated with improved biomechanical properties. The PDGF-BB-coated scaffold will be promising for clinical translation to healing of meniscus tears. STATEMENT OF SIGNIFICANCE: Meniscus tears are the most common injury of the knee joint. The most prevalent forms that occur in the inner third typically do not spontaneously heal and represent a major risk factor for the development of knee osteoarthritis. The goal of this project was to develop an approach that is readily applicable for clinical use. We selected a natural and readily available decellularized meniscus scaffold and conjugated it with PDGF, which we had previously found to have strong chemotactic activity for chondrocytes and progenitor cells. The present results show that insertion of the PDGF-conjugated scaffold in defects in avascular meniscus led to endogenous cell migration and proliferation into the defect zone with tissue integration between the scaffold and injured explants and improved tensile properties. This PDGF-conjugated scaffold will be promising for a translational approach to healing of meniscus tears.

FoxO transcription factors modulate autophagy and proteoglycan 4 in cartilage homeostasis and osteoarthritis.

Aging is a main risk factor for osteoarthritis (OA). FoxO transcription factors protect against cellular and organismal aging, and FoxO expression in cartilage is reduced with aging and in OA. To investigate the role of FoxO in cartilage, Col2Cre-FoxO1, 3, and 4 single knockout (KO) and triple KO mice (Col2Cre-TKO) were analyzed. Articular cartilage in Col2Cre-TKO and Col2Cre-FoxO1 KO mice was thicker than in control mice at 1 or 2 months of age. This was associated with increased proliferation of chondrocytes of Col2Cre-TKO mice in vivo and in vitro. OA-like changes developed in cartilage, synovium, and subchondral bone between 4 and 6 months of age in Col2Cre-TKO and Col2Cre-FoxO1 KO mice. Col2Cre-FoxO3 and FoxO4 KO mice showed no cartilage abnormalities until 18 months of age when Col2Cre-FoxO3 KO mice had more severe OA than control mice. Autophagy and antioxidant defense genes were reduced in Col2Cre-TKO mice. Deletion of FoxO1/3/4 in mature mice using Aggrecan(Acan)-CreERT2 (AcanCreERT-TKO) also led to spontaneous cartilage degradation and increased OA severity in a surgical model or treadmill running. The superficial zone of knee articular cartilage of Col2Cre-TKO and AcanCreERT-TKO mice exhibited reduced cell density and markedly decreased Prg4 In vitro, ectopic FoxO1 expression increased Prg4 and synergized with transforming growth factor-ß stimulation. In OA chondrocytes, overexpression of FoxO1 reduced inflammatory mediators and cartilage-degrading enzymes, increased protective genes, and antagonized interleukin-1ß effects. Our observations suggest that FoxO play a key role in postnatal cartilage development, maturation, and homeostasis and protect against OA-associated cartilage damage.

Transthyretin deposition promotes progression of osteoarthritis.

Deposition of amyloid is a common aging-associated phenomenon in several aging-related diseases. Osteoarthritis (OA) is the most prevalent joint disease, and aging is its major risk factor. Transthyretin (TTR) is an amyloidogenic protein that is deposited in aging and OA-affected human cartilage and promotes inflammatory and catabolic responses in cultured chondrocytes. Here, we investigated the role of TTR in vivo using transgenic mice overexpressing wild-type human TTR (hTTR-TG). Although TTR protein was detected in cartilage in hTTR-TG mice, the TTR transgene was highly overexpressed in liver, but not in chondrocytes. OA was surgically induced by destabilizing the medial meniscus (DMM) in hTTR-TG mice, wild-type mice of the same strain (WT), and mice lacking endogenous Ttr genes. In the DMM model, both cartilage and synovitis histological scores were significantly increased in hTTR-TG mice. Further, spontaneous degradation and OA-like changes in cartilage and synovium developed in 18-month-old hTTR mice. Expression of cartilage catabolic (Adamts4, Mmp13) and inflammatory genes (Nos2, Il6) was significantly elevated in cartilage from 6-month-old hTTR-TG mice compared with WT mice as was the level of phospho-NF-κB p65. Intra-articular injection of aggregated TTR in WT mice increased synovitis and significantly increased expression of inflammatory genes in synovium. These findings are the first to show that TTR deposition increases disease severity in the murine DMM and aging model of OA.