Sex-specific effects of injury and beta-adrenergic activation on metabolic and inflammatory mediators in a murine model of post-traumatic osteoarthritis.

OBJECTIVE: Metabolic processes are intricately linked to the resolution of innate inflammation and tissue repair, two critical steps for treating post-traumatic osteoarthritis (PTOA). Based on lipolytic and immunoregulatory actions of norepinephrine, we hypothesized that intra-articular ß-adrenergic receptor (ßAR) stimulation would suppress PTOA-associated inflammation in the infrapatellar fat pad (IFP) and synovium. DESIGN: We used the ßAR agonist isoproterenol to perturb intra-articular metabolism 3.5 weeks after applying a non-invasive single-load compression injury to knees of 12-week-old male and female mice. We examined the acute effects of intra-articular isoproterenol treatment relative to saline on IFP histology, multiplex gene expression of synovium-IFP tissue, synovial fluid metabolomics, and mechanical allodynia. RESULTS: Injured knees developed PTOA pathology characterized by heterotopic ossification, articular cartilage loss, and IFP atrophy and fibrosis. Isoproterenol suppressed the upregulation of pro-fibrotic genes and downregulated the expression of adipose genes and pro-inflammatory genes (Adam17, Cd14, Icam1, Csf1r, and Casp1) in injured joints of female (but not male) mice. Analysis of published single-cell RNA-seq data identified elevated catecholamine-associated gene expression in resident-like synovial-IFP macrophages after injury. Injury substantially altered synovial fluid metabolites by increasing amino acids, peptides, sphingolipids, phospholipids, bile acids, and dicarboxylic acids, but these changes were not appreciably altered by isoproterenol. Intra-articular injection of either isoproterenol or saline increased mechanical allodynia in female mice, whereas neither substance affected male mice. CONCLUSIONS: Acute ßAR activation altered synovial-IFP transcription in a sex and injury-dependent manner, suggesting that women with PTOA may be more sensitive than men to treatments targeting sympathetic neural signaling pathways.

Imaging mass cytometry reveals tissue-specific cellular immune phenotypes in the mouse knee following ACL injury.

Objective: To develop an imaging mass cytometry method for identifying complex cell phenotypes, inter-cellular interactions, and population changes in the synovium and infrapatellar fat pad (IFP) of the mouse knee following a non-invasive compression injury. Design: Fifteen male C57BL/6 mice were fed a high-fat diet for 8 weeks prior to random assignment to sham, 0.88 â€‹mm, or 1.7 â€‹mm knee compression displacement at 24 weeks of age. 2-weeks after loading, limbs were prepared for histologic and imaging mass cytometry analysis, focusing on myeloid immune cell populations in the synovium and IFP. Results: 1.7 â€‹mm compression caused anterior cruciate ligament (ACL) rupture, development of post-traumatic osteoarthritis, and a 2- to 3-fold increase in cellularity of synovium and IFP tissues compared to sham or 0.88 â€‹mm compression. Imaging mass cytometry identified 11 myeloid cell subpopulations in synovium and 7 in IFP, of which approximately half were elevated 2 weeks after ACL injury in association with the vasculature. Notably, two monocyte/macrophage subpopulations and an MHC IIhi population were elevated 2-weeks post-injury in the synovium but not IFP. Vascular and immune cell interactions were particularly diverse in the synovium, incorporating 8 unique combinations of 5 myeloid cell populations, including a monocyte/macrophage population, an MHC IIhi population, and 3 different undefined F4/80+ myeloid populations. Conclusions: Developing an imaging mass cytometry method for the mouse enabled us to identify a diverse array of synovial and IFP vascular-associated myeloid cell subpopulations. These subpopulations were differentially elevated in synovial and IFP tissues 2-weeks post injury, providing new details on tissue-specific immune regulation.

Effects of Leptin and Body Weight on Inflammation and Knee Osteoarthritis Phenotypes in Female Rats.

Leptin is a proinflammatory adipokine that contributes to obesity-associated osteoarthritis (OA), especially in women. However, the extent to which leptin causes knee OA separate from the effect of increased body weight is not clear. We hypothesized that leptin is necessary to induce knee OA in obese female rats but not sufficient to induce knee OA in lean rats lacking systemic metabolic inflammation. The effect of obesity without leptin signaling was modeled by comparing female lean Zucker rats to pair fed obese Zucker rats, which possess mutant fa alleles of the leptin receptor gene. The effect of leptin without obesity was modeled in female F344BN F1 hybrid rats by systemically administering recombinant rat leptin versus saline for 23 weeks via osmotic pumps. Primary OA outcomes included cartilage histopathology and subchondral bone micro-computed tomography. Secondary outcomes included targeted cartilage proteomics, serum inflammation, and synovial fluid inflammation following an acute intra-articular challenge with interleukin-1ß (IL-1ß). Compared to lean Zucker rats, obese Zucker rats developed more severe tibial osteophytes and focal cartilage lesions in the medial tibial plateau, with modest changes in proximal tibial epiphysis trabecular bone structure. In contrast, exogenous leptin treatment, which increased plasma leptin sixfold without altering body weight, caused mild generalized cartilage fibrillation and reduced Safranin O staining compared to vehicle-treated animals. Leptin also significantly increased subchondral and trabecular bone volume and bone mineral density in the proximal tibia. Cartilage metabolic and antioxidant enzyme protein levels were substantially elevated with leptin deficiency and minimally suppressed with leptin treatment. In contrast, leptin treatment induced greater changes in systemic and local inflammatory mediators compared to leptin receptor deficiency, including reduced serum IL-6 and increased synovial fluid IL-1ß. In conclusion, rat models that separately elevate leptin or body weight develop distinct OA-associated phenotypes, revealing how obesity increases OA pathology through both leptin-dependent and independent pathways. © 2023 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Age-Associated Changes in Knee Osteoarthritis, Pain-Related Behaviors, and Dorsal Root Ganglia Immunophenotyping of Male and Female Mice.

OBJECTIVE: Osteoarthritis (OA) is a leading cause of chronic pain, yet OA pain management remains poor. Age is the strongest predictor of OA development, and mechanisms driving OA pain are unclear. We undertook this study to characterize age-associated changes in knee OA, pain-related behaviors, and dorsal root ganglion (DRG) molecular phenotypes in mice of both sexes. METHODS: Male or female C57BL/6 mice 6 or 20 months of age were evaluated for histopathologic knee OA, pain-related behaviors, and L3-L5 DRG immune characterization via flow cytometry. DRG gene expression in older mice and humans was also examined. RESULTS: Male mice at 20 months of age had worse cartilage degeneration than 6-month-old mice. Older female mouse knees showed increased cartilage degeneration but to a lesser degree than those of male mice. Older mice of both sexes had worse mechanical allodynia, knee hyperalgesia, and grip strength compared to younger mice. For both sexes, DRGs from older mice showed decreased CD45+ cells and a significant increase in F4/80+ macrophages and CD11c+ dendritic cells. Older male mouse DRGs showed increased expression of Ccl2 and Ccl5, and older female mouse DRGs showed increased Cxcr4 and Ccl3 expression compared to 6-month-old mouse DRGs, among other differentially expressed genes. Human DRG analysis from 6 individuals >80 years of age revealed elevated CCL2 in men compared to women, whereas CCL3 was higher in DRGs from women. CONCLUSION: We found that aging in male and female mice is accompanied by mild knee OA, mechanical sensitization, and changes to immune cell populations in the DRG, suggesting novel avenues for development of OA therapies.

RELATIONSHIP BETWEEN REPRODUCTIVE AND BONE BIOMARKERS AND OSTEOARTHRITIS IN ZOO ASIAN (ELEPHAS MAXIMUS) AND AFRICAN (LOXODONTA AFRICANA) ELEPHANTS.

Osteoarthritis (OA) is common in zoo Asian (Elephas maximus) and African (Loxodonta africana) elephants. This study investigated the relationship between confirmed or suspected OA with ovarian cyclicity, gonadotropins, progestagens, luteinizing hormone (LH), follicle-stimulating hormone (FSH), and collagen type I (CTX-I) in zoo elephants. In Asian elephants, odds of having confirmed or suspected OA decreased with cycling (OR = 0.22, P = 0.016; OR = 0.29, P = 0.020, respectively), however, not when adjusted for age (odds ratio [OR] = 0.31, P = 0.112; OR = 0.58, P = 0.369, respectively). In African elephants, none of the models between confirmed OA and cycling status were significant (P > 0.060), while the odds of having suspected OA decreased with cycling (OR = 0.12, P = 0.001), even after adjusting for age (OR = 0.15, P = 0.005). Progestagens (Asian elephants P > 0.096; African elephants P > 0.415), LH (Asian P > 0.129; African P > 0.359), and FSH (Asian P > 0.738; African P > 0.231) did not differ with confirmed or suspected OA status, unadjusted. CTX-I concentrations were not related to OA status (P > 0.655). This study concluded hormonal changes may not have a strong impact on OA, so additional investigation into other serologic biomarkers is warranted.

In vivo mechanotransduction: Effect of acute exercise on the metabolomic profiles of mouse synovial fluid.

Objective: Exercise is known to induce beneficial effects in synovial joints. However, the mechanisms underlying these are unclear. Synovial joints experience repeated mechanical loading during exercise. These mechanical stimuli are transduced into biological responses through cellular mechanotransduction. Mechanotransduction in synovial joints is typically studied in tissues. However, synovial fluid directly contacts all components of the joint, and thus may produce a whole-joint picture of the mechanotransduction response to loading. The objective of this study was to determine if metabolic phenotypes are present in the synovial fluid after acute exercise as a first step to understanding the beneficial effects of exercise on the joint. Material and methods: Mice underwent a single night of voluntary wheel running or standard housing and synovial fluid was harvested for global metabolomic profiling by LC-MS. Hierarchical unsupervised clustering, partial least squares discriminant, and pathway analysis provided insight into exercise-induced mechanotransduction. Results: Acute exercise produced a distinct metabolic phenotype in synovial fluid. Mechanosensitive metabolites included coenzyme A derivatives, prostaglandin derivatives, phospholipid species, tryptophan, methionine, vitamin D3, fatty acids, and thiocholesterol. Enrichment analysis identified several pathways previously linked to exercise including amino acid metabolism, inflammatory pathways, citrulline-nitric oxide cycle, catecholamine biosynthesis, ubiquinol biosynthesis, and phospholipid metabolism. Conclusion: To our knowledge, this is the first study to investigate metabolomic profiles of synovial fluid during in vivo mechanotransduction. These profiles indicate that exercise induced stress-response processes including both pro- and anti-inflammatory pathways. Further research will expand these results and define the relationship between the synovial fluid and the serum.

Sirt3 Promotes Chondrogenesis, Chondrocyte Mitochondrial Respiration and the Development of High-Fat Diet-Induced Osteoarthritis in Mice.

Understanding how obesity-induced metabolic stress contributes to synovial joint tissue damage is difficult because of the complex role of metabolism in joint development, maintenance, and repair. Chondrocyte mitochondrial dysfunction is implicated in osteoarthritis (OA) pathology, which motivated us to study the mitochondrial deacetylase enzyme sirtuin 3 (Sirt3). We hypothesized that combining high-fat-diet (HFD)-induced obesity and cartilage Sirt3 loss at a young age would impair chondrocyte mitochondrial function, leading to cellular stress and accelerated OA. Instead, we unexpectedly found that depleting cartilage Sirt3 at 5 weeks of age using Sirt3-flox and Acan-CreERT2 mice protected against the development of cartilage degeneration and synovial hyperplasia following 20 weeks of HFD. This protection was associated with increased cartilage glycolysis proteins and reduced mitochondrial fatty acid metabolism proteins. Seahorse-based assays supported a mitochondrial-to-glycolytic shift in chondrocyte metabolism with Sirt3 deletion. Additional studies with primary murine juvenile chondrocytes under hypoxic and inflammatory conditions showed an increased expression of hypoxia-inducible factor (HIF-1) target genes with Sirt3 deletion. However, Sirt3 deletion impaired chondrogenesis using a murine bone marrow stem/stromal cell pellet model, suggesting a context-dependent role of Sirt3 in cartilage homeostasis. Overall, our data indicate that Sirt3 coordinates HFD-induced changes in mature chondrocyte metabolism that promote OA. © 2022 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

Insulin-like growth factor 1 receptor mediates photoreceptor neuroprotection.

Insulin-like growth factor I (IGF-1) is a neurotrophic factor and is the ligand for insulin-like growth factor 1 receptor (IGF-1R). Reduced expression of IGF-1 has been reported to cause deafness, mental retardation, postnatal growth failure, and microcephaly. IGF-1R is expressed in the retina and photoreceptor neurons; however, its functional role is not known. Global IGF-1 KO mice have age-related vision loss. We determined that conditional deletion of IGF-1R in photoreceptors and pan-retinal cells produces age-related visual function loss and retinal degeneration. Retinal pigment epithelial cell-secreted IGF-1 may be a source for IGF-1R activation in the retina. Altered retinal, fatty acid, and phosphoinositide metabolism are observed in photoreceptor and retinal cells lacking IGF-1R. Our results suggest that the IGF-1R pathway is indispensable for photoreceptor survival, and activation of IGF-1R may be an essential element of photoreceptor and retinal neuroprotection.

An In Vivo Stable Isotope Labeling Method to Investigate Individual Matrix Protein Synthesis, Ribosomal Biogenesis, and Cellular Proliferation in Murine Articular Cartilage.

Targeting chondrocyte dynamics is a strategy for slowing osteoarthritis progression during aging. We describe a stable-isotope method using in vivo deuterium oxide labeling and mass spectrometry to measure protein concentration, protein half-life, cell proliferation, and ribosomal biogenesis in a single sample of murine articular cartilage. We hypothesized that a 60-d labeling period would capture age-related declines in cartilage matrix protein content, protein synthesis rates, and cellular proliferation. Knee cartilage was harvested to the subchondral bone from 25- to 90-wk-old female C57BL/6J mice treated with deuterium oxide for 15, 30, 45, and 60 d. We measured protein concentration and half-lives using targeted high resolution accurate mass spectrometry and d2ome data processing software. Deuterium enrichment was quantified in isolated DNA and RNA to measure cell proliferation and ribosomal biogenesis, respectively. Most collagen isoforms were less abundant in aged animals, with negligible collagen synthesis at either age. In contrast, age altered the concentration and half-lives of many proteoglycans and other matrix proteins, including several with greater concentration and half-lives in older mice such as proteoglycan 4, clusterin, and fibronectin-1. Cellular proteins were less abundant in older animals, consistent with reduced cellularity. Nevertheless, deuterium was maximally incorporated into 60% of DNA and RNA by 15 d of labeling in both age groups, suggesting the presence of two large pools of either rapidly (<15 d) or slowly (>60 d) proliferating cells. Our findings indicate that age-associated changes in cartilage matrix protein content and synthesis occur without detectable changes in the relative number of proliferating cells.

Determining the contributions of protein synthesis and breakdown to muscle atrophy requires non-steady-state equations.

BACKGROUND: Ageing and cachexia cause a loss of muscle mass over time, indicating that protein breakdown exceeds protein synthesis. Deuterium oxide (D2 O) is used for studies of protein turnover because of the advantages of long-term labelling, but these methods introduce considerations that have been largely overlooked when studying conditions of protein gain or loss. The purpose of this study was to demonstrate the importance of accounting for a change in protein mass, a non-steady state, during D2 O labelling studies while also exploring the contribution of protein synthesis and breakdown to denervation-induced muscle atrophy. METHODS: Adult (6 months) male C57BL/6 mice (n = 14) were labelled with D2 O for a total of 7 days following unilateral sciatic nerve transection to induce denervation of hindlimb muscles. The contralateral sham limb and nonsurgical mice (n = 5) were used as two different controls to account for potential crossover effects of denervation. We calculated gastrocnemius myofibrillar and collagen protein synthesis and breakdown assuming steady-state or using non-steady-state modelling. We measured RNA synthesis rates to further understand ribosomal turnover during atrophy. RESULTS: Gastrocnemius mass was less in denervated muscle (137 ± 9 mg) compared with sham (174 ± 15 mg; P < 0.0001) or nonsurgical control (162 ± 5 mg; P < 0.0001). With steady-state calculations, fractional synthesis and breakdown rates (FSR and FBR) were lower in the denervated muscle (1.49 ± 0.06%/day) compared with sham (1.81 ± 0.09%/day; P < 0.0001) or nonsurgical control (2.27 ± 0.04%/day; P < 0.0001). When adjusting for change in protein mass, FSR was 4.21 ± 0.19%/day in denervated limb, whereas FBR was 4.09 ± 0.22%/day. When considering change in protein mass (ksyn ), myofibrillar synthesis was lower in denervated limb (2.44 ± 0.14 mg/day) compared with sham (3.43 ± 0.22 mg/day; P < 0.0001) and non-surgical control (3.74 ± 0.12 mg/day; P < 0.0001), whereas rate of protein breakdown (kdeg, 1/t) was greater in denervated limb (0.050 ± 0.003) compared with sham (0.019 ± 0.001; P < 0.0001) and nonsurgical control (0.023 ± 0.000; P < 0.0001). Muscle collagen breakdown was completely inhibited during denervation. There was a strong correlation (r = 0.83, P < 0.001) between RNA and myofibrillar protein synthesis in sham but not denervated muscle. CONCLUSIONS: We show conflicting results between steady- and non-steady-state calculations on myofibrillar protein synthesis and breakdown during periods of muscle loss. We also found that collagen accumulation was largely from a decrease in collagen breakdown. Comparison between sham and non-surgical control demonstrated a crossover effect of denervation on myofibrillar protein synthesis and ribosomal biogenesis, which impacts study design for unilateral atrophy studies. These considerations are important because not accounting for them can mislead therapeutic attempts to maintain muscle mass.

A Novel Stable Isotope Approach Demonstrates Surprising Degree of Age-Related Decline in Skeletal Muscle Collagen Proteostasis.

Age-related deterioration in turnover of collagen proteins accelerates extracellular matrix fibrosis and hinders adaptation to external stimuli. This project sought to understand factors that increase skeletal muscle fibrosis with age by studying what we term the dynamic protein pool. We hypothesized that the dynamic protein pool size of muscle collagen decreases with age, thus indicating a decrease in proteostatic maintenance (ie, ability to maintain proteostasis), and that failure to account for these changes impacts the interpretation of tracer-measured synthesis rates. We used deuterium oxide (D2O) labeling for up to 60 days in adult (6 months) and old (23 months) mice. The dynamic protein pool in adult skeletal muscle was 65% in tibialis anterior (TA), but only 28% in gastrocnemius (Gastroc). In aged muscle, the dynamic protein pool was further decreased to only 35% and 14% for TA and Gastroc, respectively. We showed that this loss in dynamic pool size was associated with increases in markers of fibrosis and decreased proteostatic maintenance. We demonstrate that aged muscle has higher rates of collagen protein synthesis and lower rates of collagen protein breakdown, which causes collagen accumulation. We further demonstrated that the normal assumption of complete protein renewal and the standard practice of taking a single sample with isotope labeling have profound impacts on interpretation of the genesis of fibrosis. Strategies to maintain muscle function with aging should focus on the dynamic protein pool with attention to methodological strategies to assess those changes.

PFKFB3-dependent glucose metabolism regulates 3T3-L1 adipocyte development.

Proliferation and differentiation of preadipocytes, and other cell types, is accompanied by an increase in glucose uptake. Previous work showed that a pulse of high glucose was required during the first 3 days of differentiation in vitro, but was not required after that. The specific glucose metabolism pathways required for adipocyte differentiation are unknown. Herein, we used 3T3-L1 adipocytes as a model system to study glucose metabolism and expansion of the adipocyte metabolome during the first 3 days of differentiation. Our primary outcome measures were GLUT4 and adiponectin, key proteins associated with healthy adipocytes. Using complete media with 0 or 5 mM glucose, we distinguished between developmental features that were dependent on the differentiation cocktail of dexamethasone, insulin, and isobutylmethylxanthine alone or the cocktail plus glucose. Cocktail alone was sufficient to activate the capacity for 2-deoxglucose uptake and glycolysis, but was unable to support the expression of GLUT4 and adiponectin in mature adipocytes. In contrast, 5 mM glucose in the media promoted a transient increase in glucose uptake and glycolysis as well as a significant expansion of the adipocyte metabolome and proteome. Using genetic and pharmacologic approaches, we found that the positive effects of 5 mM glucose on adipocyte differentiation were specifically due to increased expression of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3), a key regulator of glycolysis and the ancillary glucose metabolic pathways. Our data reveal a critical role for PFKFB3 activity in regulating the cellular metabolic remodeling required for adipocyte differentiation and maturation.

Sirt5 Deficiency Causes Posttranslational Protein Malonylation and Dysregulated Cellular Metabolism in Chondrocytes Under Obesity Conditions.

OBJECTIVE: Obesity accelerates the development of osteoarthritis (OA) during aging and is associated with altered chondrocyte cellular metabolism. Protein lysine malonylation (MaK) is a posttranslational modification (PTM) that has been shown to play an important role during aging and obesity. The objective of this study was to investigate the role of sirtuin 5 (Sirt5) in regulating MaK and cellular metabolism in chondrocytes under obesity-related conditions. METHODS: MaK and SIRT5 were immunostained in knee articular cartilage of obese db/db mice and different aged C57BL6 mice with or without destabilization of the medial meniscus surgery to induce OA. Primary chondrocytes were isolated from 7-day-old WT and Sirt5-/- mice and treated with varying concentrations of glucose and insulin to mimic obesity. Sirt5-dependent effects on MaK and metabolism were evaluated by western blot, Seahorse Respirometry, and gas/chromatography-mass/spectrometry (GC-MS) metabolic profiling. RESULTS: MaK was significantly increased in cartilage of db/db mice and in chondrocytes treated with high concentrations of glucose and insulin (GluhiInshi). Sirt5 was increased in an age-dependent manner following joint injury, and Sirt5 deficient primary chondrocytes had increased MaK, decreased glycolysis rate, and reduced basal mitochondrial respiration. GC-MS identified 41 metabolites. Sirt5 deficiency altered 13 distinct metabolites under basal conditions and 18 metabolites under GluhiInshi treatment. Pathway analysis identified a wide range of Sirt5-dependent altered metabolic pathways that include amino acid metabolism, TCA cycle, and glycolysis. CONCLUSION: This study provides the first evidence that Sirt5 broadly regulates chondrocyte metabolism. We observed changes in SIRT5 and MaK levels in cartilage with obesity and joint injury, suggesting that the Sirt5-MaK pathway may contribute to altered chondrocyte metabolism that occurs during OA development.

Proximal colon-derived O-glycosylated mucus encapsulates and modulates the microbiota.

Colon mucus segregates the intestinal microbiota from host tissues, but how it organizes to function throughout the colon is unclear. In mice, we found that colon mucus consists of two distinct O-glycosylated entities of Muc2: a major form produced by the proximal colon, which encapsulates the fecal material including the microbiota, and a minor form derived from the distal colon, which adheres to the major form. The microbiota directs its own encapsulation by inducing Muc2 production from proximal colon goblet cells. In turn, O-glycans on proximal colon-derived Muc2 modulate the structure and function of the microbiota as well as transcription in the colon mucosa. Our work shows how proximal colon control of mucin production is an important element in the regulation of host-microbiota symbiosis.

Mosaic Mutant Analysis Identifies PDGFRα/PDGFRß as Negative Regulators of Adipogenesis.

Adipocyte progenitors (APs) express platelet-derived growth factor receptors (PDGFRs), PDGFRα and PDGFRß. Elevated PDGFRα signaling inhibits adipogenesis and promotes fibrosis; however, the function of PDGFRs in APs remains unclear. We combined lineage tracing and functional analyses in a sequential dual-recombinase approach that creates mosaic Pdgfr mutant cells by Cre/lox recombination with a linked Flp/frt reporter to track individual cell fates. Using mosaic lineage labeling, we show that adipocytes are derived from the Pdgfra lineage during postnatal growth and adulthood. In contrast, adipocytes are only derived from the mosaic Pdgfrb lineage during postnatal growth. Functionally, postnatal mosaic deletion of PDGFRα enhances adipogenesis and adult deletion enhances ß3-adrenergic-receptor-induced beige adipocyte formation. Mosaic deletion of PDGFRß also enhances white, brown, and beige adipogenesis. These data show that both PDGFRs are cell-autonomous inhibitors of adipocyte differentiation and implicate downregulation of PDGF signaling as a critical event in the transition from AP to adipocyte.

Correlation network analysis shows divergent effects of a long-term, high-fat diet and exercise on early stage osteoarthritis phenotypes in mice.

Background: Obesity increases knee osteoarthritis (OA) risk through metabolic, inflammatory, and biomechanical factors, but how these systemic and local mediators interact to drive OA pathology is not well understood. We tested the effect of voluntary running exercise after chronic diet-induced obesity on knee OA-related cartilage and bone pathology in mice. We then used a correlation-based network analysis to identify systemic and local factors associated with early-stage knee OA phenotypes among the different diet and exercise groups. Methods: Male C57BL/6J mice were fed a defined control (10% kcal fat) or high fat (HF) (60% kcal fat) diet from 6 to 37 weeks of age. At 25 weeks, one-half of the mice from each diet group were housed in cages with running wheels for the remainder of the study. Histology, micro computed tomography, and magnetic resonance imaging were used to evaluate changes in joint tissue structure and OA pathology. These local variables were then compared to systemic metabolic (body mass, body fat, and glucose tolerance), inflammatory (serum adipokines and inflammatory mediators), and functional (mechanical tactile sensitivity and grip strength) outcomes using a correlation-based network analysis. Diet and exercise effects were evaluated by two-way analysis of variance. Results: An HF diet increased the infrapatellar fat pad size and posterior joint osteophytes, and wheel running primarily altered the subchondral cortical and trabecular bone. Neither HF diet nor exercise altered average knee cartilage OA scores compared to control groups. However, the coefficient of variation was ≥25% for many outcomes, and some mice in both diet groups developed moderate OA (≥33% maximum score). This supported using correlation-based network analyses to identify systemic and local factors associated with early-stage knee OA phenotypes. In wheel-running cohorts, an HF diet reduced the network size compared to the control diet group despite similar running distances, suggesting that diet-induced obesity dampens the effects of exercise on systemic and local OA-related factors. Each of the 4 diet and activity groups showed mostly unique networks of local and systemic factors correlated with early-stage knee OA. Conclusion: Despite minimal group-level effects of chronic diet-induced obesity and voluntary wheel running on knee OA pathology under the current test durations, diet and exercise substantially altered the relationships among systemic and local variables associated with early-stage knee OA. These results suggest that distinct pre-OA phenotypes may exist prior to the development of disease.

Bridging Disciplines as a pathway to Finding New Solutions for Osteoarthritis a collaborative program presented at the 2019 Orthopaedic Research Society and the Osteoarthritis Research Society International.

Objective: To stimulate future research directions that seek solutions for osteoarthritis (OA) at the interface between diverse disciplines and address osteoarthritis (OA) as a serious disease with a complexity that has presented a barrier to finding safe effective solutions. Methods: Sessions were conducted at the 2019 meetings of the Orthopaedic Research Society (ORS) and Osteoarthritis Research Society International (OARSI) that included presentations and questions/comments submitted from leading OA researchers representing imaging, mechanics, biomarkers, phenotyping, clinical, epidemiology, inflammation and exercise. Results: Solutions for OA require a paradigm shift in research and clinical methods in which OA is contextualized as a complex whole-body/person disease. New OA definition(s)/phenotype(s) and OA markers/signals are needed to address the interplay between genetic and environmental factors of the disease as well as capture the mechanosensitivity of the disease. The term "Mechanokines" was proposed to highlight the importance of incorporating whole body mechanics as a marker of early OA. New interventions and apparent paradoxical observations/questions (e.g. exercise vs. load modification) were also discussed in the context of considering OA as a complex system. Conclusion: To advance new OA treatments that are safe and effective, OA should be considered as a "Whole Person" disease. This approach requires a concerted effort to bridge disciplines and include interactions across scales from the molecule to the whole body, including psychosocial aspects.

Glutathione as a mediator of cartilage oxidative stress resistance and resilience during aging and osteoarthritis.

Purpose: An underlying cause of osteoarthritis (OA) is the inability of chondrocytes to maintain homeostasis in response to changing stress conditions. The purpose of this article was to review and experimentally evaluate oxidative stress resistance and resilience concepts in cartilage using glutathione redox homeostasis as an example. This framework may help identify novel approaches for promoting chondrocyte homeostasis during aging and obesity.Materials and Methods: Changes in glutathione content and redox ratio were evaluated in three models of chondrocyte stress: (1) age- and tissue-specific changes in joint tissues of 10 and 30-month old F344BN rats, including ex vivo patella culture experiments to evaluate N-acetylcysteine dependent resistance to interleukin-1beta; (2) effect of different durations and patterns of cyclic compressive loading in bovine cartilage on glutathione stress resistance and resilience pathways; (3) time-dependent changes in GSH:GSSG in primary chondrocytes from wild-type and Sirt3 deficient mice challenged with the pro-oxidant menadione.Results: Glutathione was more abundant in cartilage than meniscus or infrapatellar fat pad, although cartilage was also more susceptible to age-related glutathione oxidation. Glutathione redox homeostasis was sensitive to the duration of compressive loading such that load-induced oxidation required unloaded periods to recover and increase total antioxidant capacity. Exposure to a pro-oxidant stress enhanced stress resistance by increasing glutathione content and GSH:GSSG ratio, especially in Sirt3 deficient cells. However, the rate of recovery, a marker of resilience, was delayed without Sirt3.Conclusions: OA-related models of cartilage stress reveal multiple mechanisms by which glutathione provides oxidative stress resistance and resilience.

Innate inflammation and synovial macrophages in osteoarthritis pathophysiology.

Although osteoarthritis (OA) was historically referred to as the non-inflammatory arthritis, it is now considered a condition involving persistent low-grade inflammation and activation of innate inflammatory pathways. Synovitis increases the risk of OA onset and progression and involves the recruitment of monocytes, lymphocytes, and other leukocytes. In particular, macrophages are important mediators of synovial inflammatory activity and pathologic cartilage and bone responses that are characteristic of OA. Advances in understanding how damage-associated molecular patterns (DAMPs) trigger monocyte/macrophage recruitment and activation in joints provide opportunities for disease-modifying therapies. However, the complexity and plasticity of macrophage phenotypes that exist in vivo have thus far prevented the successful development of macrophage-targeted treatments. Current studies show that synovial macrophages are derived from distinct cellular lineages, which correspond to unique functional roles for maintaining joint homeostasis. An improved understanding of the aetiology of synovial inflammation in specific OA-subtypes, such as with obesity or genetic risk, is a potential strategy for developing patient selection criteria for future precision therapies.