Osteoarthritis Synovium Research Articles

Ribosomal RNA-based epitranscriptomic regulation of chondrocyte translation and proteome in osteoarthritis

Osteoarthritis-related cartilage extracellular matrix remodelling is dependent on changes in chondrocyte protein expression. Yet, the role of ribosomes in chondrocyte translation regulation is unknown. In this exploratory study, we investigated ribosomal RNA (rRNA) epitranscriptomic-based ribosome heterogeneity in human articular chondrocytes and its relevance for osteoarthritis. Sequencing-based rRNA 2'-O-methylation profiling analysis (RiboMethSeq) was performed on non-OA primary human articular chondrocytes (n=5) exposed for 14 days to osteoarthritic synovial fluid (14 donors, pooled, 20% v/v). The SW1353 SNORD71 KO cell pool was generated using LentiCRISPRv2/Cas9. The mode of translation initiation and fidelity were determined by dual-luciferase reporters. The cellular proteome was analysed by LC-MS/MS and collagen type I protein expression was evaluated by immunoblotting. Loading of COL1A1 mRNA into polysomes was determined by sucrose gradient ultracentrifugation and fractionation. We discovered that osteoarthritic synovial fluid instigates site-specific changes in the rRNA 2'-O-me profile of primary human articular chondrocytes. We identified five sites with differential 2'-O-me levels. The 2'-O-me status of 5.8S-U14 (one of identified differential 2'-O-me sites; decreased by 7.7%, 95% CI [0.9% to 14.5%]) was targeted by depleting the level of its guide snoRNA SNORD71 (50% decrease, 95% CI [33% to 64%]). This resulted in an altered ribosome translation modus (e.g CrPV IRES, FC 3, 95% CI [2.2 to 4.1]) and promoted translation of COL1A1 mRNA which led to increased levels of COL1A1 protein (FC 1.7, 95% CI [1.3 to 2.0]). Our data identify a novel concept suggesting that articular chondrocytes employ rRNA epitranscriptomic mechanisms in osteoarthritis development.

Syntenin-1-mediated arthritogenicity is advanced by reprogramming RA metabolic macrophages and Th1 cells

ObjectivesSyntenin-1, a novel endogenous ligand, was discovered to be enriched in rheumatoid arthritis (RA) specimens compared with osteoarthritis synovial fluid and normal synovial tissue (ST). However, the cellular origin, immunoregulation...

Inhibition of fibroblast activation protein ameliorates cartilage matrix degradation and osteoarthritis progression

Fibroblast activation protein (Fap) is a serine protease that degrades denatured type I collagen, α2-antiplasmin and FGF21. Fap is highly expressed in bone marrow stromal cells and functions as an osteogenic suppressor and can be inhibited by the bone growth factor Osteolectin (Oln). Fap is also expressed in synovial fibroblasts and positively correlated with the severity of rheumatoid arthritis (RA). However, whether Fap plays a critical role in osteoarthritis (OA) remains poorly understood. Here, we found that Fap is significantly elevated in osteoarthritic synovium, while the genetic deletion or pharmacological inhibition of Fap significantly ameliorated posttraumatic OA in mice. Mechanistically, we found that Fap degrades denatured type II collagen (Col II) and Mmp13-cleaved native Col II. Intra-articular injection of rFap significantly accelerated Col II degradation and OA progression. In contrast, Oln is expressed in the superficial layer of articular cartilage and is significantly downregulated in OA. Genetic deletion of Oln significantly exacerbated OA progression, which was partially rescued by Fap deletion or inhibition. Intra-articular injection of rOln significantly ameliorated OA progression. Taken together, these findings identify Fap as a critical pathogenic factor in OA that could be targeted by both synthetic and endogenous inhibitors to ameliorate articular cartilage degradation.

Cartilage Wear in Healthy and Osteoarthritis Joints

This study is designed to determine whether the outermost layer of articular cartilage is deficient in Osteoarthritis (OA). Phospholipids present in healthy and osteoarthritis (OA) synovial fluid show significant differences in their concentration. While examining the surface properties of OA joints, we found that OA PLs molecules cannot support lubrication, and increased friction was observed. Our lubrication mechanism was based on a surface active phospholipids (SAPL) multibilayer which in OA condition was deactivated and removed from the cartilage surface under OA conditions. Cartilage wettability study clearly demonstrated a significant decrease in hydrophobicity, the contact angle, θ (theta), dropping from 103° from bovine healthy cartilage to 65° in surface partially depleted and 35.1° for completely depleted surface. These results are discussed in the context that surface active phospholipid (SAPL) and lubricin, each has specific roles in a lamellar-repulsive lubrication system. However, deactivated phospholipid molecules are major indicator of cartilage wear (model) introduced in this study.

Engineered M2a macrophages for the treatment of osteoarthritis.

Macrophage is a central regulator of innate immunity. Its M2 subsets, such as interstitial synovial macrophages, have been found to play critical roles in suppressing chronic inflammation and maintaining homeostasis within the joint. These macrophages have great potential as a disease-modifying cell therapy for osteoarthritis (OA). However, this has not yet been studied. Macrophages were isolated from the bone marrow of rats. We constructed a stable macrophage that "locked" in anti-inflammatory and pro-regenerative M2a polarity (L-M2a) by simultaneously knocking out tumor necrosis factor receptor 1 (TNFR1) and overexpressing IL-4 using Cas9-ribonuclear proteins (Cas9-RNP) and electroporation. In vitro, these L-M2a macrophages were treated with OA synovial fluid or co-cultured with OA chondrocytes or fibroblast-like synoviocytes (FLS). In vivo, L-M2a macrophages were injected intra-articularly to evaluate their homing and engrafting abilities and therapeutic effects on OA progression using a rat model. L-M2a macrophages displayed a typical anti-inflammatory phenotype similar to that of M2 macrophages in vitro. In OA microenvironment, L-M2a macrophages maintained a stable anti-inflammatory phenotype, whereas unmodified M2 macrophages lost their phenotype and switched to M1 polarity. L-M2a macrophages demonstrated a potent anti-inflammatory effect in crosstalk with OA-FLSs and an anti-degenerative effect in crosstalk with senescent OA chondrocytes. In vivo, compared with M2 macrophages and exosomes, L-M2a macrophages exhibited significantly superior therapeutic effects in OA by successfully resolving inflammation, restoring tissue homeostasis, and promoting cartilage regeneration. The engineered L-M2a macrophages maintained a superior anti-inflammatory and pro-regenerative capacity in the inflammatory OA microenvironment and represents an ideal new strategy for the disease-modifying therapy of OA.

Molecular crosstalk between articular cartilage, meniscus, synovium, and subchondral bone in osteoarthritis.

Osteoarthritis (OA) is a common degenerative joint disease worldwide, which is characterized by articular cartilage lesions. With more understanding of the disease, OA is considered to be a disorder of the whole joint. However, molecular communication within and between tissues during the disease process is still unclear. In this study, we used transcriptome data to reveal crosstalk between different tissues in OA. We used four groups of transcription profiles acquired from the Gene Expression Omnibus database, including articular cartilage, meniscus, synovium, and subchondral bone, to screen differentially expressed genes during OA. Potential crosstalk between tissues was depicted by ligand-receptor pairs. During OA, there were 626, 97, 1,060, and 2,330 differentially expressed genes in articular cartilage, meniscus, synovium, and subchondral bone, respectively. Gene Ontology enrichment revealed that these genes were enriched in extracellular matrix and structure organization, ossification, neutrophil degranulation, and activation at different degrees. Through ligand-receptor pairing and proteome of OA synovial fluid, we predicted ligand-receptor interactions and constructed a crosstalk atlas of the whole joint. Several interactions were reproduced by transwell experiment in chondrocytes and synovial cells, including TNC-NT5E, TNC-SDC4, FN1-ITGA5, and FN1-NT5E. After lipopolysaccharide (LPS) or interleukin (IL)-1β stimulation, the ligand expression of chondrocytes and synovial cells was upregulated, and corresponding receptors of co-culture cells were also upregulated. Each tissue displayed a different expression pattern in transcriptome, demonstrating their specific roles in OA. We highlighted tissue molecular crosstalk through ligand-receptor pairs in OA pathophysiology, and generated a crosstalk atlas. Strategies to interfere with these candidate ligands and receptors may help to discover molecular targets for future OA therapy.Cite this article: Bone Joint Res2022;11(12):862-872.

Blocking TRPV4 Ameliorates Osteoarthritis by Inhibiting M1 Macrophage Polarization via the ROS/NLRP3 Signaling Pathway.

Osteoarthritis (OA) is a low-level inflammatory disease in which synovial macrophage M1 polarization exacerbates the progression of synovitis and OA. Notedly, the ROS (reactive oxygen species) level in macrophages is intimately implicated in macrophage M1 polarization. TRPV4 (transient receptor potential channel subfamily V member 4), as an ion channel, plays a pivotal role in oxidative stress and inflammation. In this study, we investigated the role of TRPV4 in OA progression and M1 macrophage polarization. Male adult Sprague-Dawley (SD) rats underwent a medial meniscus radial transection operation to create an OA model in vivo and RAW 264.7 cells were intervened with 100 ng/mL LPS (lipopolysaccharide) to induce M1-polarized macrophages in vitro. We demonstrated that the infiltration of M1 synovial macrophages and the expression of TRPV4 were increased significantly in OA synovium. In addition, intra-articular injection of HC067074 (a specific inhibitor of TRPV4) alleviated the progression of rat OA and significantly decreased synovial macrophage M1 polarization. Further mechanisms suggested that ROS production by M1 macrophages was decreased after TRPV4 inhibition. In addition, NLRP3 (pyrin domain containing protein 3) as a downstream effector of ROS in M1-polarized macrophage, was significantly suppressed following TRPV4 inhibition. In conclusion, this study discovered that inhibition of TRPV4 delays OA progression by inhibiting M1 synovial macrophage polarization through the ROS/NLRP3 pathway.

Could Osteopontin be a useful biomarker in the diagnosis and severity assessment of osteoarthritis? A systematic review and meta-analysis of recent evidence

PurposeOsteoarthritis (OA) patients demonstrated higher Osteopontin (OPN) plasma, serum, and synovial fluid concentrations than healthy individuals. In the present study, we aimed to investigate whether OPN could be used as a diagnostic or prognostic marker for OA symptom/disease severity. MethodsUsing Web of Science, PubMed, Scopus, and Embase, we conducted a systematic review and meta-analysis of studies that measured OPN levels in OA patients' plasma, serum, or synovial fluid. After setting the eligibility criteria, data extraction, and quality assessment of the identified studies, we performed statistical analysis using Revman 5.4 and Open Meta analyst. ResultsOPN has been found to be associated with advanced knee joint damage in OA patients. In addition, higher expression of OPN is thought to be associated with disease progression. Nevertheless, further studies should examine the role of other markers of chronic bone damage, such as leptin and sclerostin. This systematic review and meta-analysis included 14 studies with a total of 776 cases and 530 controls. OPN was significantly elevated in osteoarthritis patients' plasma, serum, and synovial fluid samples, with significant heterogeneity between studies. ConclusionWe recommend that OPN plasma and synovial fluid levels be measured as a diagnostic and prognostic marker to determine the severity of OA symptoms.

1H-NMR metabolomic profile of healthy and osteoarthritic canine synovial fluid before and after UC-II supplementation

The aim of the study was to compare the metabolomic synovial fluid (SF) profile of dogs affected by spontaneous osteoarthritis (OA) and supplemented with undenatured type II collagen (UC-II), with that of healthy control dogs. Client-owned dogs were enrolled in the study and randomized in two different groups, based on the presence/absence of OA (OA group and OA-free group). All dogs were clinically evaluated and underwent SF sampling for 1H-Nuclear Magnetic Resonance spectroscopy (1H-NMR) analysis at time of presentation. All dogs included in OA group were supplemented with UC-II orally administered for 30 days. After this period, they were reassessed (OA-T30). The differences in the 1H-NMR metabolic SFs profiles between groups (OA-free, OA-T0 and OA-T30) were studied. The multivariate statistical analysis performed on SFs under different conditions (OA-T0 vs OA-T30 SFs; OA-T0 vs OA-free SFs and OA-T30 vs OA-free SFs) gave models with excellent goodness of fit and predictive parameters, revealed by a marked separation between groups. β-Hydroxybutyrate was identified as a characteristic compound of osteoarthritic joints, showing the important role of fat metabolism during OA. The absence of β-hydroxybutyrate after UC-II supplementation suggests the supplement’s effectiveness in rebalancing the metabolism inside the joint. The unexpectedly high level of lactate in the OA-free group suggests that lactate could not be considered a good marker for OA. These results prove that 1H-NMR-based metabolomic analysis is a valid tool to study and monitor OA and that UC-II improves clinical symptoms and the SF metabolic profile in OA dogs.

Exosome modification to better alleviates endoplasmic reticulum stress induced chondrocyte apoptosis and osteoarthritis

Osteoarthritis (OA) is characterized by cartilage matrix degeneration and chondrocyte apoptosis. Prolonged endoplasmic reticulum (ER) stress participates in chondrocyte apoptosis and cartilage degeneration in OA progression. miR-486-5p could suppress the apoptosis of nucleus pulposus cells and cardiomyocyte, yet whether miR-486-5p modified exosomes could modulate ER stress and apoptosis of chondrocytes remain unknown. We validated the increased inflammation and ER stress in OA synovium and cartilage, and the inhibition of ER stress could attenuate the IL-1β induced chondrocyte apoptosis. Administration of exogenous miR-486-5p could inhibit the ER stress, alleviate chondrocytes apoptosis and promote matrix regeneration. In comparison with direct administration of miR-486-5p and miR-486-5p overexpressing ADSCs, miR-486-5p modified exosomes indicated a better effect in modulating chondrocyte homeostasis. MiR-486-5p containing exosomes could also regulate macrophage polarization. Our IVIS imaging data validated that intraarticular injection of miR-486-5p containing exosomes could sustain for at least 7 days. MiR-486-5p containing exosomes showed a better effect on alleviating rats OA compared with direct administration of miR-486-5p and miR-486-5p overexpressing ADSCs. Our data demonstrated that miR-486-5p modified exosomes have a better effect on alleviating chondrocyte apoptosis and osteoarthritis. This study provides evidence of this efficient strategy of exosomal miRNA delivery and the miRNA-based therapy for OA.

Rheological and interface adhesive properties of osteoarthritic synovial fluids

Lubrication by synovial fluid is vital in maintaining the physiological operation of diarthrodial joints, which can be compromised due to changes in synovial fluid as osteoarthritis progresses, resulting in a lubrication deficiency. We studied the rheological and interface adhesive properties of osteoarthritic synovial fluid samples (49 in total), aiming to establish the correlation with medical conditions experienced by patients who underwent knee or hip Total Joint Replacement due to osteoarthritis. The steady-state rheology and the interfacial adhesion energy of those samples were examined, of which the results were compared across various patient characteristics. Seniority (60–79 years old) and obesity (Body Mass Index above 30) were identified as the dominant risk factors for the degeneration of synovial fluid and, thus, the potential development of osteoarthritis. At low shear rates where the interactions are more prominent than high shear rates, synovial fluid viscosity decreased by 58% with age and 38% with Body Mass Index, while the shear-thinning index increased by 40% and 7%, respectively. Additionally, synovial fluid interfacial adhesion properties deteriorate, as shown by the adhesion energy results. The adhesion energy, which is positively correlated to the coefficient of friction, increased by 172% with age and by 234% with Body Mass Index. The changes in both rheological and interface adhesion properties of synovial fluid samples support the relationship between the physico-chemical properties and the clinical observation. The AFM based surface adhesion method could be considered as a new approach to evaluate the tribological characteristics of natural synovial fluids as it requires small volume of samples.

Counts of hyaluronic acid-containing extracellular vesicles decrease in naturally occurring equine osteoarthritis

Osteoarthritis (OA) is a degenerative joint disease with inadequately understood pathogenesis leading to pain and functional limitations. Extracellular vesicles (EVs) released by synovial joint cells can induce both pro- and anti-OA effects. Hyaluronic acid (HA) lubricates the surfaces of articular cartilage and is one of the bioactive molecules transported by EVs. In humans, altered EV counts and composition can be observed in OA synovial fluid (SF), while EV research is in early stages in the horse—a well-recognized OA model. The aim was to characterize SF EVs and their HA cargo in 19 horses. SF was collected after euthanasia from control, OA, and contralateral metacarpophalangeal joints. The SF HA concentrations and size distribution were determined with a sandwich-type enzyme-linked sorbent assay and size-exclusion chromatography. Ultracentrifugation followed by nanoparticle tracking analysis (NTA) were utilized to quantify small EVs, while confocal laser scanning microscopy (CLSM) and image analysis characterized larger EVs. The number and size distribution of small EVs measured by NTA were unaffected by OA, but these results may be limited by the lack of hyaluronidase pre-treatment of the samples. When visualized by CLSM, the number and proportion of larger HA-containing EVs (HA–EVs) decreased in OA SF (generalized linear model, count: p = 0.024, %: p = 0.028). There was an inverse association between the OA grade and total EV count, HA–EV count, and HA–EV % (rs = – 0.264 to – 0.327, p = 0.012–0.045). The total HA concentrations were also lower in OA (generalized linear model, p = 0.002). To conclude, the present study discovered a potential SF biomarker (HA–EVs) for naturally occurring equine OA. The roles of HA–EVs in the pathogenesis of OA and their potential as a joint disease biomarker and therapeutic target warrant future studies.

Anti-Inflammatory and Chondro-Protective Effects of Acidic Polysaccharide from Enteromorpha Prolifera in Experimental Models of Osteoarthritis In-Vitro and In-Vivo.

Osteoarthritis (OA) progression has been shown to increase the expression of inflammatory cytokines in joints, leading to the destruction of cartilage matrix. Interleukin (IL)-1β is a potent inflammatory cytokine associated with osteoarthritic synovial fluid. The protective effects of polysaccharides from Enteromorpha prolifera against acute hepatic injury was reported. In this study, we examined the effects of Enteromorpha polysaccharide extracts (EPEs) in the treatment of OA. The effects of the EPEs were assessed using an IL-1β-stimulated SW1353 and SW982 cells. The expression levels of specific mRNA and proteins were evaluated using semi-quantitative reverse transcription polymerase chain reaction (sqRT-PCR) and western immunoblotting. An OA animal study involving C57BL/6J mice was also conducted to assess the effects on tactile sensitivity and anterior cruciate ligament transection (ACLT). Acidic polysaccharide extract (APE) was shown to significantly reduce cytokine and chemokine mRNA levels in IL-1β-stimulated SW1353 and SW982 cells and attenuate the expression of proinflammatory cytokines and p38/AP-1 in SW1353 cells. APE was also shown to minimize the effect of osteolytic lesions in the knee joints of ACLT-induced osteoarthritic mice. APE is a potent inhibitor of joint degeneration associated with OA.

Activation of the kynurenine-aryl hydrocarbon receptor axis impairs the chondrogenic and chondroprotective effects of human umbilical cord-derived mesenchymal stromal cells in osteoarthritis rats.

It has been proven that intra-articular injection of mesenchymal stromal cells (MSCs) can alleviate cartilage damage in osteoarthritis (OA) by differentiating into chondrocytes and protecting inherent cartilage. However, the mechanism by which the OA articular microenvironment affects MSCs' therapeutic efficiency is yet to be fully elucidated. The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor involved in various cellular processes, such as osteogenesis and immune regulation. Tryptophan (Trp) metabolites, most of which are endogenous ligand for AHR, are abnormally increased in synovial fluid (SF) of OA and rheumatoid arthritis (RA) patients. In this study, the effects of kynurenine (KYN), one of the most important metabolites of Trp, were evaluated on the chondrogenic and chondroprotective effects of human umbilical cord-derived mesenchymal stromal cells (hUC-MSCs). hUC-MSCs were cultured in conditioned medium containing different proportions of OA/RA SF, or stimulated with KYN directly, and then, AHR activation, proliferation, and chondrogenesis of hUC-MSCs were measured. Moreover, the chondroprotective efficiency of short hairpin-AHR-UC-MSC (shAHR-UC-MSC) was determined in a rat surgical OA model (right hind joint). OA SF could activate AHR signaling in hUC-MSCs in a concentration-dependent manner and inhibit the chondrogenic differentiation and proliferation ability of hUC-MSCs. Similar results were observed in hUC-MSCs stimulated with KYN in vitro. Notably, shAHR-UC-MSC exhibited superior therapeutic efficiency in OA rat upon intra-articular injection. Taken together, this study indicates that OA articular microenvironment is not conducive to the therapeutic effect of hUC-MSCs, which is related to the activation of the AHR pathway by tryptophan metabolites, and thus impairs the chondrogenic and chondroprotective effects of hUC-MSCs. AHR might be a promising modification target for further improving the therapeutic efficacy of hUC-MSCs on treatment of cartilage-related diseases such as OA.

Gene Transcription in End Stage Ankle Osteoarthritis Differs Markedly From Knee Arthritis and Non- Arthritic Ankles

Category: Ankle Arthritis; Arthroscopy; Basic Sciences/Biologics; Trauma Introduction/Purpose: Post-traumatic Osteoarthritis (OA) of the ankle is a debilitating condition that impacts millions of people. However, little is known about the genetic and epigenetic risk factors for progression to OA following injury. In order to better understand these factors, a thorough knowledge of the molecular signature in ankle OA relative to non-arthritic ankles is critical. The objective of this study was to perform RNA-sequencing of ankle joint synovial samples from OA and non-OA ankles to better characterize the synovial transcriptome. We compared synovial gene expression patterns in patients with end-stage OA with ankle synovium from non-OA ankles to investigate the unique biological pathways altered in ankle OA. Methods: Patients undergoing total ankle arthroplasty or ankle arthrodesis for end stage OA and patients undergoing ankle arthroscopic surgery for non-arthritic conditions (negative control), were consented for participation. Patients with immune disorders or inflammatory arthropathies were excluded. Patient demographics and medical history were collected. Synovial samples were harvested during surgery and RNA was isolated. Bulk 150 bp, paired-end RNA seq was performed using the Illumina NovaSeq6000 platform. Differential expression was determined using the edgeR (v3.16.5) package for R, after adjustment using a scaling normalization factor and P-values were corrected using the Benjamini Hochberg method. Similarities between samples were determined using Spearman Correlation and degree of similarity was visualized considering the first three Principal Components. Gene Ontology terms were identified to classify gene properties. Results: A total of 8 synovial samples from total ankle OA and 24 synovial samples from ankle non-OA were available for analysis. We found 808 genes uniquely expressed in OA synovium and another 786 different genes expressed in synovium from the control group. GO enrichment term analysis revealed an upregulation in endomembrane system organization, ubiquitin system/proteasomal degradation, and membrane transport processes including cytoskeletal, endosomal, and vacuolar in ankle OA samples compared to controls (Figure 1). These signatures differ from those previously identified in knee OA. Reactome analysis revealed pathways to those previously identified in OA of other joints, including TGF-beta signaling modulation. However, several signatures unique to ankle OA included extracellular matrix organization, integrin signaling, and AKT signaling modulation, among others. In addition, unlike in knee OA, reactome analyses of ankle OA synovium did not present with an inflammatory component associated with M1 macrophage infiltration. Conclusion: Ankle synovium in patients with end-stage ankle OA exhibit a transcriptomic signature distinct from ankle synovium collected from non-OA patients. More intriguing, we find that the ankle OA synovial transcriptome signature is distinct from previously published datasets studying synovium from patients with OA in other joints, particularly the knee. GO term and Reactome analyses identified signatures in bulk membrane transport, proteasome, matrix organization, integrin signaling, and AKT signaling, which are different from phenotypes previously reported in the knee. We conclude that development of effective disease modifying treatments for OA require attention to joint-specific biology and disease progression.

Direct comparison of non-osteoarthritic and osteoarthritic synovial fluid-induced intracellular chondrocyte signaling and phenotype changes

Since the joint microenvironment and tissue homeostasis are highly dependent on synovial fluid, we aimed to compare the essential chondrocyte signaling signatures of non-osteoarthritic vs end-stage osteoarthritic knee synovial fluid. Moreover, we determined the phenotypic consequence of the distinct signaling patterns on articular chondrocytes. Protein profiling of synovial fluid was performed using antibody arrays. Chondrocyte signaling and phenotypic changes induced by non-osteoarthritic and osteoarthritic synovial fluid were analyzed using a phospho-kinase array, luciferase-based transcription factor activity assays, and RT-qPCR. The origin of osteoarthritic synovial fluid signaling was evaluated by comparing the signaling responses of conditioned media from cartilage, synovium, infrapatellar fat pad and meniscus. Osteoarthritic synovial fluid induced pathway-phenotype relationships were evaluated using pharmacological inhibitors. Compared to non-osteoarthritic synovial fluid, osteoarthritic synovial fluid was enriched in cytokines, chemokines and growth factors that provoked differential MAPK, AKT, NFκB and cell cycle signaling in chondrocytes. Functional pathway analysis confirmed increased activity of these signaling events upon osteoarthritic synovial fluid stimulation. Tissue secretomes of osteoarthritic cartilage, synovium, infrapatellar fat pad and meniscus activated several inflammatory signaling routes. Furthermore, the distinct pathway signatures of osteoarthritic synovial fluid led to accelerated chondrocyte dedifferentiation via MAPK/ERK signaling, increased chondrocyte fibrosis through MAPK/JNK and PI3K/AKT activation, an elevated inflammatory response mediated by cPKC/NFκB, production of extracellular matrix-degrading enzymes by MAPK/p38 and PI3K/AKT routes, and enabling of chondrocyte proliferation. This study provides the first mechanistic comparison between non-osteoarthritic and osteoarthritic synovial fluid, highlighting MAPKs, cPKC/NFκB and PI3K/AKT as crucial OA-associated intracellular signaling routes.

RNA-seq characterization of histamine-releasing mast cells as potential therapeutic target of osteoarthritis

ObjectiveMast cells in the osteoarthritis (OA) synovium correlate with disease severity. This study aimed to further elucidate the role of mast cells in OA by RNA-Seq analysis and pharmacological blockade of the activity of histamine, a key mast cell mediator, in murine OA. MethodsWe examined OA synovial tissues and fluids by flow cytometry, immunostaining, single-cell and bulk RNA-Seq, qPCR, and ELISA. Cetirizine, a histamine H1 receptor (H1R) antagonist, was used to treat the destabilization of the medial meniscus (DMM) mouse model of OA. ResultsFlow cytometry and immunohistology analysis of OA synovial cells revealed KIT+ FcεRI+ and TPSAB1+ mast cells. Single-cell RNA-Seq of OA synovial cells identified the expression of prototypical mast cell markers KIT, TPSAB1, CPA3 and HDC, as well as distinctive markers HPGD, CAVIN2, IL1RL1, PRG2, and CKLF, confirmed by bulk RNA-Seq and qPCR. A mast cell prototypical marker expression score classified 40 OA patients into three synovial pathotypes: mast cell-high, −medium, and -low. Additionally, we detected mast cell mediators including histamine, tryptase AB1, CPA3, PRG2, CAVIN2, and CKLF in OA synovial fluids. Elevated H1R expression was detected in human OA synovium, and treatment of mice with the H1 receptor antagonist cetirizine reduced the severity and OA-related mediators in DMM. ConclusionBased on differential expression of prototypical and distinct mast cell markers, human OA joints can be stratified into mast cell-high, −medium, and -low synovial tissue pathotypes. Pharmacologic blockade of histamine activity holds the potential to improve OA disease outcome.

Synovial membrane-derived mesenchymal progenitor cells from osteoarthritic joints in dogs possess lower chondrogenic-, and higher osteogenic capacity compared to normal joints

BackgroundSynovial membrane-derived mesenchymal progenitor cells (SM-MPCs) are a promising candidate for the cell-based treatment of osteoarthritis (OA) considering their in vitro and in vivo capacity for cartilage repair. However, the OA environment may adversely impact their regenerative capacity. There are no studies for canine (c)SM-MPCs that compare normal to OA SM-MPCs, even though dogs are considered a relevant animal model for OA. Therefore, this study compared cSM-MPCs from normal and OA synovial membrane tissue to elucidate the effect of the OA environment on MPC numbers, indicated by CD marker profile and colony-forming unit (CFU) capacity, and the impact of the OA niche on tri-lineage differentiation.MethodsNormal and OA synovial membrane were collected from the knee joints of healthy dogs and dogs with rupture of the cruciate ligaments. The synovium was assessed by histopathological OARSI scoring and by RT-qPCR for inflammation/synovitis-related markers. The presence of cSM-MPCs in the native tissue was further characterized with flow cytometry, RT-qPCR, and immunohistochemistry, using the MPC markers; CD90, CD73, CD44, CD271, and CD34. Furthermore, cells isolated upon enzymatic digestion were characterized by CFU capacity, and a population doublings assay. cSM-MPCs were selected based on plastic adherence, expanded to passage 2, and evaluated for the expression of MPC-related surface markers and tri-lineage differentiation capacity.ResultsSynovial tissue collected from the OA joints had a significantly higher OARSI score compared to normal joints, and significantly upregulated inflammation/synovitis markers S100A8/9, IL6, IL8, and CCL2. Both normal and OA synovial membrane contained cells displaying MPC properties, including a fibroblast-like morphology, CFU capacity, and maintained MPC marker expression over time during expansion. However, OA cSM-MPCs were unable to differentiate towards the chondrogenic lineage and had low adipogenic capacity in contrast to normal cSM-MPCs, whereas they possessed a higher osteogenic capacity. Furthermore, the OA synovial membrane contained significantly lower percentages of CD90+, CD44+, CD34+, and CD271+ cells.ConclusionsThe OA environment had adverse effects on the regenerative potential of cSM-MPCs, corroborated by decreased CFU, population doubling, and chondrogenic capacity compared to normal cSM-MPCs. OA cSM-MPCs may be a less optimal candidate for the cell-based treatment of OA than normal cSM-MPCs.

Typical response of CD14++CD16– monocyte to knee synovial derived mediators as a key target to overcome the onset and progression of osteoarthritis

ObjectiveSynovitis with increased infiltration of immune cells is observed in osteoarthritis (OA). Given the inflammatory condition of synovitis, we explored the protein profile of OA synovium (OAS) and its effect on circulating monocytes activation, migration, and functional commitments.MethodsKnee-synovium was acquired from end-stage OA (N = 8) and trauma patients (Trauma baseline control: TBC; N = 8) for characterization using H&E histology, IHC (iNOS), LCMS-QTOF, and MALDI-imaging. Response of peripheral blood monocytes to OAS conditioned-media (OACM) was observed using transwell (n = 6). The migrated cells were captured in SEM, quantified using phase-contrast microphotographs, and their activation receptors (CCR2, CXCR2, CX3CR1, and CD11b), pro-inflammatory genes, and phagocytic potential were studied using flow cytometry, gene expression array/qPCR, and latex beads (LB) phagocytosis assay, respectively.ResultsThe Venn diagram displayed 119 typical proteins in OAS, while 55 proteins in TBCS. The STRING protein network analysis indicated distinctive links between proteins and gene ontology (GO) and revealed proteins associated with leukocyte-mediated immunity in OAS as compared to TBC. The MALDI-imaging showed typical localized proteins at 2234.97, 2522.61, 2627.21, 3329.50, and 3539.69 m/z and IHC confirmed pro-inflammatory iNOS expression in OA synovium. CD14++CD16– classical monocytes significantly migrated in OACM and expressed CCR2, CXCR2, and CD11b receptors, TNFRSF11A, MAPK1, S100A8, HSPB1, ITGAL, NFATC1, IL13RA1, CD93, IL-1β, TNF-α, and MYD88 genes and increased LB uptake as compared to SFM.ConclusionOur findings suggest that the differential protein profile of OA synovium and the classical monocytes migrated, activated, and functionally committed in response to these mediators could be of therapeutic advantage.

Classifying Integrated Signature Molecules in Macrophages of Rheumatoid Arthritis, Osteoarthritis, and Periodontal Disease: An Omics-Based Study.

Rheumatoid arthritis (RA), osteoarthritis (OA), and periodontal disease (PD) are chronic inflammatory diseases that are globally prevalent, and pose a public health concern. The search for a potential mechanism linking PD to RA and OA continues, as it could play a significant role in disease prevention and treatment. Recent studies have linked RA, OA, and PD to Porphyromonas gingivalis (PG), a periodontal bacterium, through a similar dysregulation in an inflammatory mechanism. This study aimed to identify potential gene signatures that could assist in early diagnosis as well as gain insight into the molecular mechanisms of these diseases. The expression data sets with the series IDs GSE97779, GSE123492, and GSE24897 for macrophages of RA, OA synovium, and PG stimulated macrophages (PG-SM), respectively, were retrieved and screened for differentially expressed genes (DEGs). The 72 common DEGs among RA, OA, and PG-SM were further subjected to gene–gene correlation analysis. A GeneMANIA interaction network of the 47 highly correlated DEGs comprises 53 nodes and 271 edges. Network centrality analysis identified 15 hub genes, 6 of which are DEGs (API5, ATE1, CCNG1, EHD1, RIN2, and STK39). Additionally, two significantly up-regulated non-hub genes (IER3 and RGS16) showed interactions with hub genes. Functional enrichment analysis of the genes showed that “apoptotic regulation” and “inflammasomes” were among the major pathways. These eight genes can serve as important signatures/targets, and provide new insights into the molecular mechanism of PG-induced RA, OA, and PD.