Synovial macrophage M1 polarisation exacerbates experimental osteoarthritis partially through R-spondin-2

2018 ◽  
Vol 77 (10) ◽  
pp. 1524-1534 ◽  
Author(s):  
Haiyan Zhang ◽  
Chuangxin Lin ◽  
Chun Zeng ◽  
Zhenyu Wang ◽  
Hua Wang ◽  
...  
Keyword(s):  
Cartilage Damage ◽  
Research Society ◽  
M1 Macrophages ◽  
Myeloid Lineage ◽  
Synovial Macrophage ◽  
Osteoarthritis Research Society ◽  
Synovial Tissues ◽  
Experimental Osteoarthritis ◽  
And Control

ObjectivesTo investigate the roles and regulatory mechanisms of synovial macrophages and their polarisation in the development of osteoarthritis (OA).MethodsSynovial tissues from normal patients and patients with OA were collected. M1 or M2-polarised macrophages in synovial tissues of patients with OA and OA mice were analysed by immunofluorescence and immunohistochemical staining. Mice with tuberous sclerosis complex 1 (TSC1) or Rheb deletion specifically in the myeloid lineage were generated and subjected to intra-articular injection of collagenase (collagenase-induced osteoarthritis, CIOA) and destabilisation of the medial meniscus (DMM) surgery to induce OA. Cartilage damage and osteophyte size were measured by Osteoarthritis Research Society International score and micro-CT, respectively. mRNA sequencing was performed in M1 and control macrophages. Mice and ATDC5 cells were treated with R-spondin-2 (Rspo2) or anti-Rspo2 to investigate the role of Rspo2 in OA.ResultsM1 but not M2-polarised macrophages accumulated in human and mouse OA synovial tissue. TSC1 deletion in the myeloid lineage constitutively activated mechanistic target of rapamycin complex 1 (mTORC1), increased M1 polarisation in synovial macrophages and exacerbated experimental OA in both CIOA and DMM models, while Rheb deletion inhibited mTORC1, enhanced M2 polarisation and alleviated CIOA in mice. The results show that promoting the macrophage M1 polarisation leads to exacerbation of experimental OA partially through secretion of Rspo2 and activation of β-catenin signalling in chondrocytes.ConclusionsSynovial macrophage M1 polarisation exacerbates experimental CIOA partially through Rspo2. M1 macrophages and Rspo2 are potential therapeutic targets for OA treatment.

Asporin regulated by miR-26b-5p mediates chondrocyte senescence and exacerbates osteoarthritis progression via TGF-β1/Smad2 pathway

Rheumatology ◽  
2021 ◽  
Author(s):  
Liangliang Liu ◽  
Chang Zhao ◽  
Haiyan Zhang ◽  
Yuheng Lu ◽  
Bingsheng Luo ◽  
...  
Keyword(s):  
Therapeutic Strategy ◽  
Medial Meniscus ◽  
Articular Chondrocytes ◽  
Cartilage Damage ◽  
Cartilage Degeneration ◽  
Research Society ◽  
Osteoarthritis Progression ◽  
Osteoarthritis Research Society ◽  
Tgf Β1

Abstract Objectives This study aimed to investigate the role and mechanism of asporin in modulating chondrocyte senescence in osteoarthritis (OA) pathology. Methods Asporin and senescence-related hallmark expression were examined in human and experimental OA mouse cartilage samples. Twelve-week-old male C57 mice were administered with recombinant protein (rm-asporin)- or asporin-siRNA-expressing lentiviruses via intra-articular injection once a week after destabilization of the medial meniscus (DMM) surgery to induce OA. Cartilage damage was measured using the Osteoarthritis Research Society International score. Senescence-associated β-galactosidase (SA-βGal) staining, γH2AX, p21, and p16INK4a were analyzed by immunofluorescence staining and western blot to assess the specific role of asporin in chondrocyte senescence. The TGF-β1/Smad2 signaling pathway and miR-26b-5p were further evaluated to explore the mechanism of asporin in OA. Results Asporin was upregulated in articular chondrocytes of OA patients and DMM mice and accompanied by accumulation of senescent cells. Asporin overexpression exaggerated OA progression, whereas silencing asporin restored chondrocyte homeostasis and deferred chondrocyte senescence, leading to markedly attenuated DMM-induced OA. Cellular and molecular analyses showed that asporin can be inhibited by miR-26b-5p, which was significantly downregulated in OA cartilage, leading to exacerbation of experimental OA partially through inhibition of TGF-β1/Smad2 signaling in chondrocytes. Conclusions Our findings indicate that asporin plays an essential role in chondrocyte senescence and OA pathogenesis. Upregulated by miR-26b-5p, asporin inhibits the TGF-β1/Smad2 pathway to accelerate chondrocyte senescence and exacerbate cartilage degeneration. Targeting the miR-26b-5p/asporin/Smad2 axis may serve as a practical therapeutic strategy to delay chondrocyte senescence and OA development.

scholarly journals Comparison of properties determined using electromechanical assessment (Arthro-BST™) with macroscopic and histological properties in symptomatic human articular cartilage of the hip

2021 ◽  
Vol 23 (1) ◽  
Author(s):  
Taku Ukai ◽  
Masato Sato ◽  
Shiho Wasai ◽  
Takumi Takahashi ◽  
Haruka Omura ◽  
...  
Keyword(s):  
Histological Grade ◽  
Cartilage Damage ◽  
Hip Osteoarthritis ◽  
Cartilage Degeneration ◽  
Research Society ◽  
Cartilage Tissue ◽  
Human Articular Cartilage ◽  
Mankin Score ◽  
Osteoarthritis Research Society ◽  
Noninvasive Assessment

Abstract Background Cartilage degeneration is assessed using various methods. Although macroscopic evaluation can directly measure cartilage degeneration, it cannot accurately assess cartilage properties. Histological examination is one of the most accurate methods for evaluating cartilage degeneration. However, it is invasive and requires collection of cartilage tissue. In contrast, the Arthro-BST™ probe can assess cartilage properties noninvasively. This study aimed to evaluate the effectiveness of the Arthro-BST in assessing cartilage degeneration by comparing macroscopic (International Cartilage Repair Society [ICRS] classification) and histological evaluations (modified Mankin score and Osteoarthritis Research Society International [OARSI] histological grade). Methods Fourteen femoral heads were excised from 13 patients during surgery to treat hip osteoarthritis or femoral fracture. The ICRS score was used for macroscopic evaluation of cartilage degeneration. The Arthro-BST was applied at sites matching the areas of cartilage damage. The sites assessed using the ICRS classification and Arthro-BST were evaluated histologically (modified Mankin score and OARSI histological grade), and these were compared with the Arthro-BST results. Results The ICRS classification identified significant differences between grades 1 and 3 (p < 0.01), between grades 1 and 4 (p < 0.01), between grades 2 and 3 (p < 0.01), and between grades 2 and 4 (p < 0.01). Significant correlations were observed between the Arthro-BST results and the ICRS score, modified Mankin score (structure, cellularity, matrix staining, total score), and OARSI histological grade. Conclusions In the assessment of hip osteoarthritis, the Arthro-BST results correlated with those of macroscopic and histological evaluations. The Arthro-BST is useful for assessing hip osteoarthritis and may be helpful for noninvasive assessment of cartilage degeneration.

Comparison of Properties Determined Using Electromechanical Assessment (Arthro-BSTTM) With Macroscopic and Histological Properties in Symptomatic Human Articular Cartilage of the Hip

2021 ◽  
Author(s):  
Taku Ukai ◽  
Masato Sato ◽  
Shiho Wasai ◽  
Takumi Takahashi ◽  
Haruka Omura ◽  
...  
Keyword(s):  
Histological Grade ◽  
Cartilage Damage ◽  
Hip Osteoarthritis ◽  
Cartilage Degeneration ◽  
Research Society ◽  
Cartilage Tissue ◽  
Histological Evaluation ◽  
Human Articular Cartilage ◽  
Mankin Score ◽  
Osteoarthritis Research Society

Abstract Background: Cartilage degeneration is assessed using various methods. Although macroscopic evaluation can measure cartilage degeneration directly, it cannot accurately assess cartilage properties. Histological examination is one of the most accurate methods for evaluating cartilage degeneration. However, it is invasive and requires collection of cartilage tissue. By contrast, the Arthro-BSTTM probe can assess cartilage properties noninvasively. This study aimed to evaluate the effectiveness of the Arthro-BST for assessing cartilage degeneration by comparing macroscopic evaluation (International Cartilage Repair Society [ICRS] classification) and histological evaluation (modified Mankin score and Osteoarthritis Research Society International [OARSI] histological grade).Methods: Fourteen femoral heads were excised from 13 patients during surgery to treat hip osteoarthritis or femoral fracture. The ICRS score was used for macroscopic evaluation of cartilage degeneration. The Arthro-BST was applied at sites matching the areas of cartilage damage. The sites assessed using the ICRS classification and Arthro-BST were evaluated histologically (modified Mankin score and OARSI histological grade), and these were compared with the Arthro-BST results.Results: ICRS classification identified significant differences between grades 1 and 3 (p < 0.01), grades 1 and 4 (p < 0.01), grades 2 and 3 (p < 0.01), and grades 2 and 4 (p < 0.01). Significant correlations were observed between the Arthro-BST results and the ICRS score, modified Mankin score (structure, cellularity, matrix staining, total score), and OARSI histological grade.Conclusions: In the assessment of hip osteoarthritis, Arthro-BST results correlated with those of macroscopic and histological evaluation. The Arthro-BST is useful for assessing hip osteoarthritis and may be helpful for the noninvasive assessment of cartilage degeneration.

Precise targeting of miR-141/200c cluster in chondrocytes attenuates osteoarthritis development

2020 ◽  
pp. annrheumdis-2020-218469
Author(s):  
Ming-Liang Ji ◽  
Hua Jiang ◽  
Fei Wu ◽  
Rui Geng ◽  
Li kun Ya ◽  
...  
Keyword(s):  
Ectopic Expression ◽  
Joint Space ◽  
Research Society ◽  
Mass Spectrometry Analysis ◽  
Human Cartilage ◽  
Spectrometry Analysis ◽  
Ct Analysis ◽  
Osteoarthritis Research Society ◽  
Dynamics Simulations

ObjectivesDespite preclinical studies involving miRNA therapeutics conducted in osteoarthritis (OA) over the years, none of these miRNAs have yet translated to clinical applications, owing largely to the lack of efficient intra-articular (IA) delivery systems. Here, we investigated therapeutic efficacy of the chondrocyte-specific aptamer-decorated PEGylated polyamidoamine nanoparticles (NPs)-based miRNAs delivery for OA.MethodsThe role of miR-141/200c cluster during skeletal and OA development was examined by miR-141/200cflox/flox mice and Col2a1-CreERT2; miR-141/200cflox/flox mice. Histological analysis was performed in mouse joints and human cartilage specimens. Chondrocyte-specific aptamer-decorated NPs was designed, and its penetration, stability and safety were evaluated. OA progression was assessed by micro-CT analysis, X-ray and Osteoarthritis Research Society International scores after destabilising the medial meniscus surgery with miR-141/200c manipulation by NPs IA injection. Mass spectrometry analysis, molecular docking and molecular dynamics simulations were performed to investigate the interaction between aptamer and receptor.ResultsIncreased retention of NPs inside joint space is observed. The NPs are freely and deeply penetrant to mice and human cartilage, and unexpectedly persist in chondrocytes for at least 5 weeks. OA chondrocytes microenviroment improves endo/lysosomal escape of microRNAs (miRNAs). Therapeutically, IA injection of miR-141/200c inhibitors provides strong chondroprotection, whereas ectopic expression of miR-141/200c exacerbates OA. Mechanistically, miR-141/200c promotes OA by targeting SIRT1, which acetylates histone in the promoters of interleukin 6 (IL-6), thereby activating IL-6/STAT3 pathway.ConclusionsOur findings indicate that this nanocarrier can optimise the transport kinetics of miR-141/200c into chondrocytes, fostering miRNA-specific disease-modifying OA drugs development.

Early Role of NFKB2 on the Myeloid Differentiation of CD34+ Hematopoietic Cells

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1276-1276
Author(s):  
Greice A. Molfetta ◽  
Dalila L. Zanette ◽  
Rodrigo A. Panepucci ◽  
Wilson A. Silva ◽  
Marco A. Zago
Keyword(s):  
Gene Expression ◽  
Molecular Mechanisms ◽  
Rna Extraction ◽  
Myeloid Differentiation ◽  
Signaling Proteins ◽  
Myeloid Lineage ◽  
Before And After ◽  
A Subunit ◽  
And Control

Abstract Efforts to understand the molecular mechanisms underlying the differentiation of hematopoietic progenitor cells into mature blood cells have focused mainly on late events that largely reflect the differentiated state of the cells. In order to evaluate early changes of the gene expression profile of HSPC subjected to differentiation stimuli, transcriptional profiles of immuno-magnetically sorted bone marrow CD34+ HSPC were generated before and after 12 and 40 hours of culture with supplemented media favoring myeloid or erythroid commitment. Four independent samples were pooled and submitted to each treatment, and cells aliquots were subjected to RNA extraction and to methylcellulose cultures. CD34+ HSPC without previous stimulation generated about equal percentages (50% each) of erythroid (BFU-E) and myeloid (CFU-GM) colonies, whereas upon erythroid stimulation the median percentages of BFU-E, CFU-GM and mixed colonies (CFU-Mix) were, respectively, 61%, 38% and 1% for the12 hour treatment and 83%, 17% and 0% for 40 hours treatment. Conversely, upon myeloid stimulation, respectively, 37%, 61% and 2% colonies were observed after 12 hours and 23%, 61% and 14% for 40 hours treatment. These results indicate that after 12 and 40 hours of treatment, the genetic program of those cells were shifted towards the desired phenotype. Serial analysis of gene expression (SAGE) was employed to generate four independent libraries, each with more than 60,000 tags sequenced, representing more than 12,000 annotated transcripts. By analyzing the differentially regulated transcripts between the control CD34+ HSPC and the stimulated cells, we observed a set of genes that were initially up-regulated at 12 hours but were then down-regulated at 40 hours, exclusively after myeloid stimuli. Among those we found transcripts for IL1B, LTB, TNFRSF4 and NFKB2. Additionally, the receptor for LTB and the inhibitor of NF-κB signaling NFKBIA (IKBA) were respectively, up and down modulated at both time points. All those transcripts code for signaling proteins of the nuclear factor kappa B (NF-κB) pathway. More specifically, NFKB2 is a subunit of the NF-κB transcription factor (TF) that together with RELB mediates the non-canonical NF-κB pathway. The up-regulation followed by a down regulation was confirmed for NFKB2 and also demonstrated for RELB and NFKB1, indicating that the NF-κB pathway could be involved in the early commitment of CD34+ HSPC towards the myeloid lineage. To test this hypothesis, interference RNA (RNAi) against NFKB2 and control RNAi were transfected into BM CD34+ HSPC. Cells submitted to transfection with RNAi were stimulated towards the myeloid lineage and subjected to evaluation on methylcellulose cultures. Transcript levels of NFKB2 and RELB (a transcription target of NFKB2) were shown to be down-modulated, confirming the successful inhibition of NFKB2. After inhibition of NFKB2, the percentage of CFU-GM and BFU-E colonies shifted from 53% and 47% on control cells, respectively, to 22% and 78%. Altogether, our results indicate that NFKB2 has a role in the early commitment of CD34+ HSPC towards the myeloid lineage, directly inducing the differentiation program or, alternatively, protecting early myeloid progenitors from apoptosis.

METTL3-mediated m6A modification of ATG7 regulates autophagy-GATA4 axis to promote cellular senescence and osteoarthritis progression

2021 ◽  
pp. annrheumdis-2021-221091
Author(s):  
Xiang Chen ◽  
Wang Gong ◽  
Xiaoyan Shao ◽  
Tianshu Shi ◽  
Lei Zhang ◽  
...  
Keyword(s):  
Cellular Senescence ◽  
Cartilage Damage ◽  
Rna Stability ◽  
Cartilage Destruction ◽  
Autophagic Flux ◽  
Senescent Phenotype ◽  
Rna Immunoprecipitation ◽  
Potential Strategy ◽  
Synovial Tissues

ObjectiveThe aim of the study was to investigate the role and regulatory mechanisms of fibroblast-like synoviocytes (FLSs) and their senescence in the progression of osteoarthritis (OA).MethodsSynovial tissues from normal patients and patients with OA were collected. Synovium FLS senescence was analysed by immunofluorescence and western blotting. The role of methyltransferase-like 3 (METTL3) in autophagy regulation was explored using N6-methyladenosine (m6A)-methylated RNA and RNA immunoprecipitation assays. Mice subjected to destabilisation of the medial meniscus (DMM) surgery were intra-articularly injected with or without pAAV9 loaded with small interfering RNA (siRNA) targeting METTL3. Histological analysis was performed to determine cartilage damage.ResultsSenescent FLSs were markedly increased with the progression of OA in patients and mouse models. We determined that impaired autophagy occurred in OA-FLS, resulting in the upregulation of senescence-associated secretory phenotype (SASP). Re-establishment of autophagy reversed the senescent phenotype by suppressing GATA4. Further, we observed for the first time that excessive m6A modification negatively regulated autophagy in OA-FLS. Mechanistically, METTL3-mediated m6A modification decreased the expression of autophagy-related 7, an E-1 enzyme crucial for the formation of autophagosomes, by attenuating its RNA stability. Silencing METTL3 enhanced autophagic flux and inhibited SASP expression in OA-FLS. Intra-articular injection of synovium-targeted METTL3 siRNA suppressed cellular senescence propagation in joints and ameliorated DMM-induced cartilage destruction.ConclusionsOur study revealed the important role of FLS senescence in OA progression. Targeted METTL3 inhibition could alleviate the senescence of FLS and limit OA development in experimental animal models, providing a potential strategy for OA therapy.

scholarly journals Targeted deletion of Atg5 in chondrocytes promotes age-related osteoarthritis

2015 ◽  
Vol 75 (3) ◽  
pp. 627-631 ◽  
Author(s):  
Thibault Bouderlique ◽  
Karuna K Vuppalapati ◽  
Phillip T Newton ◽  
Lei Li ◽  
Björn Barenius ◽  
...  
Keyword(s):  
Cell Death ◽  
Research Society ◽  
Targeted Deletion ◽  
Nucleotidyl Transferase ◽  
Medial Meniscectomy ◽  
Age Related ◽  
Osteoarthritis Research Society ◽  
Post Traumatic ◽  
Safranin O ◽  
And Control

ObjectivesIt has been suggested that the lysosomal recycling process called macro-autophagy plays a role in osteoarthritis development. We thus decided to genetically ablate the autophagy-indispensable Atg5 gene specifically in chondrocytes and analyse the development of osteoarthritis upon aging and in a post-traumatic model.MethodsMice lacking the Atg5 gene in their chondrocytes (Atg5cKO) were generated by crossing Atg5-floxed mice with transgenic mice that expressed cre recombinase driven by the collagen type 2 promoter. Animals were analysed at the age of 2, 6 and 12 months for age-related osteoarthritis or underwent mini-open partial medial meniscectomy at 2 months of age and were analysed 1 or 2 months after surgery. We evaluated osteoarthritis using the Osteoarthritis Research Society International (OARSI) scoring on safranin-O-stained samples. Cell death was evaluated by terminal deoxy-nucleotidyl-transferase-mediated deoxy-UTP nick end labelling (TUNEL) and by immunostaining of cleaved caspases.ResultsWe observed the development of osteoarthritis in Atg5cKO mice with aging including fibrillation and loss of proteoglycans, which was particularly severe in males. The ablation of Atg5 was associated with an increased cell death as assessed by TUNEL, cleaved caspase 3 and cleaved caspase 9. Surprisingly, no difference in the development of post-traumatic osteoarthritis was observed between Atg5cKO and control mice.ConclusionsAutophagy protects from age-related osteoarthritis by facilitating chondrocyte survival.

scholarly journals CCL2/CCR2, but not CCL5/CCR5, mediates monocyte recruitment, inflammation and cartilage destruction in osteoarthritis

2016 ◽  
Vol 76 (5) ◽  
pp. 914-922 ◽  
Author(s):  
Harini Raghu ◽  
Christin M Lepus ◽  
Qian Wang ◽  
Heidi H Wong ◽  
Nithya Lingampalli ◽  
...  
Keyword(s):  
Chemokine Receptors ◽  
Cartilage Damage ◽  
Cartilage Destruction ◽  
Synthesis Inhibitor ◽  
Promising Therapeutic Approach ◽  
Concomitant Reduction ◽  
Synovial Tissues ◽  
And Control ◽  
Microarray Analyses ◽  
And Cartilage

ObjectivesWhile various monocyte chemokine systems are increased in expression in osteoarthritis (OA), the hierarchy of chemokines and chemokine receptors in mediating monocyte/macrophage recruitment to the OA joint remains poorly defined. Here, we investigated the relative contributions of the CCL2/CCR2 versus CCL5/CCR5 chemokine axes in OA pathogenesis.MethodsCcl2-, Ccr2-, Ccl5- and Ccr5-deficient and control mice were subjected to destabilisation of medial meniscus surgery to induce OA. The pharmacological utility of blocking CCL2/CCR2 signalling in mouse OA was investigated using bindarit, a CCL2 synthesis inhibitor, and RS-504393, a CCR2 antagonist. Levels of monocyte chemoattractants in synovial tissues and fluids from patients with joint injuries without OA and those with established OA were investigated using a combination of microarray analyses, multiplexed cytokine assays and immunostains.ResultsMice lacking CCL2 or CCR2, but not CCL5 or CCR5, were protected against OA with a concomitant reduction in local monocyte/macrophage numbers in their joints. In synovial fluids from patients with OA, levels of CCR2 ligands (CCL2, CCL7 and CCL8) but not CCR5 ligands (CCL3, CCL4 and CCL5) were elevated. We found that CCR2+ cells are abundant in human OA synovium and that CCR2+ macrophages line, invade and are associated with the erosion of OA cartilage. Further, blockade of CCL2/CCR2 signalling markedly attenuated macrophage accumulation, synovitis and cartilage damage in mouse OA.ConclusionsOur findings demonstrate that monocytes recruited via CCL2/CCR2, rather than by CCL5/CCR5, propagate inflammation and tissue damage in OA. Selective targeting of the CCL2/CCR2 system represents a promising therapeutic approach for OA.

Morphogenesis of a New Human Herpes-Type Virus Isolate (Sasha Virus)

1973 ◽  
Vol 31 ◽  
pp. 592-593
Author(s):  
R. F. Zeigel ◽  
W. Munyon
Keyword(s):  
Virus Isolate ◽  
Calf Serum ◽  
Uranyl Acetate ◽  
Human Herpes ◽  
Human Embryonic Lung ◽  
Human Herpes Virus ◽  
Intracytoplasmic Inclusions ◽  
Hel Cells ◽  
And Control

In continuing studies on the role of viruses in biochemical transformation, Dr. Munyon has succeeded in isolating a highly infectious human herpes virus. Fluids of buccal pustular lesions from Sasha Munyon (10 mo. old) uiere introduced into monolayer sheets of human embryonic lung (HEL) cell cultures propagated in Eagles’ medium containing 5% calf serum. After 18 hours the cells exhibited a dramatic C.P.E. (intranuclear vacuoles, peripheral patching of chromatin, intracytoplasmic inclusions). Control HEL cells failed to reflect similar changes. Infected and control HEL cells were scraped from plastic flasks at 18 hrs. of incubation and centrifuged at 1200 × g for 15 min. Resultant cell packs uiere fixed in Dalton's chrome osmium, and post-fixed in aqueous uranyl acetate. Figure 1 illustrates typical hexagonal herpes-type nucleocapsids within the intranuclear virogenic regions. The nucleocapsids are approximately 100 nm in diameter. Nuclear membrane “translocation” (budding) uias observed.

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