MicroRNA-146a is linked to pain-related pathophysiology of osteoarthritis

Because miR-146a is linked to osteoarthritis (OA) and cartilage degeneration is associated with pain, we have characterized the functional role of miR-146a in the regulation of human articular cartilage homeostasis and pain-related factors. Expression of miRNA 146a was analyzed in human articular cartilage and synovium, as well as in dorsal root ganglia (DRG) and spinal cord from a rat model for OA-related pain assessment. The functional effects of miR-146a on human chondrocytic, synovial, and microglia cells were studied in cells transfected with miR-146a. Using real-time PCR, we assessed the expression of chondrocyte metabolism-related genes in chondrocytes, genes for inflammatory factors in synovial cells, as well as pain-related proteins and ion channels in microglial cells. Previous studies showed that miR-146a is significantly upregulated in human peripheral knee OA joint tissues. Transfection of synthetic miR-146a significantly suppresses extracellular matrix-associated proteins (e.g., Aggrecan, MMP-13, ADAMTS-5, collagen II) in human knee joint chondrocytes and regulates inflammatory cytokines in synovial cells from human knee joints. In contrast, miR-146a is expressed at reduced levels in DRGs and dorsal horn of the spinal cords isolated from rats experiencing OA-induced pain. Exogenous supplementation of synthetic miR-146a significantly modulates inflammatory cytokines and pain-related molecules (e.g., TNFα, COX-2, iNOS, IL-6, IL8, RANTS and ion channel, TRPV1) in human glial cells. Our findings suggest that miR-146a controls knee joint homeostasis and OA-associated algesia by balancing inflammatory responses in cartilage and synovium with pain-related factors in glial cells. Hence, miR-146a may be useful for the treatment of both cartilage regeneration and pain symptoms caused by OA.     

TGF β-induced cartilage repair is maintained but fibrosis is blocked in the presence of Smad7

Cartilage damage in osteoarthritis (OA) is considered an imbalance between catabolic and anabolic factors, favoring the catabolic side. We assessed whether adenoviral overexpression of transforming growth factor-β (TGFβ) enhanced cartilage repair and whether TGFβ-induced fibrosis was blocked by local expression of the intracellular TGFβ inhibitor Smad7. We inflicted cartilage damage by injection of interleukin-1 (IL-1) into murine knee joints. After 2 days, we injected an adenovirus encoding TGFβ. On day 4, we measured proteoglycan (PG) synthesis and content. To examine whether we could block TGFβ-induced fibrosis and stimulate cartilage repair simultaneously, we injected Ad-TGFβ and Ad-Smad7. This was performed both after IL-1-induced damage and in a model of primary OA. In addition to PG in cartilage, synovial fibrosis was measured by determining the synovial width and the number of procollagen I-expressing cells. Adenoviral overexpression of TGFβ restored the IL-1-induced reduction in PG content and increased PG synthesis. TGFβ-induced an elevation in PG content in cartilage of the OA model. TGFβ-induced synovial fibrosis was strongly diminished by simultaneous synovial overexpression of Smad7 in the synovial lining. Of great interest, overexpression of Smad7 did not reduce the repair-stimulating effect of TGFβ on cartilage. Adenoviral overexpression of TGFβ stimulated repair of IL-1- and OA-damaged cartilage. TGFβ-induced synovial fibrosis was blocked by locally inhibiting TGFβ signaling in the synovial lining by simultaneously transfecting it with an adenovirus overexpressing Smad7.     

Identification of Novel Adipokines in the Joint. Differential Expression in Healthy and Osteoarthritis Tissues

Objectives

Emerging data suggest that several metabolic factors, released mainly by white adipose tissue (WAT) and joint tissues, and collectively named adipokines, might have a role in the pathophysiology of OA. Recently, novel adipokines such as SERPINE2, WISP2, GPNMB and ITIH5 have been identified in WAT. The main goal of this study was to analyse the expression of these novel adipokines in synovium, infrapatellar fat pad and chondrocytes and to compare the expression of these molecules in healthy and OA tissues.

Methods

Synovial tissues, infrapatellar fat pad and chondrocytes were obtained from 36 OA patients (age 52–85; mean BMI 28.9) who underwent total knee replacement surgery. Healthy synovial tissues and infrapatellar fat pad were obtained from 15 traumatic knee patients (age 23–53; mean BMI 23.5). mRNA and protein expression were determined by qRT-PCR and western blot analysis respectively.

Results

All the novel adipokines, matter of our study, are expressed in OA synovium, infrapatellar fat pad and chondrocytes. Moreover, we detected a differential expression of SERPINE2 and ITIH5 in OA synovial tissues as compared to healthy samples. Finally, we also observed an increased expression of WISP2 in OA infrapatellar fat pad in comparison to healthy controls.

Conclusions

In this study we demonstrated for the first time the expression of four novel adipokines in different joint tissues and how these molecules are differentially expressed in healthy and OA joint tissues.     

Stimulation of α7-nAChRs coordinates autophagy and apoptosis signaling in experimental knee osteoarthritis

Osteoarthritis (OA) is the most common chronic joint disease in the elderly population. Growing evidence indicates that a balance between autophagy and apoptosis in chondrocytes plays a key role in OA’s cartilage degradation. Thus, drugs targeting the balance between apoptosis and autophagy are potential therapeutic approaches for OA treatment. In previous studies, we found that the activation of α7 nicotinic acetylcholine receptors (α7-nAChRs) alleviated monosodium iodoacetate (MIA)-induced joint degradation and osteoarthritis pain. To explore the potential functions of α7-nAChRs in autophagy and apoptosis signaling in knee OA, we compared the expression of α7-nAChRs in human knee articular cartilage tissues from normal humans and OA patients. We found that knee joint cartilage tissues of OA patients showed decreased α7-nAChRs and an imbalance between autophagy and apoptosis. Next, we observed that α7-nAChRs deficiency did not affect cartilage degradation in OA development but reversed the beneficial effects of nicotine on mechanical allodynia, cartilage degradation, and an MIA-induced switch from autophagy to apoptosis. Unlike in vivo studies, we found that primary chondrocytes from α7-nAChRs knockout (KO) mice showed decreased LC3 levels under normal conditions and were more sensitive toward MIA-induced apoptosis. Finally, we found that α7-nAChRs deficiency increased the phosphorylation of mTOR after MIA treatment, which can also be observed in OA patients’ tissues. Thus, our findings not only confirmed that nicotine alleviated MIA-induced pain behavior and cartilage degradation via stimulating the α7-nAChRs/mTOR signal pathway but found the potential role of α7-nAChRs in mediating the balance between apoptosis and autophagy.Subject terms: Autophagy, Apoptosis     

Relationships between biomarkers of cartilage, bone, synovial metabolism and knee pain provide insights into the origins of pain in early knee osteoarthritis

Introduction

We tested the hypothesis that there exist relationships between the onset of early stage radiographically defined knee osteoarthritis (OA), pain and changes in biomarkers of joint metabolism.

Methods

Using Kellgren-Lawrence (K/L) grading early radiographic knee OA (K/L 2) was detected in 16 of 46 patients. These grades (K/L 1 is no OA and K/L 2 is early OA) were divided into two groups according to the presence or absence of persistent knee pain. Sera (s) and urines (u) were analysed with biomarkers for cartilage collagen cleavage (sC2C and uCTX-II) and synthesis (sCPII), bone resorption (uNTx) and synovitis (hyaluronic acid: sHA).

Results

sCPII decreased and sC2C/sCPII, uCTX-II/sCPII and sHA increased with onset of OA (K/L 2 versus K/L 1) irrespective of joint pain. In contrast, sC2C and uCTX-II remained unchanged in early OA patients. Of the patients with K/L grades 1 and 2 sC2C, sCPII, sHA, uNTX and uCTX-II were all significantly increased in patients with knee pain independent of grade. Among the K/L grade 2 subjects, only uCTX-II and uCTX-II/sCPII were increased in those with knee pain. In grade 1 patients both sC2C and sCPII were increased in those with knee pain. No such grade specific changes were seen for the other biomarkers including sHA.

Conclusions

These results suggest that changes in cartilage matrix turnover detected by molecular biomarkers may reflect early changes in cartilage structure that account directly or indirectly for knee pain. Also K/L grade 1 patients with knee pain exhibit biomarker features of early OA.     

Matrix Metalloproteases and Tissue Inhibitors of Metalloproteinases in Medial Plica and Pannus-like Tissue Contribute to Knee Osteoarthritis Progression

Osteoarthritis (OA) is characterized by degradation of the cartilage matrix, leading to pathologic changes in the joints. However, the pathogenic effects of synovial tissue inflammation on OA knees are not clear. To investigate whether the inflammation caused by the medial plica is involved in the pathogenesis of osteoarthritis, we examined the expression of matrix metalloproteinases (MMPs), tissue inhibitors of metalloproteinases (TIMPs), interleukin (IL)-1β, and tumor necrosis factor (TNF)-α in the medial plica and pannus-like tissue in the knees of patients with medial compartment OA who underwent either arthroscopic medial release (stage II; 15 knee joints from 15 patients) or total knee replacement (stage IV; 18 knee joints from 18 patients). MMP-2, MMP-3, MMP-9, IL-1β, and TNF-α mRNA and protein levels measured, respectively, by quantitative real-time PCR and Quantibody human MMP arrays, were highly expressed in extracts of medial plica and pannus-like tissue from stage IV knee joints. Immunohistochemical staining also demonstrated high expression of MMP-2, MMP-3, and MMP-9 in plica and pannus-like tissue of stage IV OA knees and not in normal cartilage. Some TIMP/MMP ratios decreased significantly in both medial plica and pannus-like tissue as disease progressed from stage II to stage IV. Furthermore, the migration of cells from the pannus-like tissue was enhanced by IL-1β, while plica cell migration was enhanced by TNF-α. The results suggest that medial plica and pannus-like tissue may be involved in the process of cartilage degradation in medial compartment OA of the knee.     

Plasma proteins present in osteoarthritic synovial fluid can stimulate cytokine production via Toll-like receptor 4

Introduction

Osteoarthritis (OA) is a degenerative disease characterized by cartilage breakdown in the synovial joints. The presence of low-grade inflammation in OA joints is receiving increasing attention, with synovitis shown to be present even in the early stages of the disease. How the synovial inflammation arises is unclear, but proteins in the synovial fluid of affected joints could conceivably contribute. We therefore surveyed the proteins present in OA synovial fluid and assessed their immunostimulatory properties.

Methods

We used mass spectrometry to survey the proteins present in the synovial fluid of patients with knee OA. We used a multiplex bead-based immunoassay to measure levels of inflammatory cytokines in serum and synovial fluid from patients with knee OA and from patients with rheumatoid arthritis (RA), as well as in sera from healthy individuals. Significant differences in cytokine levels between groups were determined by significance analysis of microarrays, and relations were determined by unsupervised hierarchic clustering. To assess the immunostimulatory properties of a subset of the identified proteins, we tested the proteins' ability to induce the production of inflammatory cytokines by macrophages. For proteins found to be stimulatory, the macrophage stimulation assays were repeated by using Toll-like receptor 4 (TLR4)-deficient macrophages.

Results

We identified 108 proteins in OA synovial fluid, including plasma proteins, serine protease inhibitors, proteins indicative of cartilage turnover, and proteins involved in inflammation and immunity. Multiplex cytokine analysis revealed that levels of several inflammatory cytokines were significantly higher in OA sera than in normal sera, and levels of inflammatory cytokines in synovial fluid and serum were, as expected, higher in RA samples than in OA samples. As much as 36% of the proteins identified in OA synovial fluid were plasma proteins. Testing a subset of these plasma proteins in macrophage stimulation assays, we found that Gc-globulin, α₁-microglobulin, and α₂-macroglobulin can signal via TLR4 to induce macrophage production of inflammatory cytokines implicated in OA.

Conclusions

Our findings suggest that plasma proteins present in OA synovial fluid, whether through exudation from plasma or production by synovial tissues, could contribute to low-grade inflammation in OA by functioning as so-called damage-associated molecular patterns in the synovial joint.     

Acupuncture reduces pain in rats with osteoarthritis by inhibiting MCP2/CCR2 signaling pathway

Acupuncture is an emerging alternative therapy that has been beneficial for the pain of osteoarthritis (OA). However, the underlying mechanism of protective effect remains unclear. MCP1/CCR2 axis can be stimulated in various periods of OA, and we hypothesize that acupuncture may treat OA by regulating the MCP1/CCR2 axis. This study aimed to explore the effect of acupuncture at points ST35 and ST36 on the effects of hyperalgesia and cartilage in OA rats including the expression of chemokines, nerve growth factor (NGF), and inflammatory-related proteins. OA was induced in male Sprague–Dawley rats by anterior cruciate ligament transection at the right knee. The first acupuncture intervention was performed on the seventh day after surgery and once a day for seven weeks. The knee-pain-related behaviors, histology, and related protein were examined in this study. We have found that electroacupuncture at ST35 and ST36 can significantly alleviate the hyperalgesia and cartilage degeneration as well as reducing nerve sprouting in OA knee joint. Moreover, acupuncture treatment may inhibit the MCP1/CCR2 axis as well as down-regulate inflaming factor and NGF in cartilage and synovial tissue. The data presented here indicate that acupuncture exerts a protective effect against hyperalgesia and cartilage degeneration, and the mechanism might involve in chemokines and NGF pathway.     

Targeting Vascular Endothelial Growth Factor Receptors as a Therapeutic Strategy for Osteoarthritis and Associated Pain

Pain is the major reason that patients suffering from osteoarthritis (OA) seek medical care. We found that vascular endothelial growth factors (VEGFs) mediate signaling in OA pain pathways. To determine the specific contributions of VEGFs and their receptors (VEGFRs) to joint pathology and pain transmission during OA progression, we studied intra-articular (IA) injections of VEGF ligands into murine knee joints. Only VEGF ligands specific for the activation of VEGFR1, but not VEGFR2, induced allodynia within 30 min. Interventions in OA by inhibitors of VEGFRs were done in vivo using a preclinical murine OA model by IA injections of selective inhibitors of VEGFR1/VEGFR2 kinase (pazopanib) or VEGFR2 kinase (vandetanib). OA phenotypes were evaluated using pain-associated murine behavioral tests and histopathologic analyses. Alterations in VEGF/VEGFR signaling by drugs were determined in knee joints, dorsal root ganglia, and spinal cord by immunofluorescence microscopy. Pazopanib immediately relieved OA pain by interfering with pain transmission pathways. Pain reduction by vandetanib was mainly due to the inhibition of cartilage degeneration by suppressing VEGFR2 expression. In conclusion, IA administration of pazopanib, which simultaneously inhibits VEGFR1 and VEGFR2, can be developed as an ideal OA disease-modifying drug that rapidly reduces joint pain and simultaneously inhibits cartilage degeneration.     

Decrease in expression of bone morphogenetic proteins 4 and 5 in synovial tissue of patients with osteoarthritis and rheumatoid arthritis

Bone morphogenetic proteins (BMPs) have been identified as important morphogens with pleiotropic functions in regulating the development, homeostasis and repair of various tissues. The aim of this study was to characterize the expression of BMPs in synovial tissues under normal and arthritic conditions. Synovial tissue from normal donors (ND) and from patients with osteoarthritis (OA) and rheumatoid arthritis (RA) were analyzed for BMP expression by using microarray hybridization. Differential expression of BMP-4 and BMP-5 was validated by semiquantitative RT-PCR, in situ hybridization and immunohistochemistry. Activity of arthritis was determined by routine parameters for systemic inflammation, by histological scoring of synovitis and by semiquantitative RT-PCR of IL-1β, TNF-α, stromelysin and collagenase I in synovial tissue. Expression of BMP-4 and BMP-5 mRNA was found to be significantly decreased in synovial tissue of patients with RA in comparison with ND by microarray analysis (p < 0.0083 and p < 0.0091). Validation by PCR confirmed these data in RA (p < 0.002) and also revealed a significant decrease in BMP-4 and BMP-5 expression in OA compared with ND (p < 0.015). Furthermore, histomorphological distribution of both morphogens as determined by in situ hybridization and immunohistochemistry showed a dominance in the lining layer of normal tissues, whereas chronically inflamed tissue from patients with RA revealed BMP expression mainly scattered across deeper layers. In OA, these changes were less pronounced with variable distribution of BMPs in the lining and sublining layer. BMP-4 and BMP-5 are expressed in normal synovial tissue and were found decreased in OA and RA. This may suggest a role of distinct BMPs in joint homeostasis that is disturbed in inflammatory and degenerative joint diseases. In comparison with previous reports, these data underline the complex impact of these factors on homeostasis and remodeling in joint physiology and pathology.     

Prevalence of Radiographic Osteoarthritis of the Knee and Its Relationship to Self-Reported Pain

Background and Aim

Osteoarthritis (OA) of the knee is one of the most common skeletal disorders, yet little data are available in Asian populations. We sought to assess the prevalence and pattern of radiographic OA of the knee, and its relationship to self-reported pain in a Vietnamese population.

Methods

The study was based on a sample of 170 men and 488 women aged ≥40 years who were randomly sampled from the Ho Chi Minh City (Vietnam). Radiographs of the knee were graded from 0 to 4 according to the Kellgren and Lawrence scale. Osteoarthritis was defined as being present in a knee if radiographic grades of 2 or higher were detected. Knee pain and symptoms were ascertained by direct interview using a structured questionnaire.

Results

The point prevalence of radiographic OA of the knee was 34.2%, with women having higher rate than men (35.3% vs 31.2%). The prevalence of knee OA increased with advancing age: 8% among those aged 40–49 years, 30% in those aged 50–59 years, and 61.1% in those aged ≥60 years. Greater BMI was associated with higher risk of knee OA. Self-reported knee pain was found in 35% of men and 62% of women. There was a statistically significant association between self-reported knee pain and knee OA (prevalence ratio 3.1; 95% CI 2.0 to 4.6).

Conclusions

These data indicate that approximately a third of Vietnamese men and women have radiographic OA in the knee, and that self-reported knee pain may be used as an indicator of knee osteoarthritis.     

Androgen conversion in osteoarthritis and rheumatoid arthritis synoviocytes – androstenedione and testosterone inhibit estrogen formation and favor production of more potent 5α-reduced androgens

In synovial cells of patients with osteoarthritis (OA) and rheumatoid arthritis (RA), conversion products of major anti-inflammatory androgens are as yet unknown but may be proinflammatory. Therefore, therapy with androgens in RA could be a problem. This study was carried out in order to compare conversion products of androgens in RA and OA synoviocytes. In 26 OA and 24 RA patients, androgen conversion in synovial cells was investigated using radiolabeled substrates and analysis by thin-layer chromatography and HPLC. Aromatase expression was studied by immunohistochemistry. Dehydroepiandrosterone (DHEA) was converted into androstenediol, androstenedione (ASD), 16αOH-DHEA, 7αOH-DHEA, testosterone, estrone (E1), estradiol (E2), estriol (E3), and 16αOH-testosterone (similar in OA and RA). Surprisingly, levels of E2, E3, and 16α-hydroxylated steroids were as high as levels of testosterone. In RA and OA, 5α-dihydrotestosterone increased conversion of DHEA into testosterone but not into estrogens. The second androgen, ASD, was converted into 5α-dihydro-ASD, testosterone, and negligible amounts of E1, E2, E3, or 16αOH-testosterone. 5α-dihydro-ASD levels were higher in RA than OA. The third androgen, testosterone, was converted into ASD, 5α-dihydro-ASD, 5α-dihydrotestosterone, and negligible quantities of E1 and E2. 5α-dihydrotestosterone was higher in RA than OA. ASD and testosterone nearly completely blocked aromatization of androgens. In addition, density of aromatase-positive cells and concentration of released E2, E3, and free testosterone from superfused synovial tissue was similar in RA and OA but estrogens were markedly higher than free testosterone. In conclusion, ASD and testosterone might be favorable anti-inflammatory compounds because they decrease aromatization and increase anti-inflammatory 5α-reduced androgens. In contrast, DHEA did not block aromatization but yielded high levels of estrogens and proproliferative 16α-hydroxylated steroids. Androgens were differentially converted to pro- and anti-inflammatory steroid hormones via diverse pathways.     

Extracellular localization of galectin-3 has a deleterious role in joint tissues

In this study we examine the extracellular role of galectin-3 (gal-3) in joint tissues. Following intra-articular injection of gal-3 or vehicle in knee joints of mice, histological evaluation of articular cartilage and subchondral bone was performed. Further studies were then performed using human osteoarthritic (OA) chondrocytes and subchondral bone osteoblasts, in which the effect of gal-3 (0 to 10 μg/ml) was analyzed. Osteoblasts were incubated in the presence of vitamin D₃ (50 nM), which is an inducer of osteocalcin, encoded by an osteoblast terminal differentiation gene. Genes of interest mainly expressed in either chondrocytes or osteoblasts were analyzed with real-time RT-PCR and enzyme immunoassays. Signalling pathways regulating osteocalcin were analyzed in the presence of gal-3. Intra-articular injection of gal-3 induced knee swelling and lesions in both cartilage and subchondral bone. On human OA chondrocytes, gal-3 at 1 μg/ml stimulated ADAMTS-5 expression in chondrocytes and, at higher concentrations (5 and 10 μg/ml), matrix metalloproteinase-3 expression. Experiments performed with osteoblasts showed a weak but bipolar effect on alkaline phosphatase expression: stimulation at 1 μg/ml or inhibition at 10 μg/ml. In the absence of vitamin D₃, type I collagen alpha 1 chain expression was inhibited by 10 μg/ml of gal-3. The vitamin D₃induced osteocalcin was strongly inhibited in a dose-dependent manner in the presence of gal-3, at both the mRNA and protein levels. This inhibition was mainly mediated by phosphatidylinositol-3-kinase. These findings indicate that high levels of extracellular gal-3, which could be encountered locally during the inflammatory process, have deleterious effects in both cartilage and subchondral bone tissues.     

CXCL12 is displayed by rheumatoid endothelial cells through its basic amino-terminal motif on heparan sulfate proteoglycans

The chemokine CXCL12 (also known as stromal cell-derived factor, SDF-1) is constitutively expressed by stromal resident cells and is involved in the homeostatic and inflammatory traffic of leukocytes. Binding of CXCL12 to glycosaminoglycans on endothelial cells (ECs) is supposed to be relevant to the regulation of leukocyte diapedesis and neoangiogenesis during inflammatory responses. To improve our understanding of the relevance of this process to rheumatoid arthritis (RA), we have studied the mechanisms of presentation of exogenous CXCL12 by cultured RA ECs. RA synovial tissues had higher levels of CXCL12 on the endothelium than osteoarthritis (OA) tissues; in both, CXCL12 colocalized to heparan sulfate proteoglycans (HSPGs) and high endothelial venules. In cultured RA ECs, exogenous CXCL12α was able to bind in a CXCR4-independent manner to surface HSPGs. Desulfation of RA EC HSPGs by pretreatment with sodium chlorate, or by replacing in a synthetic CXCL12α the residues Lys24 and Lys27 by Ser (CXCL12α-K2427S), decreased or abrogated the ability of the chemokine to bind to RA ECs. Ex vivo, synovial ECs from patients with either OA or RA displayed a higher CXCL12-binding capacity than human umbilical vein ECs (HUVECs), and in HUVECs the binding of CXCL12 was increased on exposure to tumor necrosis factor-α or lymphotoxin-α₁β₂. Our findings indicate that CXCL12 binds to HSPGs on ECs of RA synovium. The phenomenon relates to the interaction of HSPGs with a CXCL12 domain with net positive surface charge located in the first β strand, which encompasses a canonical BXBB HSPG-binding motif. Furthermore, we show that the attachment of CXCL12 to HSPGs is upregulated by inflammatory cytokines. Both the upregulation of a constitutive chemokine during chronic inflammation and the HSPG-dependent immobilization of CXCL12 in EC surfaces are potential sites for therapeutic intervention.