Cartilage Homeostasis Research Articles

Increased Ca2+ signaling through CaV 1.2 induces tendon hypertrophy with increased collagen fibrillogenesis and biomechanical properties.

Tendons are tension-bearing tissues transmitting force from muscle to bone for body movement. This mechanical loading is essential for tendon development, homeostasis, and healing after injury. While Ca2+ signaling has been studied extensively for its roles in mechanotransduction, regulating muscle, bone, and cartilage development and homeostasis, knowledge about Ca2+ signaling and the source of Ca2+ signals in tendon fibroblast biology are largely unknown. Here, we investigated the function of Ca2+ signaling through CaV 1.2 voltage-gated Ca2+ channel in tendon formation. Using a reporter mouse, we found that CaV 1.2 is highly expressed in tendon during development and downregulated in adult homeostasis. To assess its function, we generated ScxCre;CaV 1.2TS mice that express a gain-of-function mutant CaV 1.2 in tendon. We found that mutant tendons were hypertrophic, with more tendon fibroblasts but decreased cell density. TEM analyses demonstrated increased collagen fibrillogenesis in the hypertrophic tendons. Biomechanical testing revealed that the hypertrophic tendons display higher peak load and stiffness, with no changes in peak stress and elastic modulus. Proteomic analysis showed no significant difference in the abundance of type I and III collagens, but mutant tendons had about two-fold increase in other ECM proteins such as tenascin C, tenomodulin, periostin, type XIV and type VIII collagens, around 11-fold increase in the growth factor myostatin, and significant elevation of matrix remodeling proteins including Mmp14, Mmp2, and cathepsin K. Taken together, these data highlight roles for increased Ca2+ signaling through CaV 1.2 on regulating expression of myostatin growth factor and ECM proteins for tendon collagen fibrillogenesis during tendon formation.

Expression of Bone Morphogenetic Protein 14 in Liver Disease and Cancer

The activation of hepatic stellate cells (HSCs) is the key event of hepatic fibrosis. Furthermore, activated HSCs also play an important role in the progression of hepatocellular cancer (HCC). Bone morphogenetic protein 14 (BMP14) is a member of the TGF-β/BMP superfamily. So far, most studies have analyzed BMP14 in the context of bone and cartilage formation and homeostasis. The aim of this study was to assess the expression and function of BMP14 in liver fibrosis and HCC. The BMP14 expression increased during the in vitro activation of primary human HSCs and also in mouse models of liver fibrosis. In human HCC, as well as non-tumorous liver tissues, there was a significant correlation between the expression of BMP14 and alpha-smooth-muscle actin (α-SMA), an established marker for HSC activation. RNAi-mediated BMP14 suppression in activated HSCs resulted in the reduced expression of the transcription factors inhibitor of differentiation 1 (ID1) and ID2, known targets of BMP signaling. Interestingly, α-SMA and collagen expression was also reduced in BMP14-depleted cells, while treatment with recombinant BMP14 induced ID1, ID2, α-SMA and collagen expression. In human HCC cell lines, treatment with recombinant BMP14 induced proliferation, migratory activity and colony formation. In summary, our data indicate activated HSCs as a major cellular source of enhanced BMP14 expression in fibrotic liver disease and HCC, and show that BMP14 exhibits pro-fibrogenic as well as pro-tumorigenic effects. Future analyses will reveal the potential of this soluble growth factor as a therapeutic target or prognostic marker for the progression of fibrosis and HCC in patients with chronic liver disease.

Inflammation-Mediated Aberrant Glucose Metabolism in Subchondral Bone Induces Osteoarthritis.

Osteoarthritis (OA) is an entire joint disease with pathological alteration in both articular cartilage and subchondral bone. It has been recognized recently the association between metabolic syndrome and OA, particularly glucose metabolism in regulation of articular cartilage homeostasis and joint integrity. Whereas the role of glucose metabolism in subchondral bone sclerosis remains largely unknown during pathogenesis of OA. Consistent with common OA features, we observed subchondral bone sclerosis and abnormal bone remodeling in human OA joints and murine OA joints as reflected by hyperactive bone resorption and overall bone formation which was measured via dynamic histomorphometry. Osx-CreER;tdTomato mice also displayed the similar overall bone formation under injury-induced OA condition. Immunohistochemistry further revealed increased IL-1β expression in human and murine OA subchondral bone. Given the inflammatory environment in joints under OA condition, we treated MC3T3-E1 cell, a pre-osteoblast cell line, with IL-1β in this study and demonstrated that IL-1β treatment could stimulate the cell osteogenic differentiation and meanwhile upregulate glycolysis and oxidative phosphorylation in cell cultures. More importantly, intraperitoneal injection of 2-deoxy-D-glucose (2-DG) and oligomycin (OGM), respectively, suppressed the subchondral bone glycolysis and oxidative phosphorylation in mice. Consequently, 2-DG and OGM treatment attenuated abnormal osteoblast differentiation and protected against aberrant bone formation in subchondral bone and articular cartilage degradation in wildtype mice following with joint injury. Collectively, these data strongly suggest glycolysis and oxidative may serve as important therapeutic targets for OA treatment.

Disrupting mechanical homeostasis promotes matrix metalloproteinase-13 mediated processing of neuron glial antigen 2 in mandibular condylar cartilage.

Post-traumatic osteoarthritis in the temporomandibular joint (TMJ OA) is associated dysfunctional cellmatrix mediated signalling resulting from changes in the pericellular microenvironment after injury. Matrix metalloproteinase (MMP)-13 is a critical enzyme in biomineralisation and the progression of OA that can both degrade the extracellular matrix and modify extracellular receptors. This study focused on MMP-13 mediated changes in a transmembrane proteoglycan, Neuron Glial antigen 2 (NG2/CSPG4). NG2/CSPG4 is a receptor for type VI collagen and a known substrate for MMP-13. In healthy articular layer chondrocytes, NG2/CSPG4 is membrane bound but becomes internalised during TMJ OA. The objective of this study was to determine if MMP-13 contributed to the cleavage and internalisation of NG2/CSPG4 during mechanical loading and OA progression. Using preclinical and clinical samples, it was shown that MMP-13 was present in a spatiotemporally consistent pattern with NG2/CSPG4 internalisation during TMJ OA. In vitro, it was illustrated that inhibiting MMP-13 prevented retention of the NG2/CSPG4 ectodomain in the extracellular matrix. Inhibiting MMP-13 promoted the accumulation of membrane-associated NG2/CSPG4 but did not affect the formation of mechanical-loading dependent variant specific fragments of the ectodomain. MMP- 13 mediated cleavage of NG2/CSPG4 is necessary to initiate clathrin-mediated internalisation of the NG2/ CSPG4 intracellular domain following mechanical loading. This mechanically sensitive MMP-13-NG2/CSPG4 axis affected the expression of key mineralisation and OA genes including bone morphogenetic protein 2, and parathyroid hormone-related protein. Together, these findings implicated MMP-13 mediated cleavage of NG2/CSPG4 in the mechanical homeostasis of mandibular condylar cartilage during the progression of degenerative arthropathies such as OA.

The role of mechano growth factor in chondrocytes and cartilage defects: a concise review.

Mechano growth factor (MGF), an isoform of insulin-like growth factor 1 (IGF-1), is recognized as a typical mechanically sensitive growth factor and has been shown to play an indispensable role in the skeletal system. In the joint cavity, MGF is highly expressed in chondrocytes, especially in the damaged cartilage tissue caused by trauma or degenerative diseases such as osteoarthritis (OA). Cartilage is an extremely important component of joints because it functions as a shock absorber and load distributer at the weight-bearing interfaces in the joint cavity, but it can hardly be repaired once injured due to its lack of blood vessels, lymphatic vessels, and nerves. MGF has been proven to play an important role in chondrocyte cell behaviors, including cell proliferation, migration, differentiation, inflammatory reactions and apoptosis, in and around the injury site. Moreover, under the normalized mechanical microenvironment in the joint cavity, MGF can sense and respond to mechanical stimuli, regulate chondrocyte activity, and maintain the homeostasis of cartilage tissue. Recent reports continue to explain its effects on various cell types and sport-related tissues, but its role in cartilage development, homeostasis and disease occurrence is still controversial, and its internal biological mechanism is still elusive. In this review, we summarize recent discoveries in the role of MGF in chondrocytes and cartilage defects, including tissue repair at the macroscopic level and chondrocyte activities at the microcosmic level, and discuss the current state of research and potential gaps in knowledge.

Inhibition of histone lysine demethylase 6A promotes chondrocytic activity and attenuates osteoarthritis development through repressing H3K27me3 enhancement of Wnt10a.

Histone hypermethylation represses gene transcription, which affects cartilage homeostasis or joint remodeling. Trimethylation of lysine 27 of histone 3 (H3K27me3) changes epigenome signatures, regulating tissue metabolism. This study aimed to investigate whether loss of H3K27me3 demethylase Kdm6a function affected osteoarthritis development. We revealed that chondrocyte-specific Kdm6a knockout mice developed relatively long femurs and tibiae as compared to wild-type mice. Kdm6a deletion mitigated osteoarthritis symptoms, including articular cartilage loss, osteophyte formation, subchondral trabecular bone loss, and irregular walking patterns of destabilized medial meniscus-injured knees. In vitro, loss of Kdm6a function compromised the loss in expression of key chondrocyte markers Sox9, collagen II, and aggrecan and improved glycosaminoglycan production in inflamed chondrocytes. RNA sequencing showed that Kdm6a loss changed transcriptomic profiles, which contributed to histone signaling, NADPH oxidase, Wnt signaling, extracellular matrix, and cartilage development in articular cartilage. Chromatin immunoprecipitation sequencing uncovered that Kdm6a knockout affected H3K27me3 binding epigenome, repressing Wnt10a and Fzd10 transcription. Wnt10a was, among others, functional molecules regulated by Kdm6a. Forced Wnt10a expression attenuated Kdm6a deletion-induced glycosaminoglycan overproduction. Intra-articular administration with Kdm6a inhibitor GSK-J4 attenuated articular cartilage erosion, synovitis, and osteophyte formation, improving gait profiles of injured joints. In conclusion, Kdm6a loss promoted transcriptomic landscapes contributing to extracellular matrix synthesis and compromised epigenetic H3K27me3-mediated promotion of Wnt10a signaling, preserving chondrocytic activity to attenuate osteoarthritic degeneration. We highlighted the chondroprotective effects of Kdm6a inhibitor for mitigating the development of osteoarthritic disorders.

The synovial environment steers cartilage deterioration and regeneration

Osteoarthritis (OA) was recently defined as an epidemic, and the lack of effective treatment is highly correlated to the limited knowledge regarding the underlying pathophysiology. Failure to regenerate upon trauma is thought to be one of the underlying causes for degenerative diseases, including OA. To investigate why lesions within an OA environment fail to heal, a heterogeneous cell population was isolated from the synovial fluid (SF) of OA patients. The cells' ability to undergo processes required for functional tissue regeneration was evaluated in the presence or absence of autologous SF. The obtained mechanistic findings were then used for the development of an immunomodulatory cell treatment, aimed to restore the pro-regenerative environment. Intra-articular injection in a clinical compassionate use study showed that the treatment restored the articular cartilage and joint homeostasis of OA patients. These findings confirm the role of pro-regenerative immune cells and their targeted influence on progenitor cells for degenerative joint disease therapies.

Analysis of the regulatory role of miR-34a-5p/PLCD3 in the progression of osteoarthritis

Osteoarthritis is a heterogeneous disease with a complex etiology. However, there is no effective treatment strategy at present. The purpose of this study was to explore the miRNA‒mRNA regulatory network and molecular mechanism that regulate the progression of osteoarthritis. In this article, we downloaded datasets (GSE55457, GSE82107, GSE143514 and GSE55235) from Gene Expression Omnibus (GEO) to screen differentially expressed mRNAs in osteoarthritis. Then, through weighted gene coexpression network (WGCNA), functional enrichment, protein‒protein interaction (PPI) network, miRNA‒mRNA coexpression network, ROC curve, and immune infiltration analyses and qPCR, the mRNA PLCD3, which was highly expressed in osteoarthritis and had clinical predictive value, was screened. We found that PLCD3 directly targets miR-34a-5p through DIANA and dual-luciferase experiments. The expression levels of PLCD3 and miR-34a-5p were negatively correlated. In addition, CCK-8 and wound healing assays showed that the miR-34a-5p mimic inhibited hFLS-OA cell proliferation and promoted hFLS-OA cell migration. PLCD3 overexpression showed the opposite trend. Western blotting further found that overexpression of miR-34a-5p reduced the protein expression levels of p-PI3K and p-AKT, while overexpression of PLCD3 showed the opposite trend. In addition, combined with the effect of the PI3K/AKT pathway inhibitor BIO (IC50 = 5.95μM), the results showed that overexpression of miR-34a-5p increased the inhibitory effects of BIO on p-PI3K and p-AKT protein expression, while overexpression of PLCD3 significantly reversed these inhibitory effects. Overall, the miR-34a-5p/PLCD3 axis may mediate the PI3K/AKT pathway in regulating cartilage homeostasis in synovial osteoarthritis. These data indicate that miR-34a-5p/PLCD3 may be a new prognostic factor in the pathology of synovial osteoarthritis.

EXCESSIVE WNT SIGNALLING ALTERS THE CARTILAGE RESPONSE TO PHYSIOLOGIC LOADING

Regulation of articular cartilage homeostasis is a complex process in which biologic and mechanical factors are involved. Hyperactivation of Wnt signaling, associated with osteoarthritis (OA), could jeopardize the protective anabolic effect of physiological loading. Here, we investigated the role of excessive Wnt signalling in cartilage molecular responses to loading.Human cartilage explants were harvested from hips of donors without OA. The Wnt agonist CHIR99021 was used to activate Wnt signalling 24 hours before cartilage explants were subjected to a loading protocol consisting of 2 cycles of 1 hour of 10% compression at 1 Hz, followed by 1-hour free swelling. Mechano-responsiveness was evaluated using the expression of type II collagen, aggrecan and MMP-13. Expression of known target genes TCF-1 and c-JUN was evaluated as positive control for Wnt and mechanical stimulation, respectively.In the absence of loading, CHIR99021 decreased the expression of the cartilage anabolic genes type II collagen and aggrecan, and increased the levels of MMP-13, corroborating that Wnt hyperactivation disrupts cartilage homeostasis. In the absence of Wnt hyperactivation, the applied loading protocol, representative for a physiologic stimulation by mechanical loading, led to an increase in type II collagen and aggrecan levels. However, when cartilage explants were subjected to mechanical stimulation in the presence of CHIR99021, the expression of cartilage anabolic genes was decreased, indicating changes to the cells’ mechano-responsiveness. Interestingly, mechanical stimulation was able to reduce the expression levels of MMP-13 compared to the condition of CHIR stimulation without loading.Hyperactivation of Wnt signaling switches the anabolic effect of physiologic compressive loading towards a potential catabolic effect and could contribute to the development and progression of OA.

Cbfβ Is a Novel Modulator against Osteoarthritis by Maintaining Articular Cartilage Homeostasis through TGF-β Signaling

TGF-β signaling is a vital regulator for maintaining articular cartilage homeostasis. Runx transcription factors, downstream targets of TGF-β signaling, have been studied in the context of osteoarthritis (OA). Although Runx partner core binding factor β (Cbfβ) is known to play a pivotal role in chondrocyte and osteoblast differentiation, the role of Cbfβ in maintaining articular cartilage integrity remains obscure. This study investigated Cbfβ as a novel anabolic modulator of TGF-β signaling and determined its role in articular cartilage homeostasis. Cbfβ significantly decreased in aged mouse articular cartilage and human OA cartilage. Articular chondrocyte-specific Cbfb-deficient mice (Cbfb△ac/△ac) exhibited early cartilage degeneration at 20 weeks of age and developed OA at 12 months. Cbfb△ac/△ac mice showed enhanced OA progression under the surgically induced OA model in mice. Mechanistically, forced expression of Cbfβ rescued Type II collagen (Col2α1) and Runx1 expression in Cbfβ-deficient chondrocytes. TGF-β1-mediated Col2α1 expression failed despite the p-Smad3 activation under TGF-β1 treatment in Cbfβ-deficient chondrocytes. Cbfβ protected Runx1 from proteasomal degradation through Cbfβ/Runx1 complex formation. These results indicate that Cbfβ is a novel anabolic regulator for cartilage homeostasis, suggesting that Cbfβ could protect OA development by maintaining the integrity of the TGF-β signaling pathway in articular cartilage.

WNT16 Regulation of the Articular Chondrocyte Phenotype in Mice.

The anabolic effects of WNT16 on osteoblasts are well established, however, little is known regarding the role of WNT16 in chondrocytes. In this study, we evaluated Wnt16 expression and its biological effects on mouse articular chondrocytes (ACs), since these cells are key to the development of osteoarthritis. While ACs derived from the long bone epiphysis of 7-day old C57BL/6J mice express multiple Wnts, Wnt5b and Wnt16 represent the two most highly expressed Wnts (expressed at several-fold higher levels than other Wnts). Treatment of serum-free AC cultures, with 100 ng/mL of recombinant human (rh) WNT16 for 24 h (hrs), increased proliferation (20%, p < 0.05) and expression levels of makers (Sox9 and Col2) of immature chondrocytes at both 24 h and 72 h, while Acan increased at 72 h. Expression of Mmp9, a marker of mature chondrocytes was decreased at 24 h. Additionally, WNT16 treatment regulated expression levels of Wnt ligands in a biphasic manner, inhibiting its expression at 24 h, while stimulating expression at 72 h. To determine whether WNT16 exerted anabolic effects on the AC phenotype, ex vivo cultures of tibial epiphyses were treated with rhWNT16 or vehicle for 9 days, and the articular cartilage phenotype was evaluated by safranin O cartilage staining and expression of articular cartilage marker genes. Both articular cartilage area and expression levels of AC markers were increased after rhWNT16 treatment. Our data suggest that Wnt16 expressed in ACs may play a role in regulating joint cartilage homeostasis via its direct effect, as well as through modulating the expression of other Wnt ligands.

Axial rotation and pain are associated with facet joint osteoarthritis in adolescent idiopathic scoliosis

Facet joints are crucial for spinal stability and develop premature osteoarthritis in patients with adolescent idiopathic scoliosis (AIS). Here, we evaluated the association between facet joint cartilage and subchondral bone homeostasis, perceived back pain and 3-dimensional spinal deformity to understand better the role of facet joint degeneration in AIS progression and pain. Cartilage and bone osteoarthritic states of AIS facet joint surgical samples were characterized using histological OARSI scoring, visual morphological grading and μCT analysis, respectively. Back pain was self-reported using a numerical rating scale and expressed relative to the location on the patient's back. The scoliotic curves from our patient cohort were digitally reconstructed using biplanar radiographs and the eOS system (EOS imaging). The deformity was then reduced to three intervertebral angles (coronal, sagittal and axial) for each pair of bilateral facet joints. Statistical associations between the intervertebral angles, osteoarthritis parameters and pain intensity were performed using the Spearman method and Friedman test. Facet joint cartilage degeneration was associated with decreased subchondral bone volume and quality. Most importantly, asymmetrical, and overall degeneration of facet joints was strongly correlated to intervertebral axial rotation. Additionally, kyphotic intervertebral segments in the sagittal plane were good predictors of increased facet joint degeneration and back pain. Facet joint degeneration is associated with axial deformity, kyphotic intervertebral angle and back pain intensity in AIS. These results suggest that facet joints are important features to consider for rotational instability in AIS spines and related disease progression and perceived back pain.

MiR146a modulates chondrogenesis of bone marrow mesenchymal stem cells by modulating Lsm11 expression.

MicroRNAs play a critical role in bone marrow mesenchymal stem cell (MSC) chondrogenesis and regulate the progression of joint regeneration in osteoarthritis. Our previous research confirmed that miR146a relieves osteoarthritis by modulating cartilage homeostasis. However, few studies have revealed the relationship between miR146a and the chondrogenesis of MSCs, and the exact mechanisms remain unclear. This study aimed to determine the function of miR146a in the chondrogenic differentiation of MSCs and the potential mechanisms involved. MiR146a expression increased during chondrogenesis. MiR146a knockout (KO) led to the increased chondrogenesis of MSCs compared to that in wild-type (WT) MSCs, whereas the overexpression of miR146a by mimics resulted in the decreased chondrogenesis of MSCs, as determined by the mRNA expression of collagen, type II, alpha 1 (COL2A1), aggrecan, cartilage oligomeric matrix protein (COMP), and matrix metallopeptidase 13 (MMP13). Furthermore, cartilage defects could be treated better when injected with spheres induced from miR146aKO MSCs than from WT MSCs, indicating that miR146a inhibits chondrogenesis in vivo. In addition, based on miRNA-mRNA prediction analysis and a dual-luciferase reporter assay, we observed that the deletion of miR146a led to the increased expression of Lsm11 during chondrogenesis and demonstrated that miR146a targeted Lsm11 by binding to its 3'-untranslated region (UTR) and inhibited its translation. The inhibition of Lsm11 by silencing RNA (siRNA) reversed the increased ability of chondrogenesis by knocking out miR146a both in vivo and in vitro, suggesting that miR146a inhibits chondrogenesis by directly inhibiting Lsm11 in MSCs, which may be a novel target for treating osteoarthritis.

The Role of Lubricin, Irisin and Exercise in the Prevention and Treatment of Osteoarthritis

Osteoarthritis is a chronic degenerative musculoskeletal disease that worsens with age and is defined by pathological alterations in joint components. All clinical treatment recommendations for osteoarthritis promote exercise, although precise molecular pathways are unclear. The purpose of this study was to critically analyze the research on lubricin and irisin and how they relate to healthy and diseased joint tissue. Our research focused specifically on exercise strategies and offered new perspectives for future potential osteoarthritis treatment plans. Although lubricin and irisin have only recently been discovered, there is evidence that they have an impact on cartilage homeostasis. A crucial component of cartilage lubrication and integrity, lubricin is a surface-active mucinous glycoprotein released by the synovial joint. Its expression increases with joint movement. In healthy joints, lubricin molecules cover the cartilage surface to lubricate the boundary of the joint and inhibit protein and cell attachment. Patients with joint trauma, inflammatory arthritis, or genetically mediated lubricin deficiency, who do not produce enough lubricin to protect the articular cartilage, develop arthropathy. Irisin, sometimes known as the "sports hormone", is a myokine secreted primarily by skeletal muscle. It is a physiologically active protein that can enter the circulation as an endocrine factor, and its synthesis and secretion are primarily triggered by exercise-induced muscle contraction. We searched PubMed, Web of Science, Google Scholar, and Scopus using the appropriate keywords to identify the most recent research. The studies considered advance our knowledge of the role that exercise plays in the fight against osteoarthritis, serve as a valuable resource, and support the advancement of osteoarthritis prevention and therapy.

Selective Clearance of Senescent Chondrocytes in Osteoarthritis by Targeting Excitatory Amino Acid Transporter Protein 1 to Induce Ferroptosis.

Aims: This study aimed at exploring the mechanism of ferroptosis (an iron-dependent form of nonapoptotic cell death) resistance in senescent chondrocytes (SenChos). Results: In this study, by utilizing metabolomics and single-cell RNA sequencing, we found that hyperactivation of ferroptosis metabolism was one of the most prominent metabolic features in SenChos. Interestingly, however, SenChos were able to survive in this state and were resistant to ferroptosis-induced cell death. Next, we elucidated that this survival mechanism of SenChos could be primarily attributed to overexpression of the membrane protein excitatory amino acid transporter protein 1 (EAAT1), which can increase intracellular glutamate (Glu) levels and activate the glutathione system to counteract ferroptosis. In addition, 2-amino-5,6,7,8-tetrahydro-4-(4-methoxyphenyl)-7-(naphthalen-1-yl)-5-oxo-4H-chromene-3-carbonitrile (UCPH-101) (a specific inhibitor of EAAT1) and siRNA-EAAT1 were able to substantially increase the sensitivity of SenChos to ferroptosis and to induce cell death, with no apparent effects on the normal cells. Administration of an intraarticular injection of UCPH-101 caused inhibition of EAAT1 selectively, cleared SenChos from cartilage, improved the cartilage homeostasis, and significantly delayed the progression of osteoarthritis (OA). Innovation: This work supports a relevant role for EAAT1 in ferroptosis resistance mechanism for SenChos, revealing a potential therapeutic target of OA. Conclusions: EAAT1-Glu-glutathione peroxidase 4 anti-ferroptosis axis is a key survival mechanism for SenChos, and EAAT1 is an effective and specific target for anti-senescence therapy in OA. Antioxid. Redox Signal. 39, 262-277.

Biomimetic photosynthetic system: Shedding light on the restoration of cell metabolism

Biomimetic photosynthetic system: Shedding light on the restoration of cell metabolism

MMP-2 Silencing through siRNA Loaded Positively-Charged Nanoparticles (AcPEI-NPs) Counteracts Chondrocyte De-Differentiation.

The abnormal matrix remodeling process, as well as inflammation, angiogenesis, and tumor metastasis, are related to an increase in the synthesis and secretion of matrix metalloproteinases (MMPs), the zinc-dependent proteolytic endopeptidases. Recent studies have evidenced MMPs' role in osteoarthritis (OA) development, during which chondrocytes undergo hypertrophic differentiation and exhibit enhanced catabolism. The trait of OA is extracellular matrix (ECM) progressive degradation regulated by many factors, in which MMPs play an important role, which indicates them as potential therapeutic targets. Herein, a small interfering RNA (siRNA) delivery system able to suppress MMPs' activity was synthetized. Results demonstrated that positively charged nanoparticles (AcPEI-NPs) complexed with MMP-2 siRNA are efficiently internalized by cells with endosomal escape. Moreover, avoiding lysosome degradation, MMP2/AcPEI nanocomplex increases nucleic acid delivery efficiency. Gel zymography, RT-PCR, and ELISA analyses confirmed MMP2/AcPEI nanocomplex activity even when embedded within collagen matrix resembling the natural extracellular matrix. Further, the inhibition of in vitro collagen degradation exerts a protective effect on chondrocyte dedifferentiation. The suppression of MMP-2 activity, preventing matrix degradation, protects chondrocytes against degeneration and supporting ECM homeostasis in articular cartilage. These encouraging results promote further investigation to validate the utilization of MMP-2 siRNA as ''molecular switch'' able to counteract osteoarthritis.

Hydroquinone, an Environmental Pollutant, Affects Cartilage Homeostasis through the Activation of the Aryl Hydrocarbon Receptor Pathway.

Exposure to environmental pollutants has a proven detrimental impact on different aspects of human health. Increasing evidence has linked pollution to the degeneration of tissues in the joints, although through vastly uncharacterised mechanisms. We have previously shown that exposure to hydroquinone (HQ), a benzene metabolite that can be found in motor fuels and cigarette smoke, exacerbates synovial hypertrophy and oxidative stress in the synovium. To further understand the impact of the pollutant on joint health, here we investigated the effect of HQ on the articular cartilage. HQ exposure aggravated cartilage damage in rats in which inflammatory arthritis was induced by injection of Collagen type II. Cell viability, cell phenotypic changes and oxidative stress were quantified in primary bovine articular chondrocytes exposed to HQ in the presence or absence of IL-1β. HQ stimulation downregulated phenotypic markers genes SOX-9 and Col2a1, whereas it upregulated the expression of the catabolic enzymes MMP-3 and ADAMTS5 at the mRNA level. HQ also reduced proteoglycan content and promoted oxidative stress alone and in synergy with IL-1β. Finally, we showed that HQ-degenerative effects were mediated by the activation of the Aryl Hydrocarbon Receptor. Together, our findings describe the harmful effects of HQ on articular cartilage health, providing novel evidence surrounding the toxic mechanisms of environmental pollutants underlying the onset of articular diseases.

Inhibition of SMAD3 effectively reduces ADAMTS-5 expression in the early stages of osteoarthritis

ObjectiveAs one of the most important protein-degrading enzymes, ADAMTS-5 plays an important role in the regulation of cartilage homeostasis, while miRNA-140 is specifically expressed in cartilage, which can inhibit the expression of ADAMTS-5 and delay the progression of OA (osteoarthritis). SMAD3 is a key protein in the TGF-β signaling pathway, inhibiting the expression of miRNA-140 at the transcriptional and post-transcriptional levels, and studies have confirmed the high expression of SMAD3 in knee cartilage degeneration, but whether SMAD3 can mediate the expression of miRNA-140 to regulate ADAMTS-5 remains unknown.MethodsSprague–Dawley (SD) rat chondrocytes were extracted in vitro and treated with a SMAD3 inhibitor (SIS3) and miRNA-140 mimics after IL-1 induction. The expression of ADAMTS-5 was detected at the protein and gene levels at 24 h, 48 h, and 72 h after treatment. The OA model of SD rats was created using the traditional Hulth method in vivo, with SIS3 and lentivirus packaged miRNA-140 mimics injected intra-articularly at 2 weeks, 6 weeks and 12 weeks after surgery. The expression of miRNA-140 and ADAMTS-5 in the knee cartilage tissue was observed at the protein and gene levels. Concurrently, knee joint specimens were fixed, decalcified, and embedded in paraffin prior to immunohistochemical, Safranin O/Fast Green staining, and HE staining analyses for ADAMTS-5 and SMAD3.ResultsIn vitro, the expression of ADAMTS-5 protein and mRNA in the SIS3 group decreased to different degrees at each time point. Meanwhile, the expression of miRNA-140 in the SIS3 group was significantly increased, and the expression of ADAMTS-5 in the miRNA-140 mimics group was also significantly downregulated (P < 0.05). In vivo, it was found that ADAMTS-5 protein and gene were downregulated to varying degrees in the SIS3 and miRNA-140 mimic groups at three time points, with the most significant decrease at the early stage (2 weeks) (P < 0.05), and the expression of miRNA-140 in the SIS3 group was significantly upregulated, similar to the changes detected in vitro. Immunohistochemical results showed that the expression of ADAMTS-5 protein in the SIS3 and miRNA-140 groups was significantly downregulated compared to that in the blank group. The results of hematoxylin and eosin staining showed that in the early stage, there was no obvious change in cartilage structure in the SIS3 and miRNA-140 mock groups. The same was observed in the results of Safranin O/Fast Green staining; the number of chondrocytes was not significantly reduced, and the tide line was complete.ConclusionThe results of in vitro and in vivo experiments preliminarily showed that the inhibition of SMAD3 significantly reduced the expression of ADAMTS-5 in early OA cartilage, and this regulation might be accomplished indirectly through miRNA-140.

Protein phosphatase PPM1A inhibition attenuates osteoarthritis via regulating TGF-β/Smad2 signaling in chondrocytes.

TGF-β signaling is crucial for modulating osteoarthritis (OA), and protein phosphatase magnesium-dependent 1A (PPM1A) has been reported as a phosphatase of SMAD2 and regulates TGF-β signaling, while the role of PPM1A in cartilage homeostasis and OA development remains largely unexplored. In this study, we found increased PPM1A expression in OA chondrocytes and confirmed the interaction between PPM1A and phospho-SMAD2 (p-SMAD2). Importantly, our data show that PPM1A KO substantially protected mice treated with destabilization of medial meniscus (DMM) surgery against cartilage degeneration and subchondral sclerosis. Additionally, PPM1A ablation reduced the cartilage catabolism and cell apoptosis after the DMM operation. Moreover, p-SMAD2 expression in chondrocytes from KO mice was higher than that in WT controls with DMM induction. However, intraarticular injection with SD-208, repressing TGF-β/SMAD2 signaling, dramatically abolished protective phenotypes in PPM1A-KO mice. Finally, a specific pharmacologic PPM1A inhibitor, Sanguinarine chloride (SC) or BC-21, was able to ameliorate OA severity in C57BL/6J mice. In summary, our study identified PPM1A as a pivotal regulator of cartilage homeostasis and demonstrated that PPM1A inhibition attenuates OA progression via regulating TGF-β/SMAD2 signaling in chondrocytes and provided PPM1A as a potential target for OA treatment.