Specific inhibition of FAK signaling attenuates subchondral bone deterioration and articular cartilage degeneration during osteoarthritis pathogenesis

Osteoarthritis (OA), a disease of the entire joint, is characterized by abnormal bone remodeling and coalescent degradation of articular cartilage. We have previously found that elevated levels of H-type vessels in subchondral bone correlate with OA and that focal adhesion kinase (FAK) is critical for H-type vessel formation in osteoporosis. However, the potential role of FAK in OA remains unexplored. Here, we demonstrate that the p-FAK level was dramatically elevated in subchondral bone following anterior cruciate ligament transection (ACLT) in rats. Specific inhibition of FAK signaling with Y15 in subchondral bone resulted in the suppression of subchondral bone deterioration and this effect was mediated by H-type vessel-induced ectopic bone formation. Further, articular cartilage degeneration was also alleviated after Y15 treatment. In vitro, the p-FAK level was significantly elevated in mesenchymal stem cells (MSCs) from vehicle-treated ACLT rats as compared to that in MSCs from sham controls and Y15-treated ACLT rats. Elevated p-FAK level in MSCs promoted vascular endothelial growth factor (VEGF) expression, as demonstrated from the high VEGF level in the blood, subchondral bone, and conditioned medium (CM) of MSCs from vehicle-treated ACLT rats. The CM of MSCs from vehicle-treated ACLT rats might promote the angiogenesis of endothelial cells and the catabolic response of chondrocytes through the FAK-growth factor receptor-bound protein 2-mitogen-activated protein kinase-mediated expression of VEGF. The effect of the CM from MSCs of Y15-treated ACLT rats or that treated with a VEGF-neutralizing antibody on vessel formation and the catabolic response was lowered. Thus, the specific inhibition of FAK signaling may be a promising avenue for the prevention or early treatment of OA.     

关节软骨与软骨下骨改变在不同骨关节炎动物模型中的特点

目的 探讨不同骨关节炎(osteoarthritis,OA)动物模型中软骨下骨和关节软骨的病理改变特征。方法 采用三种SD大鼠骨关节炎模型,将24只6月龄雌性大鼠随机分为4组:假手术对照组(Sham,n=6),前交叉韧带切除手术组(ACLT,n=6),木瓜蛋白酶关节腔注射组(Papain,n=6),以及卵巢切除术组(OVX,n=6)。造模后8周取膝关节,胫骨平台行Micro-CT扫描分析,关节软骨行甲苯胺蓝染色、Mankin评分,比较软骨下骨和关节软骨改变情况。结果 造模操作后8周,不同OA模型的软骨破坏程度有所不同。ACLT和Papain组软骨破坏比较严重,OVX组软骨变化较轻。所有OA模型中的软骨下松质骨均发生改变,OVX组相对于Sham对照组,软骨下骨微结构显著疏松,而ACLT组与Papain组相对于Sham对照组,软骨下骨微结构没有显著改变,但相对于OVX组,有显著性差异。三种OA模型的软骨下骨板厚度都较Sham组减少。结论 三种动物模型软骨下骨和关节软骨都发生明显病理改变,并且改变有所不同。不同OA模型代表不同病理,预示着软骨下骨所发挥的作用有所不同,这为进一步研究不同类型OA发生发展的机制,以及将软骨下骨作为OA治疗的可能靶点提供了更多的依据。     

Reduced PDGF-AA in subchondral bone leads to articular cartilage degeneration after strenuous running

To identify the effects of running on articular cartilage and subchondral bone remodeling, C57BL/6 mice were randomly divided into three groups: control, moderate-, and strenuous running. Magnetic resonance imaging showed bone marrow lesions in the knee subchondral bone in the strenuous-running group in contrast with the other two groups. The microcomputed tomography analysis showed promoted bone formation in the subchondral bone in mice subjected to strenuous running. Histological and immunohistochemistry results indicated that terminal differentiation of chondrocytes and degeneration of articular cartilage were enhanced but, synthesis of platelet-derived growth factor-AA (PDGF-AA) in the subchondral bone was suppressed after strenuous running. In vitro, excessive mechanical treatments suppressed the expression of PDGF-AA in osteoblasts, and the condition medium from mechanical-treated osteoblasts stimulated maturation and terminal differentiation of chondrocytes. These results indicate that strenuous running suppresses the synthesis of PDGF-AA in subchondral bone, leading to downregulated PDGF/Akt signal in articular cartilage and thus cartilage degeneration.     

Identification of gene expression profiles and key genes in subchondral bone of osteoarthritis using weighted gene coexpression network analysis

Osteoarthritis (OA) significantly influences the quality life of people around the world. It is urgent to find an effective way to understand the genetic etiology of OA. We used weighted gene coexpression network analysis (WGCNA) to explore the key genes involved in the subchondral bone pathological process of OA. Fifty gene expression profiles of GSE51588 were downloaded from the Gene Expression Omnibus database. The OA‐associated genes and gene ontologies were acquired from JuniorDoc. Weighted gene coexpression network analysis was used to find disease‐related networks based on 21756 gene expression correlation coefficients, hub‐genes with the highest connectivity in each module were selected, and the correlation between module eigengene and clinical traits was calculated. The genes in the traits‐related gene coexpression modules were subject to functional annotation and pathway enrichment analysis using ClusterProfiler. A total of 73 gene modules were identified, of which, 12 modules were found with high connectivity with clinical traits. Five modules were found with enriched OA‐associated genes. Moreover, 310 OA‐associated genes were found, and 34 of them were among hub‐genes in each module. Consequently, enrichment results indicated some key metabolic pathways, such as extracellular matrix (ECM)‐receptor interaction (hsa04512), focal adhesion (hsa04510), the phosphatidylinositol 3'‐kinase (PI3K)‐Akt signaling pathway (PI3K‐AKT) (hsa04151), transforming growth factor beta pathway, and Wnt pathway. We intended to identify some core genes, collagen (COL)6A3, COL6A1, ITGA11, BAMBI, and HCK, which could influence downstream signaling pathways once they were activated. In this study, we identified important genes within key coexpression modules, which associate with a pathological process of subchondral bone in OA. Functional analysis results could provide important information to understand the mechanism of OA.     

Imaging of subchondral bone by optical coherence tomography upon optical clearing of articular cartilage

Optical clearing is an effective method to reduce light scattering of biological tissues that provides significant enhancement of light penetration into the biological tissues making non‐invasive diagnosis more feasible. In current report Optical Coherence Tomography (OCT) in conjunction with optical clearing is applied for assessment of deep cartilage layers and cartilage‐bone interface. The solution of Iohexol in water has been used as an optical clearing agent. The cartilage‐bone boundary becomes visible after 15 min of optical clearing that enabling non‐invasive estimation of its roughness: S_a = 10 ± 1 µm. The results show that for 0.9 mm thick cartilage optical clearing is stopped after 50 min with an increase of refractive index from 1.386 ± 0.008 to 1.510 ± 0.009. Current approach enables more reliable detection of arthroscopically inaccessible regions, including cartilage‐bone boundary and subchondral bone, and potentially improves accuracy of the osteoarthritis diagnosis.

     

Phenotypic characterization of osteoarthritic osteocytes from the sclerotic zones: a possible pathological role in subchondral bone sclerosis

Subchondral bone sclerosis is a well-recognised manifestation of osteoarthritis (OA). The osteocyte cell network is now considered to be central to the regulation of bone homeostasis; however, it is not known whether the integrity of the osteocyte cell network is altered in OA patients. The aim of this study was to investigate OA osteocyte phenotypic changes and its potential role in OA subchondral bone pathogenesis. The morphological and phenotypic changes of osteocytes in OA samples were investigated by micro-CT, SEM, histology, immunohistochemistry, TRAP staining, apoptosis assay and real-time PCR studies. We demonstrated that in OA subchondral bone, the osteocyte morphology was altered showing rough and rounded cell body with fewer and disorganized dendrites compared with the osteocytes in control samples. OA osteocyte also showed dysregulated expression of osteocyte markers, apoptosis, and degradative enzymes, indicating that the phenotypical changes in OA osteocytes were accompanied with OA subchondral bone remodelling (increased osteoblast and osteoclast activity) and increased bone volume with altered mineral content. Significant alteration of osteocytes identified in OA samples indicates a potential regulatory role of osteocytes in subchondral bone remodelling and mineral metabolism during OA pathogenesis.     

Activating Nrf2 signalling alleviates osteoarthritis development by inhibiting inflammasome activation

Osteoarthritis (OA), which is characterized by proliferation of subchondral bone and the degeneration of articular cartilage, is the most prevalent human arthritis. Nod‐like receptor pyrin domain 3 (NLRP3) inflammasome is a hot spot in recent year and has been reported to be associated with OA synovial inflammation. However, there are few studies on NLRP3 inflammasome in chondrocyte. Licochalcone A (Lico A), a compound extracted from Glycyrrhiza species, has various biological effects such as anti‐inflammation, anti‐apoptotic, anti‐cancer and anti‐oxidation. In this study, we investigated the protective effect of Lico A on chondrocytes stimulated by lipopolysaccharide (LPS) and surgically induced OA models. In vitro, Lico A could reduce the expression of NLRP3, apoptosis‐associated speck‐like protein (ASC), Gasdermin D (GSDMD), caspase‐1, interleukin‐1beta (IL‐1β) and IL‐18, which indicated that Lico A attenuates LPS‐induced chondrocytes pyroptosis. In addition, Lico A ameliorates the degradation of extracellular matrix (ECM) by enhancing the expression of aggrecan and collagen‐II. Meanwhile, we found that Lico A inhibits NLRP3 inflammasome via nuclear factor erythroid‐2‐related factor 2 (Nrf2)/haeme oxygenase‐1(HO‐1)/nuclear factor kappa‐B (NF‐κB) axis. And the Nrf2 small interfering RNA (siRNA) could reverse the anti‐pyroptosis effects of Lico A in mouse OA chondrocytes. In vivo, Lico A mitigates progression OA in a mouse model and reduces OA Research Society International (OARSI) scores. Thus, Lico A may have therapeutic potential in OA.     

Oxymatrine exerts protective effects on osteoarthritis via modulating chondrocyte homoeostasis and suppressing osteoclastogenesis

Osteoarthritis (OA) is a common degenerative disease characterized by the progressive destruction both articular cartilage and the subchondral bone. The agents that can effectively suppress chondrocyte degradation and subchondral bone loss are crucial for the prevention and treatment of OA. Oxymatrine (OMT) is a natural compound with anti‐inflammatory and antitumour properties. We found that OMT exhibited a strong inhibitory effect on LPS‐induced chondrocyte inflammation and catabolism. To further support our results, fresh human cartilage explants were treated with LPS to establish an ex vivo degradation model, and the results revealed that OMT inhibited the catabolic events of LPS‐stimulated human cartilage and substantially attenuated the degradation of articular cartilage ex vivo. As subchondral bone remodelling is involved in OA progression, and osteoclasts are a unique cell type in bone resorption, we investigated the effects of OMT on osteoclastogenesis, and the results demonstrated that OMT suppresses RANKL‐induced osteoclastogenesis by suppressing the RANKL‐induced NFATc1 and c‐fos signalling pathway in vitro. Further, we found that the anti‐inflammatory and anti‐osteoclastic effects of oxymatrine are mediated via the inhibition of the NF‐κB and MAPK pathways. In animal studies, OMT suppressed the ACLT‐induced cartilage degradation, and TUNEL assays further confirmed the protective effect of OMT on chondrocyte apoptosis. MicroCT analysis revealed that OMT had an attenuating effect on ACLT‐induced subchondral bone loss in vivo. Taken together, these results show that OMT interferes with the vicious cycle associated with OA and may be a potential therapeutic agent for abnormal subchondral bone loss and cartilage degradation in osteoarthritis.     

Genome-wide microRNA screening reveals miR-582-5p as a mesenchymal stem cell-specific microRNA in subchondral bone of the human knee joint

Emerging evidence suggests that microRNAs (miRNAs) may be pathologically involved in osteoarthritis (OA). Subchondral bone (SCB) sclerosis is accounted for the knee osteoarthritis (KOA) development and progression. In this study, we aimed to screen the miRNA biomarkers of KOA and investigated whether these miRNAs regulate the differentiation potential of mesenchymal stem cells (MSCs) and thus contributing to SCB. We identified 48 miRNAs in the blood samples in KOA patients (n = 5) through microarray expression profiling detection. After validation with larger sample number, we confirmed hsa-miR-582-5p and hsa-miR-424-5p were associated with the pathology of SCB sclerosis. Target genes prediction and pathway analysis were implemented with online databases, indicating these two candidate miRNAs were closely related to the pathways of pluripotency of stem cells and pathology of OA. Surprisingly, mmu-miR-582-5p (homology of hsa-miR-582-5p) was downregulated in osteogenic differentiation and upregulated in adipogenic differentiation of mesenchymal progenitor C3H10T1/2 cells, whereas mmu-mir-322-5p (homology of hsa-miR-424-5p) showed no change through the in vitro study. Supplementing mmu-miR-582-5p mimics blocked osteogenic and induced adipogenic differentiation of C3H10T1/2 cells, whereas silencing of the endogenous mmu-miR-582-5p enhanced osteogenic and repressed adipogenic differentiation. Further mechanism studies showed that mmu-miR-582-5p was directly targeted to Runx2. Mutation of putative mmu-miR-582-5p binding sites in Runx2 3′ untranslated region (3′UTR) could abolish the response of the 3′UTR-luciferase construct to mmu-miR-582-5p supplementation. Generally speaking, our data suggest that miR-582-5p is an important biomarker of KOA and is able to regulate osteogenic and adipogenic differentiation of MSCs via targeting Runx2. The study also suggests that miR-582-5p may play a crucial role in SCB sclerosis of human KOA.     

Isorhamnetin attenuates osteoarthritis by inhibiting osteoclastogenesis and protecting chondrocytes through modulating reactive oxygen species homeostasis

Increasing evidence indicates that osteoarthritis (OA) is a musculoskeletal disease affecting the whole joint, including both cartilage and subchondral bone. Reactive oxygen species (ROS) have been demonstrated to be one of the important destructive factors during early‐stage OA development. The objective of this study was to investigate isorhamnetin (Iso) treatment on osteoclast formation and chondrocyte protection to attenuate OA by modulating ROS. Receptor activator of nuclear factor‐kappa B ligand (RANKL) was used to establish the osteoclast differentiation model in bone marrow macrophages (BMMs) in vivo. H₂O₂ was used to induce ROS, which could further cause chondrocyte apoptosis. We demonstrated that Iso suppressed RANKL‐induced ROS generation, which could mediate osteoclastogenesis. Moreover, we found that Iso inhibited osteoclast formation and function by suppressing the expression of osteoclastogenesis‐related genes and proteins. We proved that Iso inhibited RANKL‐induced activation of mitogen‐activated protein kinase activation of mitogen‐activated protein kinase (MAPK), nuclear factor‐kappa B (NF‐κB) and AKT signalling pathways in BMMs. In addition, Iso inhibited ROS‐induced chondrocyte apoptosis by regulating apoptosis‐related proteins. Moreover, Iso was administered to an anterior cruciate ligament transection (ACLT)‐induced OA mouse model. The results indicated that Iso exerted beneficial effects on inhibiting excessive osteoclast activity and chondrocyte apoptosis, which further remedied cartilage damage. Overall, our data showed that Iso is an effective candidate for treating OA.     

Subchondral bone response to injected adipose-derived stromal cells for treating osteoarthritis using an experimental rabbit model

Although articular cartilage is the target of osteoarthritis (OA), its deterioration is not always clearly associated with patient symptoms. Because a functional interaction between cartilage and bone is crucial, the pathophysiology of OA and its treatment strategy must focus also on subchondral bone. We investigated whether adipose-derived stromal cells (ASCs) injected into a joint at two different concentrations could prevent subchondral bone damage after the onset of mild OA in a rabbit model. We measured both volumetric and densitometric aspects of bone remodeling. Although OA can stimulate bone remodeling either catabolically or anabolically over time, the accelerated turnover does not allow complete mineralization of new bone and therefore gradually reduces its density. We measured changes in morphometric and densitometric bone parameters using micro-CT analysis and correlated them with the corresponding parameters in cartilage and meniscus. We found that ASCs promoted cartilage repair and helped counteract the accelerated bone turnover that occurs with OA.     

Upregulation of microRNA-27a inhibits synovial angiogenesis and chondrocyte apoptosis in knee osteoarthritis rats through the inhibition of PLK2

The function of microRNA-27a (miR-27a) on synovial angiogenesis and chondrocyte apoptosis in rats with knee osteoarthritis (KOA) by targeting PLK2 is explored in this present study. The rat model of KOA was conducted by anterior cruciate ligament transection. Rats were injected with miR-27a mimics, mimics NC, pcDNA3.1-PLK2 pcDNA3.1, or RLK2 RNAi plasmid via tail vein. A series of assays were used to figure out the functions of miR-27a and PLK2 in synovial angiogenesis and chondrocyte apoptosis in rats with KOA. Furthermore, the putative binding site between miR-27a and PLK2 was determined. Downregulated miR-27a was found in synovial tissues and cartilage tissues of KOA rats. Upregulated miR-27a and downregulated PLK2 inhibited synovial injury and promoted apoptosis of synovial cells, inhibited synovial angiogenesis, inhibited cartilage injury and chondrocyte apoptosis, inhibited cartilage collagen destruction, and alleviates inflammatory injury of synovial tissue and cartilage tissue in KOA rats. Overexpression of PLK2 reverses the effect of upregulation of miR-27a on synovial angiogenesis and chondrocyte injury in KOA rats. Our study suggests that upregulation of miR-27a inhibits synovial angiogenesis and chondrocyte apoptosis in KOA rats through the inhibition of PLK2.     

Wogonoside impedes the progression of acute myeloid leukemia through inhibiting bone marrow angiogenesis

Decreasing bone marrow (BM) microvessel density and circulating angiogenic cytokine levels are promising strategies for the treatment of relapsed and resistant acute myeloid leukemia (AML). Previous studies have reported that wogonoside could inhibit the progression of AML and suppress angiogenesis in a solid tumor, but the correlation of these two effects was ignored. In this research, we determined whether wogonoside could inhibit angiogenesis in this hematologic malignancy. We found that wogonoside could inhibit tumor growth and progression, and prolong the survival of nude mice inoculated with U937/MDR. Besides, reducing BM angiogenesis might cause therapeutic effect against resistant AML. Therefore, coculture between AML cells and BM stromal cells was established to imitate their crosstalk. Then, the effect of wogonoside on BM angiogenesis was tested in vitro and in vivo. We found that wogonoside could suppress microvessel formation in the chicken chorioallantoic membrane assay model and matrigel plug assay. The mechanism research revealed that wogonoside could block the JAK2-STAT3 pathway in AML cells and stromal cells to break their positive feedback. We detected several cytokines related to AML or angiogenesis and found that secreted interleukin-8 was a significant angiogenic cytokine to induce BM angiogenesis. These findings supported that new diagnostics and promising treatment strategies could be developed in relapsed and resistant AML patients.     

Sexual dimorphic impact of adult‐onset somatopause on life span and age‐induced osteoarthritis

Osteoarthritis (OA), the most prevalent joint disease, is a major cause of disability worldwide. Growth hormone (GH) has been suggested to play significant roles in maintaining articular chondrocyte function and ultimately articular cartilage (AC) homeostasis. In humans, the age‐associated decline in GH levels was hypothesized to play a role in the etiology of OA. We studied the impact of adult‐onset isolated GH deficiency (AOiGHD) on the life span and skeletal integrity including the AC, in 23‐ to 30‐month‐old male and female mice on C57/BL6 genetic background. Reductions in GH during adulthood were associated with extended life span and reductions in body temperature in female mice only. However, end‐of‐life pathology revealed high levels of lymphomas in both sexes, independent of GH status. Skeletal characterization revealed increases in OA severity in AOiGHD mice, evidenced by AC degradation in both femur and tibia, and significantly increased osteophyte formation in AOiGHD females. AOiGHD males showed significant increases in the thickness of the synovial lining cell layer that was associated with increased markers of inflammation (IL‐6, iNOS). Furthermore, male AOiGHD showed significant increases in matrix metalloproteinase‐13 (MMP‐13), p16, and β‐galactosidase immunoreactivity in the AC as compared to controls, indicating increased cell senescence. In conclusion, while the life span of AOiGHD females increased, their health span was compromised by high‐grade lymphomas and the development of severe OA. In contrast, AOiGHD males, which did not show extended life span, showed an overall low grade of lymphomas but exhibited significantly decreased health span, evidenced by increased OA severity.     

Pathological mechanism of chondrocytes and the surrounding environment during osteoarthritis of temporomandibular joint

Temporomandibular joint (TMJ) osteoarthritis is a common chronic degenerative disease of the TMJ. In order to explore its aetiology and pathological mechanism, many animal models and cell models have been constructed to simulate the pathological process of TMJ osteoarthritis. The main pathological features of TMJ osteoarthritis include chondrocyte death, extracellular matrix (ECM) degradation and subchondral bone remodelling. Chondrocyte apoptosis accelerates the destruction of cartilage. However, autophagy has a protective effect on condylar chondrocytes. Degradation of ECM not only changes the properties of cartilage but also affects the phenotype of chondrocytes. The loss of subchondral bone in the early stages of TMJ osteoarthritis plays an aetiological role in the onset of osteoarthritis. In recent years, increasing evidence has suggested that chondrocyte hypertrophy and endochondral angiogenesis promote TMJ osteoarthritis. Hypertrophic chondrocytes secrete many factors that promote cartilage degeneration. These chondrocytes can further differentiate into osteoblasts and osteocytes and accelerate cartilage ossification. Intrachondral angiogenesis and neoneurogenesis are considered to be important triggers of arthralgia in TMJ osteoarthritis. Many molecular signalling pathways in endochondral osteogenesis are responsible for TMJ osteoarthritis. These latest discoveries in TMJ osteoarthritis have further enhanced the understanding of this disease and contributed to the development of molecular therapies. This paper summarizes recent cognition on the pathogenesis of TMJ osteoarthritis, focusing on the role of chondrocyte hypertrophy degeneration and cartilage angiogenesis.