Proteomics Analysis of Knee Subchondral Bone Identifies Differentially Expressed Proteins Associated with Osteoarthritis.

Osteoarthritis (OA) is a prevalent debilitating whole-joint disorder. Currently, a growing number of proteomic studies have been performed to evaluate molecular biomarkers in several tissues from OA patients; however, little is known about the protein profiles in subchondral bone of OA. In this study, proteomic analysis was performed on subchondral bone from patients with OA to identify differentially expressed proteins (DEPs). Bioinformatics tools were used to further investigate these DEPs. Thereafter, DEPs were validated in the samples from patients with OA, as well as in bilateral ovariectomy-induced OA (OVX-OA) rats using immunohistochemistry. A comprehensive subchondral bone proteome profile of patients with OA was constructed. Additionally, biological information analysis showed that a majority of DEPs participated in the dysregulation of the complement and coagulation cascades. The validation experiments suggested that SerpinA5, the protein involved in the complement and coagulation cascades, was significantly increased in severely damaged subchondral bone of patients with OA compared to the control group. Furthermore, the increase of SerpinA5 in OVX-OA rats compared to control rats was also confirmed. Our results indicated that the dysregulation of coagulation and complement pathways plays a role in the progression of OA, and it provides a promising therapeutic target of OA.

Serum periostin level is not sufficient to serve as a clinically applicable biomarker of osteoarthritis.

BACKGROUND: Emerging knowledge has highlighted the role of periostin (POSTN) in osteoarthritis (OA) process; however, whether POSTN is suitable as a biomarker of OA remains unclear. This study aimed to investigate the potential value of POSTN as a biomarker of OA. METHODS: Ten 6-month-old female Sprague-Dawley (SD) rats were used in this study. Five rats underwent ovariectomy (OVX) operation and the others were carried out sham operation. Thirty-two patients with OA and eighteen patients who had meniscus injuries or ligament injuries but with intact articular cartilages were recruited in this study from January to July 2019 at the Peking University International Hospital. We first detected the expression of POSTN in the cartilage of OVX induced OA rats and different compartments of the knee joint in patients with OA using immunohistochemistry. Besides, serum POSTN levels in patients with or without OA were examined using enzyme-linked immunosorbent assay (ELISA). The associations among serum POSTN levels, clinical symptoms, and radiological severity were assessed according to the Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) scores and, Kellgren-Lawrence (KL) grading, respectively. Finally, multivariable cumulative link models were established to evaluate the validity of serum POSTN level as a predictor of knee OA. RESULTS: The significantly higher POSTN expression was found in OVX-OA rats than Sham rats, while, the expression of POSTN was significantly higher in the torn cartilage of patients with OA. However, the serum POSTN level did not differ significantly between patients with and without OA. Additionally, we found no remarkable associations between serum POSTN level and WOMAC scores and KL grading. Subsequent analysis revealed that serum POSTN was not a significant predictor of OA. CONCLUSION: Thus, although POSTN may be involved OA process and local POSTN is valuable in disease diagnosis and distinguishing of the severity of disease, its serum level is not sufficient to serve as a candidate biomarker of OA given the current analysis technology.

Cellular alterations and crosstalk in the osteochondral joint in osteoarthritis and promising therapeutic strategies.

Osteoarthritis (OA) is a joint disorder involving cartilage degeneration and subchondral bone sclerosis. The bone-cartilage interface is implicated in OA pathogenesis due to its susceptibility to mechanical and biological factors. The crosstalk between cartilage and the underlying subchondral bone is elevated in OA due to multiple factors, such as increased vascularization, porosity, microcracks and fissures. Changes in the osteochondral joint are traceable to alterations in chondrocytes and bone cells (osteoblasts, osteocytes and osteoclasts). The phenotypes of these cells can change with the progression of OA. Aberrant intercellular communications among bone cell-bone cell and bone cell-chondrocyte are of great importance and might be the factors promoting OA development. An appreciation of cellular phenotypic changes in OA and the mechanisms by which these cells communicate would be expected to lead to the development of targeted drugs with fewer side effects.

Hypoxia-induced Wnt/ß-catenin signaling activation in subchondral bone osteoblasts leads to an osteoarthritis-like phenotype of chondrocytes in articular cartilage.

Background: Osteoarthritis (OA) is a whole-joint disease and characterized by alterations in the articular cartilage, subchondral bone, ligaments, and synovial membrane. The crosstalk between cartilage and subchondral bone plays a crucial role in the pathogenesis and progression of OA. Hypoxia has been reported to play an important role in cartilage degradation and subchondral bone remodeling in OA. In this study, we aimed to identify the involvement of hypoxia in modifying the osteoblast phenotypes and determine whether these alterations could influence the metabolism of chondrocytes. Methods: First, the levels of Hif-1α in subchondral bone of different compartments in patients with OA were assessed using immunohistochemistry (IHC). In in vitro, human primary osteoblasts were cultured under hypoxic and normoxic conditions, and the hypoxic or normoxic conditioned media (HCM and NCM) were used to culture human primary chondrocytes. Then, phenotypic changes in osteoblasts were assessed using reverse transcription-polymerase chain reaction (RT-PCR), Western blotting, and enzyme-linked immunosorbent assay (ELISA). Furthermore, the expression of type II collagen (COL2A1), aggrecan (ACAN), SRY-related high-mobility group-box gene 9 (SOX9), matrix metalloproteinase 13 (MMP13), and matrix metalloproteinase 3 (MMP3) in chondrocytes was measured using RT-PCR. Finally, the serum levels of Wnt-related proteins were determined using ELISA. Results: Hif-1α was significantly increased in severely sclerotic subchondral bone compared to less damaged subchondral bone. ß-Catenin and SOST were identified as upregulated and downregulated in hypoxic osteoblasts, respectively. The hypoxia-induced results were confirmed by ELISA. Stimulating human primary chondrocytes with HCM significantly induced MMP13 and MMP3 and inhibited COL2A1, ACAN, and SOX9 mRNA expression. The serum levels of DKK-1 were significantly increased in human OA. Conclusion: Together, these findings revealed that hypoxia in subchondral bone is a key factor in the crosstalk between chondrocytes and osteoblasts and facilitates the shift of chondrocytes toward an OA-like phenotype probably by activating the Wnt/ß-catenin signaling pathway in osteoblasts.

Proteomic analysis of human articular cartilage unravels the dyscoagulation in osteoarthritis and the potential value of serpinA5 as a biomarker for osteoarthritis.

PURPOSE: Nowadays, there is no clinically applicable biomarker for osteoarthritis (OA). Therefore, the aim of the study is to discover a potential biomarker for OA. EXPERIMENTAL DESIGN: We performed a proteomics of eight cartilage samples (four damaged cartilage and four macroscopically intact cartilage) from four OA patients without any comorbidities to search for valuable OA biomarkers. Four rats underwent bilateral ovariectomy to induce the OA (OVX-OA) model, while another four underwent a sham procedure wherein the ovaries were exteriorized but not removed (SHAM). Selected candidate proteins were further verified in the patients and the OVX-OA animal model. RESULTS: A comprehensive cartilage proteome profile of patients with OA was constructed. Additionally, the complement and coagulation cascades were found to be significantly altered, and serpinA5 was chosen as a protein of interest based on biological information analysis. The reduction of serpinA5 in locally damaged cartilage and serum of patients with OA compared to the control group was determined. Furthermore, we found that serpinA5 was decreased in OVX-OA rats compared to that in SHAM rats. CONCLUSIONS AND CLINICAL RELEVANCE: Our results suggest that there is dyscoagulation in the OA process and that serpinA5 can serve as a potentially valuable OA biomarker.

Distinct pathological changes of osteochondral units in early OVX-OA involving TGF-ß signaling.

Introduction: Different opinions exist about the role of subchondral bone in osteoarthritis (OA), probably because subchondral bone has different effects on cartilage degeneration in OA induced by different pathologies. Animal studies to illustrate the role of subchondral bone in cartilage degeneration were mostly based on post-traumatic OA (PT-OA). Postmenopausal women experience a much higher occurrence of OA than similar-aged men. The physiological changes and pathogenesis of the osteochondral unit in ovariectomy-induced OA (OVX-OA) might be distinct from other types of OA. Methods: The osteochondral alterations of post-traumatic OA (PT-OA) and OVX-OA at week 9 after surgery were compared. Then the alterations of osteochondral units in OVX-OA rats were tracked over time for the designed groups: Sham, OVX and OVX rats treated with estrogen (OVX+E). DXA, micro-CT, and histochemical staining were performed to observe alterations in osteochondral units. Results: Rapid cartilage degeneration and increased bone formation were observed in PT-OA, while only mild cartilage erosion and significant bone loss were observed in OVX-OA at week 9 after surgery. Subchondral bone degradation preceded cartilage degeneration by 6 weeks in OVX-OA. TGF-ß expression was downregulated in the osteochondral unit of OVX rats. Estrogen supplementation inhibited subchondral bone loss, cartilage degradation and TGF-ß expression decrease. Discussion: This research demonstrated the distinct behaviors of the osteochondral unit and the critical role of subchondral bone in early OVX-OA compared with PT-OA. Inhibiting subchondral bone catabolism at the early stage of OVX-OA could be an effective treatment for post-menopausal OA. Based on the results, estrogen supplementation and TGF-ß modulation at the early stage are both potential therapies for post-menopausal OA.

Potential Value of Matrix Metalloproteinase-13 as a Biomarker for Osteoarthritis.

Background: Emerging knowledge has highlighted the role of matrix metalloproteinase (MMP)-13 in osteoarthritis (OA); however, the suitability of MMP-13 as a biomarker for OA remains unclear. Therefore, this study aimed to assess the potential value of MMP-13 as a biomarker for OA. Methods: The study enrolled 51 patients, of which 33 had advanced varus OA and 18 did not have OA. Immunohistochemistry and western blotting analyses were performed to measure MMP-13 activity in the cartilage and subchondral bone of patients with OA. Enzyme-linked immunosorbent assay was used to measure serum MMP-13 levels in patients with or without OA. The Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) was used to assess the association between serum MMP-13 levels and clinical symptoms. Furthermore, the association between serum MMP-13 levels and radiological severity of OA was evaluated using the Kellgren-Lawrence (KL) grading system. Finally, we built the proportional odds logistic regression models to evaluate serum MMP-13 levels as a potential predictor for OA. Results: MMP-13 levels were significantly higher in the severe-worn cartilage of the medial tibial plateau than in the relatively intact portion of the lateral cartilage (p < 0.05). This was contrary to the findings for MMP-13 differential expression in the subchondral bone in knee OA (p < 0.05). Patients with OA had significantly higher serum MMP-13 levels compared with patients without OA. Additionally, remarkable associations among serum MMP-13 levels, WOMAC scores, and KL grading scores were found in the end-stage OA. Furthermore, the subsequent analysis suggested that serum MMP-13 level was a significant predictor for OA. Conclusion: MMP-13 is valuable for diagnosing, measuring disease severity, and predicting OA in the advanced period of the disease, suggesting that it has potential possibility as a biomarker for OA. However, the underlying mechanisms and clinical application of MMP-13 as a biomarker for OA require to be further investigated.

Phenotype changes of subchondral plate osteoblasts based on a rat model of ovariectomy-induced osteoarthritis.

BACKGROUND: Osteoarthritis (OA) is prevalent in postmenopausal women. Subchondral bone in ovariectomized (OVX) rats might play a more important role in cartilage degeneration compared with other types of OA. How subchondral osteoblast changes in OVX rats is still unclear. Understanding of osteoblast changes obtained from OVX subchondral bone might be helpful to clarify pathogenesis of OVX-OA. METHODS: Female Sprague-Dawley rats were randomly divided into two groups: Sham (n=20) and OVX (n=20). Serum levels of Alkaline phosphatase (ALP) and C-telopeptide of type I collagen (CTX-I) were measured every one or two weeks. All rats were executed at week 9 post surgery. The weight of rats and the wet weight of uterus were assessed. Micro-computed Tomography (micro-CT) was used to analyze the knee microstructure, and toluidine blue staining was employed to evaluate cartilage erosion. Subchondral osteoblast proliferation ability by cell counting kit-8 assay, osteogenic genes expressions by reverse transcription polymerase chain reaction (RT-PCR), differentiation and mineralization ability by ALP staining and alizarin red staining were evaluated and compared between Sham and OVX. RESULTS: Ovariectomy induced significant increases of serum ALP and CTX-I as early as at week 2. At week 9 after surgery, the body weight of OVX rats was significantly increased, and uterus weight of OVX rats was remarkably decreased. OVX rats demonstrated significant subchondral bone change and cartilage erosion compared with Sham rats. mRNA levels of early markers of osteogenic differentiation (ALP, type I collagen, Runx2) were enhanced in OVX rats, but the late marker (osteocalcin) was not significantly different. ALP activity of osteoblasts increased, but the mineralization capacity decreased in OVX rats. CONCLUSIONS: Subchondral osteoblasts in OVX rats exhibited different proliferation, differentiation and mineralization abilities from normal counterparts.

Identification of Candidate Genetic Markers and a Novel 4-genes Diagnostic Model in Osteoarthritis through Integrating Multiple Microarray Data.

BACKGROUND: Osteoarthritis (OA) is a joint disease that leads to a high disability rate and a low quality of life. With the development of modern molecular biology techniques, some key genes and diagnostic markers have been reported. However, the etiology and pathogenesis of OA are still unknown. OBJECTIVE: To develop a gene signature in OA. METHOD: In this study, five microarray data sets were integrated to conduct a comprehensive network and pathway analysis of the biological functions of OA related genes, which can provide valuable information and further explore the etiology and pathogenesis of OA. RESULTS AND DISCUSSION: Differential expression analysis identified 180 genes with significantly expressed expression in OA. Functional enrichment analysis showed that the up-regulated genes were associated with rheumatoid arthritis (p < 0.01). Down-regulated genes regulate the biological processes of negative regulation of kinase activity and some signaling pathways such as MAPK signaling pathway (p < 0.001) and IL-17 signaling pathway (p < 0.001). In addition, the OA specific protein-protein interaction (PPI) network was constructed based on the differentially expressed genes. The analysis of network topological attributes showed that differentially upregulated VEGFA, MYC, ATF3 and JUN genes were hub genes of the network, which may influence the occurrence and development of OA through regulating cell cycle or apoptosis, and were potential biomarkers of OA. Finally, the support vector machine (SVM) method was used to establish the diagnosis model of OA, which not only had excellent predictive power in internal and external data sets (AUC > 0.9), but also had high predictive performance in different chip platforms (AUC > 0.9) and also had effective ability in blood samples (AUC > 0.8). CONCLUSION: The 4-genes diagnostic model may be of great help to the early diagnosis and prediction of OA.