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

Abstract

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.

Keywords: osteoarthritis; pro-coagulation; proteomic analysis; subchondral bone.

Figure 1

Mass spectrometry (MS) results of clinical samples. (A) Volcano plot of differentially expressed proteins in severely damaged subchondral bone (AS) and less damaged subchondral bone (NS) patients with osteoarthritis (OA). Red and blue represent significantly up- and downregulated proteins in AS compared to NS samples, respectively. (B) Pearson’s correlation coefficient heatmap of quantitative proteins between all samples. AAS, BAS, CAS, and DAS in the image represent four severely damaged subchondral bones in the AS group, and ANS, BNS, CNS, and DNS in the image represent four less damaged subchondral bones in the NS group. (C) RSD boxplot of quantitative proteins in repeated samples. (D) The Principal Component Analysis scatter plot of quantitative proteins between repeated samples, a complete separation of severely damaged subchondral bone (AS) and less damaged subchondral bone (NS) groups was observed. (E) The distribution of differentially expressed proteins identified in OA knee subchondral bone according to subcellular location.

Figure 2

Classification of differentially expressed proteins in severely damaged subchondral bone and less damaged subchondral bone based on Gene Ontology and Clusters of Orthologous Groups. (A) Gene Ontology annotation of differentially expressed proteins. The identified differentially expressed proteins are presented according to their categories: cellular component, biological process, and molecular function. (B) The Clusters of Orthologous Groups of proteins (COG) functionally classify differentially expressed proteins of subchondral bone.

Figure 3

Enrichment results of differentially expressed proteins in severely damaged subchondral bone and less damaged subchondral bone. (A) GO enrichment of differentially expressed proteins is divided into three categories: biological process, cellular component, and molecular function. The significantly upregulated (upper the X axis) and downregulated (below the X axis) GO terms are presented. (B) The enrichment bubble map of differentially expressed proteins in the biological process. (C) The enrichment bubble map of differentially expressed proteins in the KEGG pathway. (D) The enrichment bubble map of differentially expressed proteins in the protein domain levels. (E) The enrichment bubble map of differentially expressed proteins in a cellular component.

Figure 4

Cluster analysis and interaction network of differentially expressed proteins in severely damaged subchondral bone and less damaged subchondral bone. (A) Clustering heat map based on KEGG pathway enrichment. Red and blue represent higher and lower enrichment folds, respectively. AAS, BAS, CAS, and DAS represent four sclerotic subchondral bones in the AS group. ANS, BNS, CNS, and DNS represent four less damaged subchondral bones in the NS group. (B) Protein–protein interaction network analysis of differentially expressed proteins. The top two networks are circled. Dashed circles represent the main pathways in which differentially expressed proteins were involved. The size of circles represents the number of interacting proteins: the bigger the circle, the more proteins it interacts with, indicating the importance of that protein in the network.

Figure 5

Verification of proteomics results in clinical specimens and OVX-OA rats by using immunohistochemistry. (A) Representative and semiquantitative SerpinA5 expression in the less damaged subchondral bone (NS) and severely damaged subchondral bone (AS) groups. Panels A3 and A4 are higher resolution images of boxed areas in panels A1 and A2, respectively. Scale bar = 100 μm in A1 and A2 and scale bar = 50 μm in A3 and A4. (B) The semiquantitative results of SerpinA5 expression in NS and AS group. The IOD means integrated optical density. (C) Representative expression of SerpinA5 in the SHAM and OVX-OA groups. Panels C3 and C4 are higher resolution images of boxed areas in C1 and C2, respectively. Scale bar = 100 μm in C1 and C2, scale bar = 50 μm in C3 and C4. (D) The semiquantitative results of SerpinA5 expression in SHAM and OVX-OA groups.