Gene expression pattern of cells from inflamed and normal areas of osteoarthritis synovial membrane

Abstract

Objective: To compare the gene expression patterns of synovial cells from inflamed or normal/reactive areas of synovial membrane obtained from the same patient with osteoarthritis (OA).

Methods: At the time of total knee replacement, synovial tissues were obtained from 12 patients with knee OA. The inflammation status of the synovial membrane was characterized according to macroscopic criteria and classified as normal/reactive or inflamed. Biopsy samples were cultured separately for 7 days. Microarray gene expression profiling was performed on normal/reactive and inflamed areas. Western blot and immunohistochemistry were used to confirm the identified genes that were differentially expressed.

Results: We identified 896 genes that were differentially expressed between normal/reactive and inflamed areas. The key pathways were related to inflammation, cartilage metabolism, Wnt signaling, and angiogenesis. In the inflammation network, the genes TREM1 and S100A9 were strongly up-regulated. The genes MMP3, MMP9, CTSH (cathepsin H), and CTSS (cathepsin S) were significantly up-regulated in the cartilage catabolism pathway, while the most up-regulated anabolism enzyme gene was HAS1. In the Wnt signaling pathway, the genes for Wnt-5a and low-density lipoprotein receptor-related protein 5 were up-regulated, while the gene FZD2 and the gene for Dkk-3 were down-regulated. Finally, STC1, which codes for a protein involved in angiogenesis, was identified as the most up-regulated gene in inflamed compared with normal/reactive areas.

Conclusion: This study is the first to identify different expression patterns between 2 areas of the synovial membrane from the same patient. These differences concern several key pathways involved in OA pathogenesis. This analysis also provides information regarding new genes and proteins as potential targets of treatment.

Figure 1

Increase of triggering receptor expressed on myeloid cells 1 (TREM-1), S100A9, stanniocalcin 1 (STC1), and Wnt-5a protein production in inflamed (I) areas compared with normal/reactive (N/R) areas. Total protein extracts from normal/reactive or inflamed areas were analyzed by Western blotting with anti–TREM-1 (30/26 kd), anti-S100A9 (14 kd), anti-STC1 (31 kd), anti–Wnt-5a (42 kd), and anti–α-tubulin (55 kd; control) antibodies. Representative images are shown (independent protein extracts from 3 different patients).

Figure 2

Immunohistochemical detection of S100A9 in normal/reactive (A and C) and inflamed (B and D) synovial biopsy samples. Also shown are negative controls with mouse IgG1 in normal/reactive (E) and inflamed (F) synovial biopsy samples. In inflamed synovial biopsy samples, staining for S100A9 is observed in perivascular and sublining cells. At the time of surgery, synovial biopsy samples from normal/reactive or inflamed areas were macroscopically selected as described in Patients and Methods. Normal/reactive and inflamed synovial biopsy samples were stained with anti-S100A9 antibody. Representative images are shown. Arrows indicate intima lining; asterisks indicate blood vessels. Original magnification × 5 in A and B; × 10 in C–F.

Figure 3

Immunohistochemical detection of hyaluronan synthase 1 (HAS-1) in normal/reactive (A and C) and inflamed (B and D) synovial biopsy samples. Also shown are negative controls with normal goat IgG in normal/reactive (E) and inflamed (F) synovial biopsy samples. In inflamed synovial biopsy samples, there is an increase in HAS-1–positive cells with predominant localization in the intima lining. At the time of surgery, synovial biopsy samples from normal/reactive or inflamed areas were macroscopically selected as described in Patients and Methods. Normal/reactive and inflamed synovial biopsy samples were stained with anti–HAS-1 antibody. Representative images are shown. Arrows indicate intima lining. Original magnification × 5 in A and B; × 10 in C–F.

Figure 4

Immunohistochemical detection of stanniocalcin 1 (STC1) in normal/reactive (A and C) and inflamed (B and D) synovial biopsy samples. Also shown are negative controls with normal goat IgG in normal/reactive (E) and inflamed (F) synovial biopsy samples. In inflamed synovial biopsy samples, staining for STC1 is observed in perivascular and sublining cells. At the time of surgery, synovial biopsy samples from normal/reactive or inflamed areas were macroscopically selected as described in Patients and Methods. Normal/reactive and inflamed synovial biopsy samples were stained with anti-STC1 antibody. Representative images are shown. Arrows indicate intima lining; asterisks indicate blood vessels. Original magnification × 5 in A and B; × 10 in C–F.