Increase and regulation of synovial calcitonin gene-related peptide expression in patients with painful knee osteoarthritis

Abstract

Background: Recent studies suggest that the vasodilatory neuropeptide calcitonin gene-related peptide (CGRP) is localized in the synovial tissue and may be involved in the pathology of hip and knee osteoarthritis (OA). However, the regulation and relationship between pain and CGRP expression levels in the synovial tissue of human OA patients are not fully understood.

Methods: Synovial tissues were harvested from 74 participants with radiographic knee OA (unilateral Kellgren/Lawrence grades 3-4) during total knee arthroplasty. CGRP-expressing cells in the resected tissue were identified by immunohistochemical analyses. To examine CGRP expression levels, CD14-positive (CD14+) (macrophage-rich cell fraction) and CD14-negative (CD14-; fibroblast-rich cell fraction) cells were isolated from the synovial tissue. To investigate the involvement of prostaglandin E2 (PGE2) in the regulation of CGRP expression, cultured CD14- and CD14+ cells were stimulated with PGE2. In addition, CGRP expression levels in the synovial tissue of OA patients with strong/severe (visual analog scale [VAS]≥6) and mild/moderate pain (VAS<6) were compared.

Results: CGRP-positive cells were detected in the intimal lining layer and comprised both CD14- and CD14+ cells. CGRP expression in non-cultured CD14- fractions was significantly higher than that in CD14+ fractions. The expression levels of CGRP were significantly increased in cultured CD14- cell fractions treated with exogenous PGE2, compared to untreated CD14- cell fractions. In contrast, treatment with PGE2 did not increase CGRP regardless of whether or not CD14+ cells expressed CGRP. Furthermore, CGRP expression in the VAS≥6 group was also significantly higher than that in the VAS<6 group.

Conclusion: These findings suggest that CGRP expression in the synovial fibroblasts is regulated by the COX-2/PGE2 pathway and that elevation of synovial CGRP levels may contribute to OA pain.

Keywords: calcitonin gene-related peptide; knee osteoarthritis; pain; regulation; synovium.

Figure 1

CGRP immunolocalization and mRNA expression in synovial tissue. Notes: Synovial tissue stained with (A) DAPI (nuclei), (B) CD14 or (C) CGRP. (D) Merged image. (E) High-magnification merged image of (D). Scale bars =100 μm. (F) Real-time PCR analysis of CGRP expression in non-cultured CD14− and CD14+ fractions. All data are presented as mean ± standard error (n=5). *p<0.05. Abbreviations: CGRP, calcitonin gene-related peptide; DAPI, 4′,6-diamidino-2-phenylindole; PCR, polymerase chain reaction.

Figure 2

Effects of PGE2 on CGRP expression in cultured synovial fibroblasts and macrophages. Notes: Synovial fibroblasts and macrophages were stimulated in vitro with PGE2 (10 μM) or without PGE2 (vehicle) for 24 h prior to the extraction of total RNA for (A) CD14 and (B) CGRP expression analysis. All data are presented as mean ± standard error (n=5). *p<0.05. Abbreviations: PGE2, prostaglandin E2; CGRP, calcitonin gene-related peptide.

Figure 3

CGRP expression in OA patients with strong/severe (VAS≥6) and mild/moderate pain (VAS<6). Notes: Real-time PCR analysis of CGRP expression in synovial tissue of OA patients. Box and Whisker plot for CGRP expression in synovial tissue of OA patients with strong/severe (VAS≥6; n=44) and mild/moderate pain (VAS<6; n=30). The plot shows median (bold horizontal line), IQR (box) and range, with outliers plotted beyond 1.5 times the IQR from the box. *p<0.05. Abbreviations: CGRP, calcitonin gene-related peptide; OA, osteoarthritis; VAS, visual analog scale; PCR, polymerase chain reaction; IQR, interquartile range.