Screening of a Panel of Low Molecular Weight Compounds That Inhibit Synovial Fibroblast Invasion in Rheumatoid Arthritis

Abstract

Increased invasion of synovial fibroblasts and their involvement in cartilage damage are characteristic phenotypes of rheumatoid arthritis (RA). To identify low molecular weight compounds that suppress synovial fibroblast invasion, a panel of inhibitors (n = 330) was initially screened using a real-time cell analysis system for human synovial fibroblasts that were enzymatically isolated from surgical samples of RA patients. To evaluate the effects of the inhibitors identified in the screen, synovial fibroblast migration was measured using a wound-healing assay, and phosphorylation of intracellular signaling molecules was determined by immunoblots. Several candidate inhibitors were identified in the screen, including inhibitors against platelet-derived growth factor receptor (PDGFR), Akt, PI3K, and glycogen kinase synthetase 3 (GSK-3). These inhibitors strongly suppressed synovial fibroblast migration after 72 h and downregulated phosphorylation of Akt (Ser473) at 48 h. When the inhibitors were removed from the culture conditions, both migration and phosphorylated Akt (Ser473) levels were restored. Furthermore, all the categories of inhibitors except for PDGFR inhibitor IV decreased cell proliferation as well as IL-6 production in synovial fibroblasts. Interestingly, GSK-3 inhibitors increased anti-inflammatory cytokine IL-10 production but suppressed IL-23 production from LPS-primed macrophages obtained from healthy donors. In conclusion, blocking PDGFR, PI3K, or GSK-3 could have therapeutic value as an RA treatment that targets the invasion/migration of synovial fibroblasts.

Synovial fibroblasts as potential drug targets in rheumatoid arthritis, where do we stand and where shall we go?

“…In an RA-FLS-cartilage implantation model, where the sponge-cartilage was implanted with fibroblasts to SCID (severe combined immunodeficient) mice subcutaneously and a cell-free cartilage-sponge complex contralaterally, the cartilage destruction could not only be observed in the coimplantation (ipsilateral) site but also on the synovial fibroblast-free contralateral area, indicating that the RA-FLS were able to migrate actively from one site to the other 46. The phoyphorylation rate of the receptor tyrosine kinase PDGFR has been observed to be elevated in the rheumatoid synovium and PDGFR inhibitors were found to dose-dependently inhibit the in vitro migration of human FLS 47. Moreover, the inhibition of PI3Kδ (by INK007) could decrease PDGF-induced migration of RA-FLS (both when investigated in the wound-healing assay or in the Boyden chamber), while invasion of the cells was also affected by the presence of the PI3K inhibitors INK007 and CAL-101 48.…”

“… 46 The phoyphorylation rate of the receptor tyrosine kinase PDGFR has been observed to be elevated in the rheumatoid synovium and PDGFR inhibitors were found to dose-dependently inhibit the in vitro migration of human FLS. 47 Moreover, the inhibition of PI3Kδ (by INK007) could decrease PDGF-induced migration of RA-FLS (both when investigated in the wound-healing assay or in the Boyden chamber), while invasion of the cells was also affected by the presence of the PI3K inhibitors INK007 and CAL-101. 48 Paclitaxel, a known anticancer drug was shown to inhibit the in vitro migration of RA-FLS and could reduce the severity of joint inflammation in mice in the collagen-induced arthritis model.…”

“…However, the paclitaxel-induced decrease of the production of inflammatory mediators by RA-FLS can also contribute to the in vivo phenotype, while the inhibitor itself has a considerable toxicity, which requires further investigation 49. Meanwhile, there are more and more data on the altered metabolism of RA-FLS (see above) and its effects on the aggressive behaviour of these cells, which is nicely highlighted with the massive inhibitory effect of glycogen synthase kinase 3 (GSK-3) inhibitors on the migratory capacity of RA-FLS 47. In a recent paper, Orange and colleagues found elevated levels of a special mesenchymal cell type with shared features of inflammatory synovial fibroblasts, namely the CD45/CD31/Podoplanin triple positive preinflammatory mesenchymal (PRIME) cells in the blood by transcriptional analysis just before RA flares (and after B-cell activation in the circulation) 50.…”

“… 49 Meanwhile, there are more and more data on the altered metabolism of RA-FLS (see above) and its effects on the aggressive behaviour of these cells, which is nicely highlighted with the massive inhibitory effect of glycogen synthase kinase 3 (GSK-3) inhibitors on the migratory capacity of RA-FLS. 47 In a recent paper, Orange and colleagues found elevated levels of a special mesenchymal cell type with shared features of inflammatory synovial fibroblasts, namely the CD45/CD31/Podoplanin triple positive preinflammatory mesenchymal (PRIME) cells in the blood by transcriptional analysis just before RA flares (and after B-cell activation in the circulation). 50 These PRIME cells have been proposed to migrate to the affected joints and participate in the initiation of the inflammatory process.…”

“…A real-time cell analysis system mainly based on a wound-healing assay and intracellular phosphorylation of human FLS pointed towards the effectiveness of the inhibitors of PDGFR, Akt, PI3K or GSK-3 in controlling the invasive capacity of RA-FLS. 47 However, more detailed studies are needed to test these inhibitors in the control of experimental and human autoimmune arthritis.…”