CP-25 exerts anti-angiogenic effects on a rat model of adjuvant-induced arthritis by promoting GRK2-induced downregulation of CXCR4-ERK1/2 signaling in endothelial cells

Abstract

Angiogenesis can produce an invasive and destructive front, also named a pannus, comprised of inflammatory vascular tissue that covers and erodes articular cartilage, subchondral bone and peri-articular soft tissues in rheumatoid arthritis (RA). Paeoniflorin-6′-O-benzene sulfonate (CP-25) is a novel ester derivative of paeoniflorin. We previously demonstrated that CP-25 exerts anti-inflammatory and immunoregulatory effects. CP-25 also exhibits a marked therapeutic effect on adjuvant-induced arthritis (AA), and is able to inhibit synovial and immune cell function, according to our previous study. However, the effect of CP-25 on angiogenesis remains unclear. In the present study, AA was initiated in Sprague-Dawley rats via intradermal immunization in the right hind metatarsal footpad with heat-killed Mycobacterium butyricum in liquid paraffin, and rats were divided into four groups: Normal, AA rat model, CP-25 (50 mg/kg) and methotrexate (0.5 mg/kg) groups (n=10 rats/group). Subsequently, joint synovium in AA rats was pathologically evaluated by hematoxylin and eosin staining, synovial vascular proliferation was evaluated by immunofluorescence, the synovial expression levels of C-X-C motif chemokine ligand 12 (CXCL12) were detected by immunohistochemistry and ELISA, and synovial C-X-C chemokine receptor type 4 (CXCR4) was detected by western blotting. The results demonstrated that CP-25 ameliorated clinical signs and pannus formation in the ankle joint in rats with AA. Furthermore, there was a positive correlation between pannus score and CXCL12 and CXCR4 expression. In addition, the effects of CP-25 on endothelial cell function and CXCL12/CXCR4 signaling were studied in vitro using human umbilical vein endothelial cells (HUVECs). The results demonstrated that CXCL12 significantly promoted HUVEC proliferation, migration and tube formation, and that CP-25 could reverse these abnormalities by inhibiting plasma membrane localization of G protein-coupled receptor kinase 2 (GRK2) in HUVECs. These findings suggested that CP-25 may markedly inhibit pannus formation in AA. This effect may be associated with a reduction in the plasma membrane localization of GRK2 in endothelial cells, an enhancement of the inhibitory effect of GRK2 on ERK1/2 in the cytoplasm, a reduction in the phosphorylation of ERK1/2 and in the function of HUVECs.