Inhibition of angiogenesis by interleukin‐4 gene therapy in rat adjuvant‐induced arthritis

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Abstract
Objective: Interleukin‐4 (IL‐4) can modulate neovascularization. In this study, we used a gene therapy approach to investigate the role of IL‐4 in angiogenesis in rat adjuvant‐induced arthritis (AIA), a model for rheumatoid arthritis.Methods: Rats received an adenovirus producing IL‐4 (AxCAIL‐4), a control virus without insert, or control vehicle (phosphate buffered saline) intraarticularly before arthritis onset. At peak onset of arthritis, rats were killed. Vascularization was determined in the synovial tissue, and correlations with inflammation were assessed. Ankle homogenates were used in angiogenesis assays in vitro and in vivo, and protein levels of cytokines and growth factors were assessed by enzyme‐linked immunosorbent assay. Synovial tissue expression of αv integrins was determined by immunohistochemistry.Results: IL‐4 induced a reduction in synovial tissue vessel density, which was paralleled by a decrease in inflammation. AxCAIL‐4 joint homogenates significantly (P< 0.05) inhibited both endothelial cell (EC) migration and tube formation in vitro. Similarly, AxCAIL‐4 inhibited capillary sprouting in the rat aortic ring assay, and vessel growth in the in vivo Matrigel plug assay. The angiostatic effect occurred despite high levels of vascular endothelial growth factor (VEGF), and was associated with down‐regulation of the proangiogenic cytokines IL‐18, CXCL16, and CXCL5 and up‐regulation of the angiogenesis inhibitor endostatin. Of interest, AxCAIL‐4 also resulted in decreased EC expression of the αv and β3 integrin chains.Conclusion: In rat AIA, IL‐4 reduces synovial tissue vascularization via angiostatic effects, mediates inhibition of angiogenesis via an association with altered pro‐ and antiangiogenic cytokines, and may inhibit VEGF‐mediated angiogenesis and exert its angiostatic role in part via αvβ3 integrin. This knowledge of the specific angiostatic effects of IL‐4 may help optimize target‐oriented treatment of inflammatory arthritis.