Double-antiangiogenic protein DAAP targeting vascular endothelial growth factor A and angiopoietins attenuates collagen-induced arthritis

Abstract

Introduction: Angiogenesis plays a critical role in synovial inflammation and joint destruction in rheumatoid arthritis (RA). Vascular endothelial growth factor A (VEGF-A) and angiopoietins are two important mediators of synovial angiogenesis. We have previously developed a novel chimeric decoy receptor, namely, double-antiangiogenic protein (DAAP), which can both bind VEGF-A and angiopoietins and block their actions. This study was performed to evaluate the antiarthritic effect of DAAP and the combination effect with the tumor necrosis factor alpha (TNF-alpha) inhibitor in collagen-induced arthritis (CIA). Methods: Recombinant DAAP, VEGF-Trap, Tie2-Fc and dimeric Fc proteins were produced and purified from CHO cells in large-scale bioreactors. CIA was induced in DBA/1 mice with type II collagen. The preventive effect of DAAP was determined and compared with other decoy receptors such as VEGF-Trap or Tie2-Fc, which block VEGF-A or angiopoietins, respectively. The clinical, radiographic, pathologic and immunohistochemical analyses were performed in CIA mice. The levels of matrix metalloprotease 3 (MMP-3) and interleukin 1 beta (IL-1 beta) were quantified by enzyme-linked immunosorbent assay, and receptor activator of nuclear factor kappa B ligand (RANKL) mRNA levels were measured by polymerase chain reaction. Finally, we investigated the combination effects of DAAP with a low dose of TNF-alpha decoy receptor (etanercept 10 mg/kg). Results: On the basis of clinical and radiographic evaluation, DAAP had a much greater inhibitory effect than VEGF-Trap or Tie2-Fc on arthritis severity and bone destruction. These inhibitory effects were accompanied by significantly diminishing pathologic abnormalities, CD31-positive vasculature and synovial infiltration by F4/80-positive macrophages. The levels of MMP-3, IL-1 beta and RANKL were much lower in the DAAP-injected group than those of the control. Furthermore, DAAP showed a therapeutic effect and a combination effect with etanercept when injected after arthritis onset in established CIA. Conclusions: DAAP has not only potent prophylactic effects on both inflammation and bone destruction but also therapeutic effects, alone and in combination with a TNF-alpha inhibitor in CIA mice. These results suggest that DAAP could be used as an effective new therapeutic agent for RA.