RESEARCH ARTICLE Adenoviral delivery of soluble VEGF receptor 1 (sFlt-1) abrogates disease activity in murine collagen-induced arthritis AO Afuwape, M Feldmann and EM Paleolog Kennedy Institute of Rheumatology Division, Faculty of Medicine, Imperial College of Science, Technology and Medicine, London, UK The angiogenic factor VEGF promotes synovitis and bone erosion in rheumatoid arthritis (RA). Previously, we have demonstrated that VEGF expression correlates with disease severity in RA patients and in murine collagen-induced arthritis (CIA). In this study, we adopted an adenoviral gene delivery system expressing soluble VEGF receptor 1 (sFlt-1) to further study the role of VEGF in CIA. Arthritis was induced in DBA/1 mice by injection of bovine collagen. Adenoviruses expressing human soluble VEGF receptor 1 (AdvsFlt-1), or without transgene (Adv0), were injected intravenously on the first day of arthritis. We found that disease severity and paw swelling were significantly suppressed in mice receiving AdvsFlt-1, when compared to untreated or Adv0-treated mice. Expression of sFlt-1 peaked 24 h after injection, with protein detectable in the liver, synovial issue and serum, but rapidly decreased by 72 h. The effect of sFlt-1 expression on signs of disease was paralleled by reduced joint destruction and decreased expression of the vascular marker von Willebrand factor. In summary, adenoviral delivery of human soluble VEGF receptor type 1 significantly suppressed disease activity in CIA. The actions of AdvsFlt-1 are likely to be mediated by reduced synovial neovascularization, and these results support the concept that VEGF blockade may be an effective therapeutic adjunct for the treatment of RA. Gene Therapy (2003) 10, 1950–1960. doi:10.1038/ sj.gt.3302104 Keywords: angiogenesis; collagen-induced arthritis (CIA); adenovirus; VEGF; sFlt-1 Introduction Rheumatoid arthritis (RA) is a chronic inflammatory disease, which causes progressive deformity and destruction of the joints, thus leading to disability and even premature death. The condition is characterized by the aggressive infiltration of the synovial lining of the joints by both monocytes and T lymphocytes, together with proliferation of fibroblastic lining cells. This hyperproliferative pannus invades and subsequently destroys the underlying cartilage and bone. Synovial expansion is dependent on an adequate blood supply for nutrients and oxygen. However, with time, the increased proliferative activity of the pannus will outstrip the capacity of existing blood vessels for delivery of oxygen, nutrients and inflammatory cells to the lesion. The need for new blood vessel formation or angiogenesis, there- fore, becomes critically important. There is mounting evidence that vascular endothelial growth factor (VEGF), a potent endothelial cell mitogen and vascular permeability factor, may play a pivotal role in RA. Within the synovial environment, resident macrophages and fibroblast-like synovial cells have been found to express VEGF protein, while mRNA encoding for both VEGF receptors (Flt-1 and KDR) has been detected on microvascular endothelial cells. 1–6 Elevated levels of VEGF have been detected in the synovial fluid and serum from RA patients when compared to healthy controls, and serum VEGF levels have been shown to correlate with disease severity. 1,2,7 Following successful therapy of RA with monoclonal anti-TNFa antibody, a study from our group demonstrated significant but incomplete reduction of serum VEGF levels. 2 More recently, we have shown that serum VEGF levels at presentation with early RA correlate significantly with development of radiographic damage after 1 year. 7 Furthermore, we observed that expression of VEGF in RA synovial membrane cultures in vitro was upregulated by hypoxia and inflammatory cytokines (TNFa and IL-1), suggesting that there is both a hypoxic and an inflammatory trigger for VEGF expression. 2 In conjunction with these clinical observations, we utilized an experimental model, collagen-induced arthri- tis (CIA) in DBA/1 mice, to study the role of VEGF in arthritis. Using this model, we showed that synovial cells isolated from arthritic mice produced significantly higher levels of VEGF in vitro when compared to sham- immunized mice or healthy controls, with the amount of VEGF released correlating with the severity of arthritis and with the extent of paw swelling. 8 Intervention using a soluble form of the Flt-1 VEGF receptor (sFlt-1), which was polyethylene glycol (PEG)-linked to increase its in vivo half-life, partially abrogated disease, as indicated by a reduction in clinical scores and paw swelling relative to untreated mice. Furthermore, histological Received 17 February 2003; accepted 20 May 2003 Correspondence: Dr A Afuwape, Kennedy Institute of Rheumatology Division, Faculty of Medicine, Imperial College, Arthritis Research Campaign Building, 1, Aspenlea Rd, Hammersmith, London W6 8LH, UK Gene Therapy (2003) 10, 1950–1960 & 2003 Nature Publishing Group All rights reserved 0969-7128/03 $25.00 www.nature.com/gt