Prophylactic adenovirus‐mediated human kallistatin gene therapy suppresses rat arthritis by inhibiting angiogenesis and inflammation

Abstract

Kallistatin has been shown to be an angiogenesis inhibitor. In this study, we investigated whether adenovirus-mediated kallistatin gene delivery has a prophylactic effect in a rat arthritis model. Adenovirus containing the human kallistatin gene (AdHKBP) was injected intraarticularly into ankle joints before the onset of arthritis in a rat model. The effect of kallistatin gene transfer on endothelial cell proliferation in joint extracts was assayed. The response to kallistatin treatment was determined according to clinical parameters, including ankle circumference, articular index, and radiographic scores. Hematoxylin and eosin staining was performed in order to score joint tissues and count neutrophil numbers. In addition, small vessels were quantified by identification of von Willebrand factor-positive endothelial cells. The inflammatory responses were determined by measuring tumor necrosis factor alpha (TNFalpha) and interleukin-1beta (IL-1beta) levels in ankle homogenates. The expression of recombinant human kallistatin in rat ankle joints after gene transfer was identified by immunohistochemical analysis and Western blotting. Significant reductions in the ankle circumference, articular index, and radiographic score were observed in AdHKBP-treated rats compared with control rats treated with the adenoviral plasmid carrying green fluorescent protein. Kallistatin gene transfer also significantly ameliorated the histologic scores in ankle joints and reduced vessel density and neutrophil numbers. The inhibitory effect of kallistatin on the accumulation of inflammatory cells in ankle joints was accompanied by reduced TNFalpha and IL-1beta levels in joint homogenates. Furthermore, an in vitro experiment showed that the proliferation of endothelial cells was markedly inhibited by the addition of AdHKBP-treated joint extract to the culture media, supporting a role of kallistatin in inhibiting angiogenesis. This study demonstrates that kallistatin gene therapy has a prophylactic effect in inhibiting arthritis in the rat ankle. Kallistatin inhibits arthritis through its antiangiogenesis and antiinflammation activities. These results implicate potential therapeutic applications for suppression of arthritis by kallistatin gene therapy.