Targeting angiogenesis with Shiga-like toxin fused to vascular endothelial growth factor

Angiogenesis is a tightly controlled process of growing new blood vessels. Under normal circumstances angiogenesis occurs only during embryonic development, wound healing and development of the corpus luteum. However, in a large number of pathologies, such as solid tumor growth, various eye diseases, and chronic inflammatory states, angiogenesis is a crucial component of the disease process. Therefore, inhibitors of angiogenesis are being investigated as potential therapeutics for these pathologies. During angiogenesis endothelial cells of existing blood vessels undergo a complex process of reshaping, migration, growth, and organization into new vessels. Vascular endothelial growth factor (VEGF) is a central mediator of this process and acts via receptors whose expression is restricted almost exclusively to endothelial cells. Because of its of selectivity, VEGF represents a unique vehicle for delivery of inhibitors of angiogenesis to endothelial cells. Among potential inhibitors of angiogenesis, the Shiga-like toxin-I (SLT-I) produced by E. coli O157:H7 has the advantage that endothelial cells appear to be particularly sensitive to its action. I hypothesized that combining an SLT-I toxin with VEGF as a delivery vehicle would serve as a highly selective and active inhibitor of angiogenesis. To this end, fusion proteins containing VEGF₁₂₁ and two forms of Shiga-like toxin-I (SLT-I) were developed and tested in vitro for activities that have the potential to inhibit angiogenesis in vivo. Plasmids encoding the fusion proteins VEGF₁₂₁/A ₁ containing the catalytically active fragment of the SLT-I A subunit and VEGF₁₂₁/A containing the full length A subunit of SLT-I were constructed in pET-29a and pET-32a systems. Escherichia coli BL21(DE3)pLysS bacteria were transformed with the plasmid constructs and the expressed fusion proteins were recovered in inclusion bodies upon induction with isopropyl-B-D-thiogalactopyranoside (IPTG).{09}Both purified fusion proteins inhibited the translation of luciferase mRNA as a reporter gene in an in vitro translation system, indicating that both fusion proteins retain the N-glycosidase activity of SLT-I. However, only VEGF₁₂₁/A₁ fusion proteins displayed the ability to induce auto-phosphorylation of the VEGF receptor KDR/Flk-1. The inability of the VEGF₁₂₁/A fusion protein to induce KDR/Flk-1 autophosphorylation suggests that the long form of the fusion protein does not form a proper complex with KDR/Flk-1 receptors. In a biological assay VEGF₁₂₁/A ₁ fusion proteins displayed a strong, selective growth inhibition of cultured cells expressing KDR/Flk-1 receptors. In contrast VEGF₁₂₁/A proteins did not display growth inhibitory activities. In summary, these results indicate that VEGF/SLT fusion proteins are promising therapeutic agents that can be developed into powerful and selective inhibitors of angiogenesis.