ARTICLES 1026 VOLUME 9 | NUMBER 8 | AUGUST 2003 NATURE MEDICINE 1 Centre for Vascular Research, University of New South Wales, Sydney NSW 2052, Australia. 2 Department of Haematology, The Prince of Wales Hospital, Sydney NSW 2052, Australia. 3 Johnson & Johnson Research Laboratories, Sydney, Australia. Correspondence should be addressed to L.M.K. (L.Khachigian@unsw.edu.au). Angiogenesis, the formation of new blood vessels from pre-existing vasculature, is a complex, multistep process involving endothelial basement membrane degradation, cell migration, proliferation, canalization, branching and maturation of neovessels. Angiogenesis is mandatory for tumor progression because it supplies oxygen and nutrients to the growing tumor, a concept first proposed 30 years ago 1 . It follows, then, that agents that interfere with blood vessel for- mation could be used to block tumor progression 2 . Examples of these include antagonists of angiogenic growth factors, receptors, integrins and proteolytic enzymes; some of these are currently under evaluation in various clinical trials 3 . Egr-1, first discovered as an immediate-early gene inducibly expressed in growth-quiescent fibroblasts exposed to serum 4 , is a broadly expressed prototypical member of the Cys 2 -His 2 zinc fin- ger family of transcription factors 5 . Egr-1 is rapidly activated by multiple extracellular agonists (such as growth factors and cytokines) and environmental stresses (such as hypoxia, fluid shear stresses and vascular injury) 6 . Once activated, Egr-1 controls the expression of a diverse array of proangiogenic genes (encoding growth factors, cytokines, receptors, adhesion molecules and pro- teases) 5,7 through GC-rich, cis-acting elements in the promoter regions of these genes. Egr-1 is strongly coexpressed with prolifer- ating cell nuclear antigen in von Willebrand factor–positive blood vessels in the chorioallantoic membrane assay (data not shown). Therefore, Egr-1 seemed prima facie to be an appealing therapeutic target given its capacity to integrate extracellular stimuli with changes in gene expression. RESULTS DNAzyme inhibits Egr-1 and endothelial growth To begin to dissect the role of Egr-1 in angiogenesis, we evaluated the effect of two Egr-1 DNAzymes, DzF and DzA, on human microvascular endothelial cell proliferation. DNAzymes are cation-dependent enzy- matic molecules composed entirely of DNA that can be engineered to cleave target mRNA in a gene-specific and catalytically efficient man- ner 8 . DzF and DzA each bear a 15-nucleotide catalytic domain of the 10- 23 subtype 8 , flanked by two 9+9 nucleotide arms that target the A 301 U and G 198 U sites in human Egr-1 mRNA, respectively. DzF and DzA each inhibited serum-inducible endothelial cell proliferation at 0.1 μM, with more potent inhibition by DzF (Fig. 1a). DzF inhibition was dose dependent and detectable at concentrations as low as 40 nM (data not shown). In contrast, DzFSCR, in which the order of nucleotides in the hybridizing arms of DzF was scrambled (SCR) without altering the cat- alytic domain, had no effect (Fig. 1a). DzF also inhibited endothelial cell migration in an in vitro scraping assay. Regrowth from the wound edge into the denuded zone 2 d after injury was inhibited 65% by DzF (0.1 μM), whereas DzFSCR had no effect (Fig. 1b,c). We next evaluated the capacity of Egr-1 DNAzymes to inhibit capil- lary tubule formation by endothelial cells cultured on basement mem- brane matrices (Matrigel), wherein the cells undergo morphologic differentiation into an extensive network of capillary-like structures composed of highly organized three-dimensional cords 9 . Tubule forma- tion in this model proceeds rapidly after plating, with maximal network formation after 8 h and gradual dissociation by 24 h (Fig. 1d). DzF caused profound sequence-specific inhibition of tubule formation Transcription factor Egr-1 supports FGF-dependent angiogenesis during neovascularization and tumor growth Roger G Fahmy 1,2 , Crispin R Dass 3 , Lun-Quan Sun 3 , Colin N Chesterman 1,2 & Levon M Khachigian 1,2 Current understanding of key transcription factors regulating angiogenesis is limited. Here we show that RNA-cleaving phosphodiester-linked DNA-based enzymes (DNAzymes), targeting a specific motif in the 5′ untranslated region of early growth response (Egr-1) mRNA, inhibit Egr-1 protein expression, microvascular endothelial cell replication and migration, and microtubule network formation on basement membrane matrices. Egr-1 DNAzymes blocked angiogenesis in subcutaneous Matrigel plugs in mice, an observation that was independently confirmed by plug analysis in Egr-1-deficient animals, and inhibited MCF-7 human breast carcinoma growth in nude mice. Egr-1 DNAzymes suppressed tumor growth without influencing body weight, wound healing, blood coagulation or other hematological parameters. These agents inhibited endothelial expression of fibroblast growth factor (FGF)-2, a proangiogenic factor downstream of Egr-1, but not that of vascular endothelial growth factor (VEGF). Egr-1 DNAzymes also repressed neovascularization of rat cornea. Thus, microvascular endothelial cell growth, neovascularization, tumor angiogenesis and tumor growth are processes that are critically dependent on Egr-1. © 2003 Nature Publishing Group http://www.nature.com/naturemedicine