VEGF-mediated inflammation precedes angiogenesis in adult brain Susan D. Croll, a, * Richard M. Ransohoff, b Ning Cai, a Qing Zhang, a Francis J. Martin, a Tao Wei, b Lora J. Kasselman, a Jennifer Kintner, a Andrew J. Murphy, a George D. Yancopoulos, a and Stanley J. Wiegand a a Regeneron Pharmaceuticals, Tarrytown, NY 10591, USA b Department of Neurosciences, Cleveland Clinic Foundation, Cleveland, OH 44195, USA Received 24 November 2003; revised 4 February 2004; accepted 6 February 2004 Available online 28 March 2004 Abstract Vascular endothelial growth factor (VEGF) has been shown to induce angiogenesis when infused continuously into adult rat brain tissue. In addition, VEGF has been shown to enhance permeability in brain vasculature. Adult rats were continuously infused with mouse VEGF into neocortex for up to 7 days. We studied the development of VEGF-induced vasculature in rat neocortex and evaluated the temporal expression of a wide variety of markers for inflammation and vascular leak in relation to the angiogenic response using immunohistochemistry and Western blot analysis. We report here that VEGF-mediated inflammation in brain is characterized by upregulation of ICAM-1 and the chemokine MIP-1a, as well as a preferential extravasation of monocytes. VEGF causes a dramatic breakdown of the blood – brain barrier, which is characterized by decreased investment of the vasculature with astroglial endfeet. Perivascular cells, in contrast, increase around the newly formed cerebrovasculature. In addition, breakdown of the blood– brain barrier, leukocyte extravasation, and extracellular matrix deposition occur before vascular proliferation. Furthermore, administration of low doses of VEGF induces permeability and inflammation without appreciable vascular proliferation. D 2004 Elsevier Inc. All rights reserved. Keywords: Cytokine; Blood– brain barrier; Permeability; Vasculature; Chemokine; VEGF; Inflammation; MIP-1a; Monocytes Introduction Vascular endothelial growth factor (VEGF) has been shown to induce vascular proliferation in a variety of tissues (Bauters et al., 1994; Krum et al., 2002; Proescholdt et al., 1999; Rosenstein et al., 1998; Springer et al., 1998; Take- shita et al., 1994), and studies of VEGF null mutant mice have shown that VEGF is necessary for angiogenesis and vasculogenesis in the developing embryo (Ferrara et al., 1996). Consequently, VEGF has been evaluated in clinical trials as a pro-angiogenic factor to treat ischemic conditions (Isner, 1998; Isner et al., 1996; Losordo et al., 1998). Although VEGF’s role as an angiogenic factor is well established, it was originally discovered and characterized based on its ability to increase vascular permeability (Senger et al., 1986). VEGF has been shown to increase vascular leak of both proteins and particulates, and it has been proposed that this increase in permeability is a necessary prerequisite for the induction of angiogenesis (for reviews, see Dvorak et al., 1995, 1999). Many human diseases are characterized by pathological angiogenesis, such as proliferative vascular retinopathies, psoriasis, rheumatoid arthritis, endometriosis, and tumors. In these conditions, VEGF levels are known to be increased before and/or during the angiogenic process (Bhushan et al., 1999; Brown et al., 1993, 1995; Detmar et al., 1994; Harada et al., 1998; Jackson et al., 1997; Kikuchi et al., 1998; Lashkari et al., 2000; Mahnke et al., 2000). In addition, VEGF levels are elevated in many human diseases, or animal models of human disease, which are characterized by inflammation and vascular leak, such as cerebral ische- mia, tumor ascites, trauma, early diabetic retinopathy, pre- eclampsia, ovarian hyperstimulation syndrome, and status epilepticus (Baker et al., 1995; Boulton et al., 1998; Cobbs et al., 1998; Croll et al., 2002; Grad et al., 1998; Hayashi et al., 1997; Issa et al., 1999; Kovacs et al., 1996; Kraft et al., 0014-4886/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.expneurol.2004.02.010 * Corresponding author. Regeneron Pharmaceuticals, 777 Old Saw Mill River Road, Tarrytown, NY 10591. Fax: +1-914-347-5045. E-mail address: susan.croll@regeneron.com (S.D. Croll). www.elsevier.com/locate/yexnr Experimental Neurology 187 (2004) 388 – 402