Chronic stress in the adult dentate gyrus reduces cell proliferation near the vasculature and VEGF and Flk-1 protein expression Vivi M. Heine, Jessica Zareno, Suharti Maslam, Marian Joe ¨ls and Paul J. Lucassen Institute Neurobiology, Swammerdam Institute for Life Sciences, University of Amsterdam, Kruislaan 320, 1098 SM Amsterdam, The Netherlands Keywords: corticosteroids, hippocampus, Ki-67, neurogenesis, vasculature Abstract Recent evidence has shown that cell proliferation in the adult hippocampal dentate gyrus occurs in tight clusters located near the vasculature. Also, changes in neurogenesis often appear parallel to changes in angiogenesis. Moreover, both these processes share similar modulating factors, like vascular endothelial growth factor (VEGF) and its receptor Flk-1. In an earlier study we found that chronic stress decreased new cell proliferation in the adult dentate gyrus. We here questioned whether these effects of chronic stress are mediated through the vasculature and whether they involve an angiogenic-signaling pathway. We therefore measured the surface area covered by the vasculature, the proportion of vascular-associated newborn cells, and analysed VEGF and Flk-1 protein expression in the hippocampus of a control, chronically stressed and recovery group of rats. Our results show that 32% of the proliferating cells in the rat hippocampus is vascular associated. Chronic stress affected this population of newborn cells to a significantly larger extent than the non-associated cells. Interestingly, after 3 weeks of recovery, the decreased proliferation not associated with the vasculature was more effectively restored than vascular-associated proportion of proliferating cells. VEGF protein was expressed in high densities in GFAP-positive astrocytes located in the hilus, with VEGF-positive end feet extending into and often contacting the granule cells. After chronic stress, both VEGF and Flk-1 protein levels were significantly decreased in the granular cell layer, and again recovered after 3 weeks. This demonstrates that changes in angiogenic factors are implicated in the decreased adult proliferation found after chronic stress. Introduction Exposure to stress decreases cell proliferation in the adult hippocam- pal dentate gyrus (DG) (Gould et al., 1997, 1998; Fuchs et al., 2001; Tanapat et al., 2001; Czeh et al., 2002; Heine et al., 2004a). Circulating glucocorticoids (GCs) are believed to be instrumental in the structural and functional hippocampal changes after chronic stress (Sapolsky et al., 1985; Magarinos et al., 1996; Joe ¨ls, 2001; Lucassen et al., 2001; McEwen, 2001). However, the exact mechanisms responsible for the suppressed cell proliferation remain largely unknown. Recent evidence has shown that adult proliferation takes place near the local microvasculature of the hippocampus (Palmer et al., 2000; Louissaint et al., 2002; Fabel et al., 2003; Yamashima et al., 2004), while both angiogenesis and neurogenesis can be modulated by similar stimuli (Jin et al., 2000; Fabel et al., 2003). This suggests that regulatory signals for adult proliferation may at least partially be derived from the endothelium. The vascular endothelial growth factor (VEGF) is a major angiogenic factor (Jakeman et al., 1993; Flamme et al., 1995; Rosenstein et al., 1998) that can also exert direct neurotrophic and neuroprotective effects (Hayashi et al., 1998; Issa et al., 1999; Silverman et al., 1999; Jin et al., 2000; Sondell et al., 2000; Matsuzaki et al., 2001; Oosthuyse et al., 2001; Svensson et al., 2002; Widenfalk et al., 2003). Moreover, changes in VEGF concentration and VEGF receptor expression were shown to result in altered cell proliferation and survival rates both in vitro (Sondell et al., 1999; Jin et al., 2002) and in vivo (Sondell et al., 1999; Jin et al., 2002; Fabel et al., 2003). VEGF can interact with two receptors: the fms-like tyrosine kinase (VEGF-R1, Flt-1) and the fetal liver kinase receptor (VEGF-R2, Flk-1) (Neufeld et al., 1999). In the adult brain, Flk-1 rather than Flt-1 is abundantly present (Yang et al., 2003). Corticosteroids are well known inhibitors of angiogenesis and have also been extensively studied for, e.g. their use in anti-tumor treatment (Folkman, 1972, 1995; Auerbach & Auerbach, 1994). Furthermore, studies involving different cell types have revealed that steroids like estrogen, dexamethasone and corticosterone are all able to regulate VEGF and ⁄ or VEGF receptor mRNA expression (Cullinan-Bove & Koos, 1993; Klekamp et al., 1997; Nauck et al., 1998; D’Angio et al., 1999; Machein et al., 1999; Mueller et al., 2000; Halaby et al., 2002; Sibug et al., 2002; Mallet et al., 2003; Clerch et al., 2004). These effects seem glucocorticoid receptor (GR) mediated, as, e.g. inhibitory actions of dexamethasone could be reversed by GR antagonist application (Heiss et al., 1996; Gloddek et al., 1999). In this study, we therefore questioned whether the effects of chronic stress on new cell proliferation are (at least in part) mediated through effects on the vasculature, and whether chronic stress affects an angiogenic-signaling pathway. Therefore, we measured the surface Correspondence: Dr V. M. Heine, Department of Pediatric Oncology, Dana-Farber Cancer Institute and Harvard Medical School, Boston, MA 02115, USA. E-mail: vivi_heine@dfci.harvard.edu Received 23 April 2004, revised 3 December 2004, accepted 7 December 2004 European Journal of Neuroscience, Vol. 21, pp. 1304–1314, 2005 ª Federation of European Neuroscience Societies doi:10.1111/j.1460-9568.2005.03951.x