Role of heme oxygenase-1 in hydrogen peroxide-induced VEGF synthesis: effect of HO-1 knockout Jarosław Cisowski a , Agnieszka Łoboda a , Alicja Jo ´ zkowicz a , Sifeng Chen b , Anupam Agarwal b , Jo ´ zef Dulak a, * a Faculty of Biotechnology, Jagiellonian University, Krako ´ w, Poland b Department of Medicine, University of Alabama at Birmingham, Birmingham, AL, USA Received 29 October 2004 Available online 7 December 2004 Abstract Hydrogen peroxide is an important mediator of intracellular signaling, which potently enhances the expression of heme oxygen- ase-1 (HO-1) and upregulates synthesis of vascular endothelial growth factor (VEGF). The purpose of the present study was to explore the involvement of HO-1 in regulation of H 2 O 2 -mediated induction of VEGF synthesis. We provide genetic evidence that basal and H 2 O 2 -induced VEGF synthesis is partially dependent on HO-1. Inhibition of HO-1 activity by tin protoporphyrin (SnPPIX) resulted in downregulation of VEGF synthesis in murine fibroblasts and human keratinocytes. The relationship between HO-1 and VEGF was corroborated by using cells derived from HO-1 knockout mice, which demonstrated lower basal and H 2 O 2 - induced production of VEGF. Additionally, knock out of HO-1 gene impaired induction of VEGF by hemin, lysophosphatidylcho- line, and prostaglandin-J 2 . Our results provide confirmation for the involvement of HO-1 in regulation of angiogenesis. Ó 2004 Elsevier Inc. All rights reserved. Keywords: Heme oxygenase; Angiogenesis; Vascular endothelial growth factor; Reactive oxygen species; Superoxide dismutase; Protoporphyrins; Hypoxia inducible factor-1 Reactive oxygen species (ROS) are recently being rec- ognized as significant mediators of both physiological and pathological angiogenesis, during which blood ves- sels are formed from pre-existing capillaries. An increas- ing body of evidence implicates the involvement of ROS in regulation of the synthesis of various angiogenic growth factors and their activity [1]. These relationships may be of particular importance in angiogenesis associ- ated with inflammatory processes, when oxidative stress results in production of high amounts of ROS [2]. ROS can influence the production of angiogenic growth fac- tors either directly or indirectly, by induction of genes, the products of which can influence growth factor synthesis. Vascular endothelial growth factor (VEGF, also called VEGF-A) is one of the most important angio- genic mediators characterized to date. It is produced by many cell types under both physiologic and patho- logic conditions (for reviews, see [3,4]). Its synthesis is higher at sites of inflammation in various organs, it is necessary for cutaneous wound healing, and its produc- tion may aggravate the extent of atherosclerosis by stim- ulation of the formation of vasa vasorum and worsen diabetic retinopathy. Most importantly, it is responsible for the development of new blood vessels during tumor growth (for reviews, see [3,5]). ROS have been reported to induce VEGF synthesis in various cell types. Thus, hydrogen peroxide (H 2 O 2 ) in- creased VEGF expression in keratinocytes [6,7], retinal pigment epithelial cells [8], endothelial cells [9], skeletal myotubes [10], and human and rodent macrophages 0006-291X/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2004.11.083 * Corresponding author. Fax: +48 12 664 69 18. E-mail address: jdulak@mol.uj.edu.pl (J. Dulak). www.elsevier.com/locate/ybbrc Biochemical and Biophysical Research Communications 326 (2005) 670–676 BBRC