Current Neurovascular Research, 2005, 2, 103-111 103 1567-2026/05 $50.00+.00 ©2005 Bentham Science Publishers Ltd. Heme Oxygenase Overexpression Attenuates Glucose-Mediated Oxidative Stress in Quiescent Cell Phase: Linking Heme to Hyperglycemia Complications David Sacerdoti 1 , Rafal Olszanecki 2 , Giovanni Li Volti 3 , Claudia Colombrita 4 , Giovanni Scapagnini 5 and Nader G. Abraham 4,6, * 1 University of Padova, Department of Clinical and Experimental Medicine, Via Giustiniani 2, 3500, Padova, Italy; 2 Jagiellonian University Medical College, Chair of Pharmacology, 16 Grzegorzecka str. 31531 Cracow, Poland; 3 Department of Biological Chemistry, Medical Chemistry and Molecular Biology University of Catania, Italy; 4 The Rockefeller University Hospital, 1230 York Avenue, New York, New York 10021, USA; 5 Blanchette Rockefeller Neuroscience Institute, 9601 Medical Center Drive, Rockville, Maryland, 20850, USA; and 6 Department of Pharmacology, New York Medical College, Valhalla, New York 10595, USA Abstract: Heme oxygenase (HO-1) is a stress protein, which has been suggested to participate in defense mechanisms against glucose induced oxidative injury. The purpose of this study was to examine the role of human HO-1 in attenuating glucose-mediated oxidative stress. We investigated the effect of high ambient glucose (15, 33 and 66 mM) on HO-1 gene expression in endothelial cells grown in a serum deprived media compared to the effect of glucose on exponentially grown cells (10% FBS). High glucose at 15 and 33 mM caused significant inhibition of HO-1 protein and activity in G 0 /G 1 and in cells exponentially grown. Glucose concentration at 66mM caused a significant increase in HO-1. Addition of heme (10μM) increased HO-1 protein and bilirubin formation in G 0 /G 1 , in a time dependent manner peaking at 16h. Glucose attenuated heme mediated increase in HO-1 proteins. RT-PCR demonstrated that glucose decreased the levels of HO-1 mRNA in both G 0 /G 1 or cells grown in 10% FBS. The rate of HO-1 induction in response to heme was several fold higher in serum-starved cells compared to cells cultured in 10% FBS. Cells exposed to high glucose for up to 24 h had a significant increase in cellular heme and potentiated heme-mediated increase in generation of superoxide anion and 8-epi- isoprostane PGF 2α . HO-1 gene transduction prevented glucose-mediated elevation of 8-epi-isoprostane PGF 2α . These results imply that expression of HO-1 in G 0 /G 1 cells may be a key player in decreasing cellular heme, associated with increased generation of bilirubin, and in attenuating glucose mediated oxidative stress. Key Words: Heme oxygenase, cell cycle, oxidative stress, superoxide anion, 8-isoprostane, hyperglycemia, glucose, carbon monoxide, retrovirus. INTRODUCTION The control of endothelial function is an important aspect in numerous pathological conditions, such as diabetes, hypertension, atherosclerosis and ischemic reperfusion injury and oxidative stress has been implicated as a potential important factor in endothelial dysfunction. Exposure of endothelial cells to elevated glucose levels causes glucose oxidation, resulting in the generation of excess reactive oxygen species (ROS) damaging endothelial cells. A reduction in antioxidant reserves has been attributed to endothelial cell dysfunction in diabetes, even in patients with well-controlled glucose levels (Zou, MH et al., 2002, Baynes, JW, 1991, Karpen, CW et al., 1985). Hyperglycemia- mediated local formation of ROS is considered to be the major contributing factor to endothelial dysfunction, includ- ing abnormalities in cell cycling (Zou, MH et al., 2002, Lorenzi, M et al., 1987, Baumgartner-Parzer, SM et al., 1995), delayed replication, and these abnormalities can be reversed by antioxidant agents (Lorenzi, M et al., 1985, Curcio, F et al., 1992), and an increased expression of *Address correspondence to this author at the Department of Pharmacology, New York Medical College, Valhalla, New York 10595, USA; Tel: (914) 594-4132; Fax: (914) 594-4119; E-mail: nader_abraham@nymc.edu Received: August 4, 2004; Revised: August 29, 2004; Accepted: August 29, 2004 antioxidant enzymes (Ceriello, A et al., 1996). Oxidative stress-mediated heme oxygenase (HO-1/HO-2) gene expres- sion has been associated with endothelial cell cytoprotection and function, countering the response to a variety of oxida- tive promoting agonists (Ishikawa, K et al ., 1997, Mazza, F et al., 2003, Kushida, T et al., 2002b, Sethi, JM et al., 2002). HO-1/HO-2 are the sole enzymes responsible of degra- dation of heme to bilirubin, carbon monoxide (CO) and release of iron which is sequestered by ferritin synthesis (da Silva, JL et al., 2001). HO-2 is expressed constitutively in most cells, where it synthesizes, bilirubin, an antioxidant, and carbon monoxide, a vasoactive molecule to maintain physiological heme levels (da Silva, JL et al., 2001) (Abraham, NG et al., 1995). HO-1, however, is highly inducible, in the vasculature, in response to inflammatory stimuli and the products of HO-1 activity, biliverdin- bilirubin, have been shown to protect endothelial cells from oxidative stress generated by oxidant agents as heme, H 2 O 2 and tumor necrosis factor (TNF) (Kushida, T et al., 2002c) (Kushida, T et al ., 2002b, Yet, SF et al ., 1999, Morita, T et al., 1997). Inhibition of HO-1 enhances endothelial cell death (Otterbein, LE et al ., 2000b, Soares, MP et al ., 2002) which can be prevented by the supplementation of antioxidant bilirubin. Therefore, a delicate balance exists to maintain a physiological level of HO-1.