Abstract-Heme oxygenase-1 (HO-1) is a stress protein that has been suggested to participate in defense mechanisms against agents that may induce oxidative injury, such as heme and inflammatory molecules. Incubation of endothelial cells in a high-glucose (33 mmol/L) medium for 7 days resulted in a decrease of HO activity by 34% and a decrease in HO-1 and HO-2 proteins compared with cells exposed to low glucose (5 mmol/L) (PϽ0.05) or cells exposed to mannitol (33 mmol/L). Overexpression of HO-1 was coupled with an increase in HO activity and carbon monoxide synthesis, decreased cellular heme, and acceleration in all phases of the cell cycle (PϽ0.001). Key Words: cell cycle Ⅲ oxidative stress Ⅲ superoxide anion production Ⅲ gene transfer Ⅲ heme oxygenase E xposure of endothelial cells to elevated glucose levels causes glucose oxidation, resulting in the generation of excess reactive oxygen species (ROS) in endothelial cells. A reduction in antioxidant reserves has been attributed to endothelial cell dysfunction in diabetes, even in patients with well-controlled glucose levels. 1-3 Hyperglycemia-mediated local formation of ROS is considered to be the major contributing factor to endothelial dysfunction, including abnormalities in cell cycling 1,4,5 and delayed replication, and these abnormalities can be reversed by antioxidant agents 6,7 and an increased expression of antioxidant enzymes. 8 Du et al 9 have demonstrated that hyperglycemia stimulates the induction of apoptosis in endothelial cells by a mechanism that involves the generation of ROS and superoxide anion formation. Moreover, high glucose conditions facilitated the susceptibility of various serum proteins to oxidation, which contributes to the inhibition of endothelial cell proliferation. 10 Wolf et al 11 have reported that high glucose stimulates mitogen-activated protein kinase, which was associated with an enhancement in p27 Kip1 protein and growth arrest.We have previously shown that overexpression of the human heme oxygenase-1 (HO-1) gene in rabbit and rat endothelial cells renders the cells resistant to oxidative stress-causing agents 12 and enhances cell growth 13,14 and angiogenesis, 15 which highlights the important metabolic and cytoprotective role of the HO-1 gene. 12,15-17 Inhibition of HO activity has been shown to exacerbate the inflammatory response in the arterial wall in animal models of atherosclerosis model. 18 HO-1 is expressed, under basal conditions, at low levels in endothelial cells 12,15,19,20 and can be induced in these cells in response to oxidants, including heme, H 2 O 2 , and tumor necrosis factor. [21][22][23] It is conceivable, then, that upregulation of HO activity could function to attenuate the glucosemediated inhibition of cell-cycle progression.The objectives of this study were to determine the effects of glucose on HO activity and the expression of HO-1 and HO-2 proteins and DNA distribution and to examine the role of heme metabolism by HO on cell-cycle progression. We also examined the effect of overexpression and un...

Aims/hypothesis: Patients with diabetes mellitus are well known to be at high risk for vascular disease. Circulating endothelial cells (CECs) have been reported to be an ex vivo indicator of vascular injury. We investigated the presence of CECs in the peripheral blood of 25 patients with diabetes mellitus and in nine nondiabetic control donors. Methods: Endothelial cells were isolated from peripheral blood with anti-CD-146-coated immunomagnetic Dynabeads, and were stained with acridine orange dye and counted by fluorescence microscopy. The cells were also stained for von Willebrand factor and Ulex europaeus lectin 1. Results: Patients with diabetes mellitus had an elevated number of CECs (mean 69±30 cells/ml, range 35-126) compared with healthy controls (mean 10±5 cells/ml, range 3-18) (p<0.001). The increase in CECs did not correlate with the levels of HbA 1 c. Circulating endothelial cell numbers were elevated regardless of glucose levels, suggesting that, even with control of glucose levels, there is increased endothelial cell sloughing. Conclusions: Our study suggests that the higher number of CECs in patients with type 2 diabetes may reflect ongoing vascular injury that is not directly dependent on glucose control.

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