Background-Apolipoprotein A1 mimetic peptide, synthesized from D-amino acid (D-4F), enhances the ability of HDL to protect LDL against oxidation in atherosclerotic animals. Methods and Results-We investigated the mechanisms by which D-4F provides antioxidant effects in a diabetic model.Sprague-Dawley rats developed diabetes with administration of streptozotocin (STZ). We examined the effects of daily D-4F (100 g/100 g of body weight, intraperitoneal injection) on superoxide (O 2 Ϫ ), extracellular superoxide dismutase (EC-SOD), vascular heme oxygenase (HO-1 and HO-2) levels, and circulating endothelial cells in diabetic rats. In response to D-4F, both the quantity and activity of HO-1 were increased. O 2 Ϫ levels were elevated in diabetic rats (74.8Ϯ8ϫ10 3 cpm/10 mg protein) compared with controls (38.1Ϯ8ϫ10 3 cpm/10 mg protein; PϽ0.01). D-4F decreased O 2 Ϫ levels to 13.23Ϯ1ϫ10 3 (PϽ0.05 compared with untreated diabetics). The average number of circulating endothelial cells was higher in diabetics (50Ϯ6 cells/mL) than in controls (5Ϯ1 cells/mL) and was significantly decreased in diabetics treated with D-4F (20Ϯ3 cells/mL; PϽ0.005). D-4F also decreased endothelial cell fragmentation in diabetic rats. The impaired relaxation typical of blood vessels in diabetic rats was prevented by administration of D-4F (85.0Ϯ2.0% relaxation). Western blot analysis showed decreased EC-SOD in the diabetic rats, whereas D-4F restored the EC-SOD level. Conclusions-We conclude that an increase in circulating endothelial cell sloughing, superoxide anion, and vasoconstriction in diabetic rats can be prevented by administration of D-4F, which is associated with an increase in 2 antioxidant proteins, HO-1 and EC-SOD.

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...

Up-regulation of heme oxygenase (HO-1) by either cobalt protoporphyrin (CoPP) or human gene transfer improves vascular and renal function by several mechanisms, including increases in antioxidant levels and decreases in reactive oxygen species (ROS) in vascular and renal tissue. The purpose of the present study was to determine the effect of HO-1 overexpression on mitochondrial transporters, cytochrome c oxidase, and anti-apoptotic proteins in diabetic rats (streptozotocin, (STZ)-induced type 1 diabetes). Renal mitochondrial carnitine, deoxynucleotide, and ADP/ATP carriers were significantly reduced in diabetic compared with nondiabetic rats ( p < 0.05). The citrate carrier was not significantly decreased in diabetic tissue. CoPP administration produced a robust increase in carnitine, citrate, deoxynucleotide, dicarboxylate, and ADP/ATP carriers and no significant change in oxoglutarate and aspartate/ glutamate carriers. The increase in mitochondrial carriers (MCs) was associated with a significant increase in cytochrome c oxidase activity. The administration of tin mesoporphyrin (SnMP), an inhibitor of HO-1 activity, prevented the restoration of MCs in diabetic rats. Human HO-1 cDNA transfer into diabetic rats increased both HO-1 protein and activity, and restored mitochondrial ADP/ ATP and deoxynucleotide carriers. The increase in HO-1 by CoPP administration was associated with a significant increase in the phosphorylation of AKT and levels of BcL-XL proteins. These observations in experimental diabetes suggest that the cytoprotective mechanism of HO-1 against oxidative stress involves an increase in the levels of MCs and anti-apoptotic proteins as well as in cytochrome c oxidase activity.The heme-heme oxygenase (HO), 4 HO-1 and HO-2, isoforms, are viewed as having a major role in the formation of carbon monoxide (CO) and bilirubin, and in heme breakdown (1-3). The fact that HO-1 is strongly induced by its substrate, heme, and by oxidant stress, in conjunction with the robust ability of HO-1 to guard against oxidative insult (4, 5), suggests a countervailing system to oxidative stress injury. HO-1 is a regulator of endothelial cell integrity and oxidative stress (4 -6). Up-regulation of HO-1 by pharmacological agents, including cobalt protoporphyrin (CoPP), has been shown to increase superoxide dismutase and to decrease reactive oxygen species (ROS) and NAD(P)H oxidase activity in vitro and in vivo (7-9). In earlier studies, we, as well as others, have demonstrated that overexpression of the HO-1 gene in human, rabbit, and rat endothelial cells not only renders the cells resistant to agents that elicit oxidative stress but also enhances cell growth (6) and angiogenesis (10, 11) via HO-1-derived CO (12). More recently, up-regulation of HO-1 has been shown to prevent endothelial cell death and sloughing in diabetic rats (8).Mitochondrially generated ROS have been well documented in diabetes (13,14). Hyperglycemia-mediated local formation of ROS is considered to be a major contributing factor to renal and vascula...