ChOx are suitable biomarkers of oxidative stress and may be useful in clinical studies to follow drug effects on lipid oxidative modifications in diabetic patients.

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In order to determine the effect of antibodies against electronegative low-density lipoprotein LDL(-) on atherogenesis, five groups of LDL low receptor-deficient (LDLr-/-) mice (6 per group) were immunized with the following antibodies (100 µg each): mouse anti-LDL(-) monoclonal IgG2b, rabbit anti-LDL(-) polyclonal IgG or its Fab fragments and mouse irrelevant monoclonal IgG and non-immunized controls. Antibodies were administered intravenously one week before starting the hypercholesterolemic diet (1.25% cholesterol) and then every week for 21 days. The passive immunization with anti-LDL(-) monoclonal IgG2b, polyclonal antibody and its derived Fab significantly reduced the cross-sectional area of atherosclerotic lesions at the aortic root of LDLr-/-mice (28.8 ± 9.7, 67.3 ± 17.02, 56.9 ± 8.02 µm 2 (mean ± SD), respectively) compared to control (124.9 ± 13.2 µm 2 ). Vascular cell adhesion molecule-1 protein expression, quantified by the KS300 image-analyzing software, on endothelium and the number of macrophages in the intima was also decreased in aortas of mice treated with anti-LDL(-) monoclonal antibody (3.5 ± 0.70 per field x 10) compared to controls (21.5 ± 3.5 per field x 10). Furthermore, immunization with the monoclonal antibody decreased the concentration of LDL(-) in blood plasma (immunized: 1.0 ± 1.4; control: 20.5 ± 3.5 RLU), the amount of cholesterol oxides in plasma (immunized: 4.7 ± 2.7; control: 15.0 ± 2.0 pg COx/mg cholesterol) and liver (immunized: 2.3 ± 1.5; control: 30.0 ± 26.0 pg COx/mg cholesterol), and the hepatic content of lipid hydroperoxides (immunized: 0.30 ± 0.020; control: 0.38 ± 0.15 ng/mg protein). In conclusion, antibodies against electronegative LDL administered intravenously may play a protective role in atherosclerosis.

Objective-Nitro-fatty acids (NO 2 -FAs) are emerging as a new class of cell signaling mediators. Because NO 2 -FAs are found in the vascular compartment and their impact on vascularization remains unknown, we aimed to investigate the role of NO 2 -FAs in angiogenesis. Methods and Results-The effects of nitrolinoleic acid and nitrooleic acid were evaluated on migration of endothelial cell (EC) in vitro, EC sprouting ex vivo, and angiogenesis in the chorioallantoic membrane assay in vivo. At 10 mol/L, both NO 2 -FAs induced EC migration and the formation of sprouts and promoted angiogenesis in vivo in an NO-dependent manner. In addition, NO 2 -FAs increased intracellular NO concentration, upregulated protein expression of the hypoxia inducible factor-1␣ (HIF-1␣) transcription factor by an NO-mediated mechanism, and induced expression of HIF-1␣ target genes, such as vascular endothelial growth factor, glucose transporter-1, and adrenomedullin. Compared with typical NO donors such as spermine-NONOate and deta-NONOate, NO 2 -FAs were slightly less potent inducers of EC migration and HIF-1␣ expression. Short hairpin RNA-mediated knockdown of HIF-1␣ attenuated the induction of vascular endothelial growth factor mRNA expression and EC migration stimulated by NO 2 -FAs. Conclusion-Our data disclose a novel physiological role for NO 2 -FAs, indicating that these compounds induce angiogenesis in an NO-dependent mechanism via activation of HIF-1␣. Key Words: angiogenesis Ⅲ nitric oxide Ⅲ hypoxia inducible factor-1 Ⅲ nitro-fatty acids N itro-fatty acids (NO 2 -FAs) are nitroalkene products of polyunsaturated fatty acids, nitrated by nitric oxide (NO)-derived species. 1 NO 2 -FAs, including nitrolinoleic acid (LNO 2 ) and nitrooleic acid (OA-NO 2 ), have pluripotent cell signaling capabilities. For example, they exhibit antiinflammatory properties by inhibiting platelet aggregation and neutrophil activation 2 and upregulating heme oxygenase-1 expression. 3 Recently, the ability to nitrosate CD40 4 and potential cardioprotective effects have also been ascribed to these compounds. 5 Interestingly, OA-NO 2 was found in reperfused ischemic heart tissue, indicating that its formation might occur during the reperfusion process. Moreover, exogenous administration of OA-NO 2 also mitigates the damage induced by tissue reoxygenation. 5 NO 2 -FAs also activate the peroxisome proliferator-activated receptor-␥ 6 and might mediate reversible posttranslational protein modifications through nitroalkylation reactions both with low-molecularweight thiols and with cysteine and histidine residues of proteins. 2 Furthermore, NO 2 -FAs not only serve as reservoirs for NO but also may act as endogenous NO donors,4,7 inducing vessel relaxation 8,9 and upregulating endothelial NO synthase expression and activity, ultimately increasing NO levels. 10 NO is a well-established modulator of angiogenesis, 11 which is a crucial process during embryonic development and in several pathological conditions, including vascular diseases. 12 Angiogenesis is tight...

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Original ArticleCardiovascular diseases are less prevalent in premenopausal women and in those receiving hormone replacement therapy as compared with postmenopausal women and men 1 . This protective effect is attributed to estrogens, and one of the mechanisms of action may be related to the metabolism of plasma lipoproteins 2 .Estrogens reduce LDL-cholesterol and increase HDLcholesterol 3 . However, these changes in lipid profile only contribute to approximately 25% of the protective effect of estrogens 4 . Other potential mechanisms of action of estrogens may include protection against LDL oxidation 5 , reduction in lipoprotein(a), potentiation of fibrinolysis 6 , and increase in insulin sensitivity. In the arteries, estrogens improve vasodilating function, decrease calcification, secretion of cell adhesion molecules (E-selectin, ICAM-1, and VCAM-1), and formation of atherosclerotic lesions 7 .Direct action of estrogens on the arterial wall has also been demonstrated. The administration of estrogens for prolonged periods inhibits the deposition of cholesterol in the arteries and thickening of the intima in monkeys and rabbits fed an atherogenic diet 8 . However, the mechanisms determining the direct effects of estrogens on the arterial wall have not been completely elucidated. The hemodynamic effects may be partially measured by the activity of estrogens on the synthesis of endothelial nitric oxide 9 . Estrogens have been suggested to possibly improve endothelial function and decrease the risks of atherosclerosis in premenopausal women. Nitric oxide has its bioactivity reduced in postmenopausal women. However, it is not clear whether this occurs due to its lower production by nitric oxide synthase, or whether nitric oxide is inactivated by reaction with the superoxide radical also generated by endothelial cells, forming the peroxynitrite anion (ONOO -), which is a strong oxidizing agent.Peroxynitrite is decomposed into other reactive oxygen species ( fig. 1) 10 and reacts with tyrosine residues of proteins to form nitrotyrosine 11 . Although little is known NOx, nitrotyrosine, COx, CE 18:2 -OOH, and PC-OOH were higher in the postmenopausal period (33.8±22.3 µM; 230±130 nM; 55±19 ng/µL; 17±8.7 nM; 2775±460 nM, respectively) Objective -To assess the effect of endogenous estrogens on the bioavailability of nitric oxide (·NO) and in the formation of lipid peroxidation products in pre-and postmenopausal women. Methods -NOx and S-nitrosothiols were determined by gaseous phase chemiluminescence, nitrotyrosine was determined by ELISA, COx (cholesterol oxides) by gas chromatography, and cholesteryl linoleate hydroperoxides (CE 18:2 -OOH), trilinolein (TG 18:2 -OOH), and phospholipids (PC-OOH) by HPLC in samples of plasma. Results -The concentrations of

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