Circulating neutrophils maintain physiological blood pressure by suppressing bacteria and IFNgamma-dependent iNOS expression in the vasculature of healthy mice.

Whether leukocytes exert an influence on vascular function in vivo is not known. Here, genetic and pharmacologic approaches show that the absence of neutrophils leads to acute blood pressure dysregulation. Following neutrophil depletion, systolic blood pressure falls significantly over 3 days (88.0 +/- 3.5 vs 104.0 +/- 2.8 mm Hg, day 3 vs day 0, mean +/- SEM, P < .001), and aortic rings from neutropenic mice do not constrict properly. The constriction defect is corrected using l-nitroarginine-methyl ester (L-NAME) or the specific inducible nitric oxide synthase (iNOS) inhibitor 1400W, while acetylcholine relaxation is normal. iNOS- or IFNgamma-deficient mice are protected from neutropenia-induced hypotension, indicating that iNOS-derived nitric oxide (NO) is responsible and that its induction involves IFNgamma. Oral enrofloxacin partially inhibited hypotension, implicating bacterial products. Roles for cyclooxygenase, complement C5, or endotoxin were excluded, although urinary prostacyclin metabolites were elevated. Neutrophil depletion required complement opsinization, with no evidence for intravascular degranulation. In summary, circulating neutrophils contribute to maintaining physiological tone in the vasculature, at least in part through suppressing early proinflammatory effects of infection. The speed with which hypotension developed provides insight into early changes that occur in the absence of neutrophils and illustrates the importance of constant surveillance of mucosal sites by granulocytes in healthy mice.

Effects of vitamin E on oxidative stress and atherosclerosis in an obese hyperlipidemic mouse model.

Vitamin E is a natural antioxidant that has been used in animal and human studies to determine its potential in reducing cardiovascular risk; however, a detailed study in an established obese model of atherosclerosis has yet to be performed. In our current study, we show that obesity and hyperlipidemia cause a synergistic, age-related increase in urinary isoprostane levels in mice deficient in both leptin and low-density lipoprotein receptor (ob/ob;LDLR-/-). Based upon this observation, we hypothesized that vitamin E supplementation would induce potent antiatherogenic effects in this model. Lean and obese LDLR-/- mice were provided vitamin E (2000 IU/kg) in a Western-type high-fat diet for 12 weeks. Plasma lipid parameters, such as total cholesterol (TC), triglyceride (TG) and free fatty acid, were significantly higher in obese mice compared to lean mice at baseline (P<.001). Western-type diet (WD) feeding caused an increase in TC levels in all groups (P<.001); however, TG (P<.001) and free fatty acid (P<.01) were elevated only in lean mice following WD feeding. Vitamin E supplementation neither influenced any of these parameters nor reduced urinary isoprostanes in lean or obese mice. Vitamin E supplementation in ob/ob;LDLR-/- mice resulted in a trend toward a reduction in atherosclerotic lesion area (P=.10), although no differences in lesion area were noted in lean LDLR-/- animals. These data provide evidence that vitamin E supplementation is not sufficient to reduce extreme elevations in systemic oxidative stress due to hyperlipidemia and obesity and, thus, may not be cardioprotective in this setting.

Antioxidants significantly affect the formation of different classes of isoprostanes and neuroprostanes in rat cerebral synaptosomes.

Lipid peroxidation has been implicated in the pathogenesis of a number of diseases, including neurodegenerative disorders. Evidence that antioxidants can affect the clinical course of neurodegenerative diseases is limited. In the present study, we examined the ability of five common antioxidants or antioxidant combinations, alpha-tocopherol, gamma-tocopherol, ascorbic acid, GSH ethyl ester, and a combination of ascorbate and alpha-tocopherol, to modulate lipid peroxidation in peroxidizing rat cerebral synaptosomes, a well-characterized model of oxidant injury. In these studies, we quantified isoprostanes (IsoPs) derived from arachidonic acid as an index of whole tissue oxidation and neuroprostanes (NeuroPs) formed from docosahexaenoic acid as a marker of selective neuronal peroxidation. We report that these various antioxidants displayed markedly different capacities to inhibit IsoP and NeuroP formation with the most potent effects on IsoPs observed for ascorbate, GSH ethyl ester, and the alpha-tocopherol-ascorbate combination. alpha-Tocopherol was slightly less potent and gamma-tocopherol significantly less effective. The concentration-response relationships were significantly different for NeuroP formation with the antioxidants being significantly less potent than for IsoP generation. In particular, alpha-tocopherol did not inhibit NeuroP formation at concentrations up to 100 microM. We also determined that tocopherols, in particular alpha-tocopherol, act in vitro as reducing agents to convert IsoP and NeuroP endoperoxides to reduced F-ring compounds, a finding we have observed previously in vivo in brain. These studies are of importance because they have further defined the role of antioxidants to modulate the formation of lipid peroxidation products in peroxidizing brain tissue. In addition, they suggest that alpha-tocopherol may not be a particularly effective agent to inhibit oxidant stress in the terminal compartment of neurons in the central nervous system.

Quantification of F-ring isoprostane-like compounds (F4-neuroprostanes) derived from docosahexaenoic acid in vivo in humans by a stable isotope dilution mass spectrometric assay.

Lipid peroxidation has been implicated in the pathophysiological sequelae of human neurodegenerative disorders. It is recognized that quantification of lipid peroxidation is best assessed in vivo by measuring a series of prostaglandin (PG) F2-like compounds termed F2-isoprostanes (IsoPs) in tissues in which arachidonic acid is abundant. Unlike other organs, the major polyunsaturated fatty acid (PUFA) in the brain is docosahexaenoic acid (DHA, C22:6 omega-6), and this fatty acid is particularly enriched in neurons. We have previously reported that DHA undergoes oxidation in vitro and in vivo resulting in the formation of a series of F2-IsoP-like compounds termed F4-neuroprostanes (F4-NPs). We recently chemically synthesized one F4-NP, 17-F4c-NP, converted it to an 18O-labeled derivative, and utilized it as an internal standard to develop an assay to quantify endogenous production of F4-NPs by gas chromatography (GC)/negative ion chemical ionization (NICI) mass spectrometry (MS). The assay is highly precise and accurate. The lower limit of sensitivity is approximately 10 pg. Levels of F4-NPs in brain tissue from rodents were 8.7 +/- 2.0 ng/g wet weight (mean +/- S.D.). Levels of the F4-NPs in brains from normal humans were found to be 4.9 +/- 0.6 ng/g (mean +/- S.D.) and were 2.1-fold higher in affected regions of brains from humans with Alzheimer's disease (P = 0.02). Thus, this assay provides a sensitive and accurate method to assess oxidation of DHA in animal and human tissues and will allow for the further elucidation of the role of oxidative injury to the central nervous system in association with human neurodegenerative disorders.

Measurement of F2- isoprostanes and isofurans using gas chromatography-mass spectrometry.

F2-Isoprostanes (IsoPs) are isomers of prostaglandin F2α formed from the nonenzymatic free radical-catalyzed peroxidation of arachidonic acid. Since discovery of these molecules by Morrow and Roberts in 1990, F2-IsoPs have been shown to be excellent biomarkers as well as potent mediators of oxidative stress in vivo in humans. Isofurans (IsoFs) are also oxidation products generated from the nonenzymatic oxidation of arachidonic acid. IsoFs are preferentially formed instead of F2-IsoPs in settings of increased oxygen tension. The protocol presented herein is the current methodology that our laboratory uses to quantify F2-IsoPs and IsoFs in biological tissues and fluids using gas chromatography/mass spectrometry (GC/MS). A variety of analytical procedures to measure F2-IsoPs, including other GC/MS methods and liquid chromatography/MS and immunological approaches, are reported in the literature. This method provides a very low limit of quantitation and is suitable for analysis of both F2-IsoPs and IsoFs from a variety of biological sources including urine, plasma, tissues, cerebral spinal fluid, exhaled breath condensate, and amniotic fluid, among others.

Formation of highly reactive A-ring and J-ring isoprostane-like compounds (A4/J4-neuroprostanes) in vivo from docosahexaenoic acid.

Free radical-initiated oxidant injury and lipid peroxidation have been implicated in a number of neural disorders. Docosahexaenoic acid is the most abundant unsaturated fatty acid in the central nervous system. We have shown previously that this 22-carbon fatty acid can yield, upon oxidation, isoprostane-like compounds termed neuroprostanes, with E/D-type prostane rings (E(4)/D(4)-neuroprostanes). Eicosanoids with E/D-type prostane rings are unstable and dehydrate to cyclopentenone-containing compounds possessing A-type and J-type prostane rings, respectively. We thus explored whether cyclopentenone neuroprostanes (A(4)/J(4)-neuroprostanes) are formed from the dehydration of E(4)/D(4)-neuroprostanes. Indeed, oxidation of docosahexaenoic acid in vitro increased levels of putative A(4)/J(4)-neuroprostanes 64-fold from 88 +/- 43 to 5463 +/- 2579 ng/mg docosahexaenoic acid. Chemical approaches and liquid chromatography/electrospray ionization tandem mass spectrometry definitively identified them as A(4)/J(4)-neuroprostanes. We subsequently showed these compounds are formed in significant amounts from a biological source, rat brain synaptosomes. A(4)/J(4)-neuroprostanes increased 13-fold, from a basal level of 89 +/- 72 ng/mg protein to 1187 +/- 217 ng/mg (n = 4), upon oxidation. We also detected these compounds in very large amounts in fresh brain tissue from rats at levels of 97 +/- 25 ng/g brain tissue (n = 3) and from humans at levels of 98 +/- 26 ng/g brain tissue (n = 5), quantities that are nearly an order of magnitude higher than other classes of neuroprostanes. Because of the fact that A(4)/J(4)-neuroprostanes contain highly reactive cyclopentenone ring structures, it would be predicted that they readily undergo Michael addition with glutathione and adduct covalently to proteins. Indeed, incubation of A(4)/J(4)-neuroprostanes in vitro with excess glutathione resulted in the formation of large amounts of adducts. Thus, these studies have identified novel, highly reactive A/J-ring isoprostane-like compounds that are derived from docosahexaenoic acid in vivo.