Trans-arachidonic acids generated during nitrative stress induce a thrombospondin-1-dependent microvascular degeneration.

Nitrative stress has an important role in microvascular degeneration leading to ischemia in conditions such as diabetic retinopathy and retinopathy of prematurity. Thus far, mediators of nitrative stress have been poorly characterized. We recently described that trans-arachidonic acids are major products of NO(2)(*)-mediated isomerization of arachidonic acid within the cell membrane, but their biological relevance is unknown. Here we show that trans-arachidonic acids are generated in a model of retinal microangiopathy in vivo in a NO(*)-dependent manner. They induce a selective time- and concentration-dependent apoptosis of microvascular endothelial cells in vitro, and result in retinal microvascular degeneration ex vivo and in vivo. These effects are mediated by an upregulation of the antiangiogenic factor thrombospondin-1, independently of classical arachidonic acid metabolism. Our findings provide new insight into the molecular mechanisms of nitrative stress in microvascular injury and suggest new therapeutic avenues in the management of disorders involving nitrative stress, such as ischemic retinopathies and encephalopathies.

Sustained hypercapnia induces cerebral microvascular degeneration in the immature brain through induction of nitrative stress.

Hypercapnia is regularly observed in chronic lung disease, such as bronchopulmonary dysplasia in preterm infants. Hypercapnia results in increased nitric oxide synthase activity and in vitro formation of nitrates. Neural vasculature of the immature subject is particularly sensitive to nitrative stress. We investigated whether exposure to clinically relevant sustained high CO(2) causes microvascular degeneration in the newborn brain by inducing nitrative stress, and whether this microvascular degeneration has an impact on brain growth. Newborn rat pups were exposed to 10% CO(2) as inspired gas (Pa(CO(2)) = 60-70 mmHg) starting within 24 h of birth until postnatal day 7 (P7). Brains were notably collected at different time points to measure vascular density, determine brain cortical nitrite/nitrate, and trans-arachidonic acids (TAAs; products of nitration) levels as effectors of vessel damage. Chronic exposure of rat pups to high CO(2) (Pa(CO(2)) approximately 65 mmHg) induced a 20% loss in cerebrovascular density at P3 and a 15% decrease in brain mass at P7; at P30, brain mass remained lower in CO(2)-exposed animals. Within 24 h of exposure to CO(2), brain eNOS expression and production of nitrite/nitrate doubled, lipid nitration products (TAAs) increased, and protein nitration (3-nitrotyrosine immunoreactivity) was also coincidently augmented on brain microvessels (lectin positive). Intracerebroventricular injection of TAAs (10 microM) replicated cerebrovascular degeneration. Treatment of rat pups with NOS inhibitor (L-N(omega)-nitroarginine methyl ester) or a peroxynitrite decomposition catalyst (FeTPPS) prevented hypercapnia-induced microvascular degeneration and preserved brain mass. Cytotoxic effects of high CO(2) were reproduced in vitro/ex vivo on cultured endothelial cells and sprouting microvessels. In summary, hypercapnia at values frequently observed in preterm infants with chronic lung disease results in increased nitrative stress, which leads to cerebral cortical microvascular degeneration and curtails brain growth.

Trans-arachidonic acids: new mediators of nitro-oxidative stress.

A reaction of arachidonic acid with the nitrogen dioxide radical (*NO2) or its precursors (peroxynitrite, nitrous acid, nitrogen trioxide) generates a group of nitro lipids named nitroeicosanoids. A distinct feature of this reaction is abundant formation of four trans isomers of arachidonic acid (TAA) via reversible addition of the NO2 radical to the arachidonic acid cis double bonds. This cis-trans isomerization is biologically relevant because many pathologies that involve NO formation such as inflammation, hyperoxia, hypercapnia or exposure to cigarette smoke increase the TAA levels in cells, tissues and in the systemic circulation. Inflammatory conditions have been known to stimulate formation of a variety of oxidized lipids from unsaturated fatty acid precursors via lipid peroxidation mechanisms; however, nitration-dependent cis-trans-isomerization of arachidonic acid is a characteristic process for *NO2. TAA are likely to function as specific and selective biomarkers of the pathologic conditions that define nitro-oxidative stress. Diet independent biosynthesis of trans fatty acids as a result of disease is our new observation. In the past, experimental feeding and clinical studies have supported the concerns that dietary trans fatty acids are cardiovascular risk factors, however, clinical consequences of the endogenous formation of trans fatty acids are not known but potentially important given available studies on TAA. This review aims to summarize the emerging role of TAA as a unique group of biomarkers that target microcirculation and other systems. A biological mechanism that generates endogenous trans fatty acids poses new challenges for pharmacologic intervention and we suggest approaches that may limit TAA effects.

Inhibition of iNOS and COX-2 in human whole blood ex vivo and monocyte-macrophage J774 cells by a new group of aminothiopyrimidone derivatives.

We tested a series of 11 new aminothiopyrimidones on the activity of inducible nitric oxide synthase (iNOS) and prostaglandin G/H synthase-1 and 2 (COX-1 and COX-2) in the whole human blood and monocyte-macrophage J774 cell line. To induce COX-2 and iNOS, blood samples and J774 cells were stimulated with bacterial lipopolysaccharide (LPS) in the absence or presence of the test compounds. After incubation, the plasma and the supernatants of culture media were collected for the measurement of TxB(2) and PGE(2) by a specific enzyme-immunoassay and determination of nitrite by a colorimetric assay. Several phenylthieno derivatives of substituted pyrimidone inhibited formation of both COX-2 and iNOS derived products with one of the compounds (compound 11, N-[2-[(2,4-dinitrophenyl)thio]-4-oxo-6-phenylthieno[2,3-d]pyrimidin-3(4H)-y]methanesulfonamide) showing a complete inhibition of LPS-stimulated formation of NO and PGE(2).

The mechanism of oleic acid nitration by *NO(2).

Fatty acid nitration is a recently discovered process that generates biologically active nitro lipids; however, its mechanism has not been fully characterized. For example, some structural details such as vinyl and allyl isomers of the nitro fatty acids have not been established. To characterize lipids that originated from a biomimetic reaction of *NO(2) with oleic acid, we synthesized several isomers of nitro oleic acids and studied their chromatography and mass spectra by various techniques of mass spectrometry. LC/MS analysis performed on a high resolution micro column detected molecular carboxylic anions of various oleic acid nitro isomers (NO(2)OA). Esterification of NO(2)OA with pentafluorobenzyl bromide and diisopropylethylamine as a catalyst produced a unique isoxazole ester derivative exclusively from allyl NO(2)OA isomers via dehydration of the nitro group at ambient temperatures. This new analytical procedure revealed that *NO(2) generated two vinyl and two allyl isomers of NO(2)OA. The vinyl isomers showed high regioselectivity with the 1.8:1 preference for the 10-NO(2)OA isomer that was absent among allylic isomers. The nitration also generated elaidic acid via cis-trans isomerization and diatereoisomers of vicinal nitro hydroxy, nitro keto and alpha-nitro epoxy stearic acids with high stereo and regioselectivity. Nitration of small unilamelar phospholipid vesicles resulted in several phospholipids containing nitro lipids and elaidic acid amenable to hydrolysis by phospholipase A(2).

trans-Arachidonic acids induce a heme oxygenase-dependent vasorelaxation of cerebral microvasculature.

Nitrative stress is an important regulator of vascular tone. We have recently described that trans-arachidonic acids (TAA) are major products of NO(2)(.)-mediated isomerization of arachidonic acid in cell membranes and that nitrative stress increases TAA levels leading to neural microvascular degeneration. In the present study, we explored whether TAA exert acute effects on neuromicrovascular tone and investigated potential mechanisms thereof. TAA induced an endothelium-dependent vasorelaxation of rat brain pial microvasculature. This vasorelaxation was independent of nitric oxide, prostanoids, lipoxygenase products, and CYP(450) metabolite trans-hydroxyeicosatetraenoic acids. However, inhibition of heme oxygenase (using zinc protoporphyrin IX) and of dependent soluble guanylate cyclase (sGC; using ODQ) significantly diminished (by approximately 70%) the TAA-induced vasorelaxation. Consistent with these findings, TAA stimulated heme oxygenase (HO)-2-dependent bilirubin (using siRNA HO-2) and cGMP formation, and the HO product carbon monoxide (using CO-releasing CORM-2) reproduced the sGC-dependent cGMP formation and vasorelaxation. Further exploration revealed that TAA-induced vasorelaxation and bilirubin formation (HO activation) were nearly abrogated by large-conductance calcium-dependent potassium channels (BK(Ca)) (using TEA and iberiotoxin). Opening of BK(Ca) with the selective activator NS1619 induced a concentration-dependent vasorelaxation, which was inhibited by HO and sGC inhibitors. Coimmunoprecipitation suggested a molecular complex interaction between BK(Ca) and HO-2 (but not HO-1). Collectively, these findings identify new properties of TAA, specifically cerebral vasorelaxation through interactive activation of BK(Ca) with HO-2 and, in turn, sGC. Our findings provide new insights into the characterization of nitrative stress-derived TAA products, by showing they can act as acute mediators of nitrative stress on neurovascular tone.

Toxicity and carcinogenicity mechanisms of fibrous antigorite.

We studied the effects of fibrous antigorite on mesothelial MeT-5A and monocyte-macrophage J774 cell lines to further understand cellular mechanisms induced by asbestos fibers leading to lung damage and cancer. Antigorite is a mineral with asbestiform properties, which tends to associate with chrysotile or tremolite, and frequently occurs as the predominant mineral in the veins of several serpentinite rocks found abundantly in the Western Alps. Particles containing antigorite are more abundant in the breathing air of this region than those typically found in urban ambient air. Exposure of MeT-5A and J774 cells to fibrous antigorite at concentrations of 5-100 microg/ml for 72 hr induced dose-dependent cytotoxicity. Antigorite also stimulated the ROS production, induced the generation of nitrite and PGE2. MeT-5A cells were more sensitive to antigorite than J774 cells. The results of this study revealed that the fibrous antigorite stimulates cyclooxygenase and formation of hydroxyl and nitric oxide radicals. These changes represent early cellular responses to antigorite fibers, which lead to a host of pathological and neoplastic conditions because free radicals and PGE2 play important roles as mediators of tumor pathogenesis. Understanding the mechanisms of the cellular responses to antigorite and other asbestos particles should be helpful in designing rational prevention and treatment approaches.

Hypercapnia prevents neovascularization via nitrative stress.

Neovascularization after an ischemic insult is a beneficial attempt to salvage the injured tissue. Yet, despite the production of angiogenic factors within ischemic tissues, compensatory growth of new vessels fails to provide adequate vascularization. Thus, we hypothesized that local factors counter efficient revascularization. Whereas ischemia is often considered to be synonymous with an oxygen deficit, it is also associated with a concomitant local elevation of carbon dioxide (CO2). Although studies suggest that hypercapnia impacts tissue neovascularization, its significance relative to the abundantly described effects of hypoxia and its underlying mechanisms have yet to be elucidated. Therefore, we investigated the effects of hypercapnia on blood vessel growth in models of developmental and ischemic neovascularization. Acute and prolonged CO2 exposure inhibited developmental neovascularization of the rodent retina, as well as revascularization of the ischemic retina. Hypercapnia induced early increases in endothelial nitric oxide synthase and nitrative stress, associated with astrocyte impairment and endothelial cell death, as well as downregulation of the proangiogenic prostaglandin E2 receptor EP3. These results establish a previously unexplored means by which hypercapnia hinders efficient neovascularization, a mechanism that may contribute to ischemic tissue injury.

Cytochrome P450/NADPH-dependent biosynthesis of 5,6-trans-epoxyeicosatrienoic acid from 5,6-trans-arachidonic acid.

5,6-trans-AA (5,6-TAA, where TAA stands for trans-arachidonic acid) is a recently identified trans fatty acid that originates from the cis-trans isomerization of AA initiated by the NO2 radical. This trans fatty acid has been detected in blood circulation and we suggested that it functions as a lipid mediator of the toxic effects of NO2. To understand its role as a lipid mediator, we studied the metabolism of 5,6-TAA by liver microsomes stimulated with NADPH. Profiling of metabolites by liquid chromatography/MS revealed a complex mixture of oxidized products among which were four epoxides, their respective hydrolysis products (dihydroxyeicosatrienoic acids), and several HETEs (hydroxyeicosatetraenoic acids) resulting from allylic, bis-allylic and (omega-1)/(omega-2) hydroxylations. We found that the C5-C6 trans bond competed with the three cis bonds for oxidative metabolism mediated by CYP (cytochrome P450) epoxygenase and hydroxylase. This was evidenced by the detection of 5,6-trans-EET (where EET stands for epoxyeicosatrienoic acid), 5,6-erythro-dihydroxyeicosatrienoic acid and an isomer of 5-HETE. A standard of 5,6-trans-EET obtained by iodolactonization of 5,6-TAA was used for the unequivocal identification of the unique microsomal epoxide in which the oxirane ring was of trans configuration. Additional lipid products originated from the metabolism involving the cis bonds and thus these metabolites had the trans C5-C6 bond. The 5,6-trans-isomers of 18- and 19-HETE were likely to be products of the CYP2E1, because a neutralizing antibody partially inhibited their formation without having an effect on the formation of the epoxides. Our study revealed a novel pathway of microsomal oxidative metabolism of a trans fatty acid in which both cis and trans bonds participated. Of particular significance is the detection of the trans-epoxide of AA, which may be involved in the metabolic activation of such trans fatty acids and probably contribute to their biological activity. Unlike its cis-isomer, 5,6-trans-EET was significantly more stable and resisted microsomal hydrolysis and conjugation with glutathione catalysed by hepatic glutathione S-transferase.

5E, 8Z, 11Z, 14Z-eicosatetraenoic acid, a novel trans isomer of arachidonic acid, causes G1 phase arrest and induces apoptosis of HL-60 cells.

Trans arachidonic acid isomers (trans-AA) constitute a new group of trans fatty acids (trans-FA) generated in vivo via endogenous cis-trans isomerization stimulated by the NO2 radical. Because both NO2 and trans-FA have been implicated as causative factors in cancer, we studied the effect of the trans-AA isomers on proliferation and viability of human promyelocytic (HL-60) cells. The four trans arachidonic (trans-AA) acid isomers synthesized by us have been presently tested with respect to their competence to affect the proliferation and viability of human promyeolocytic HL-60 cells in culture. The data demonstrate that one of the isomers, 5,6-trans-AA, showed distinct activity by targeting cell progression through the cell cycle and inducing apoptosis. The effects were time- and concentration-dependent: the cytostatic effect of 5E-AA was observed at 10 microM following 72 h of treatment. This effect was manifested as a perturbation of cell progression through G1 phase, indicating the 'on' activation of the G1 checkpoint as evidenced by the flow- and laser scanning-cytometry techniques. Apoptotic cells were identified by comparison of their morphology, DNA fragmentation, caspase activation and collapse of mitochondrial potential with control cells. These observations suggested that 5E-AA induced a mitochondrial pathway of apoptosis. There was no evidence of cell-cycle phase specificity in induction of apoptosis by 5E-AA, as the cells showing highly fragmented DNA or caspase-3 activation were distributed in all phases of the cycle. The data suggest that 5E-AA may have at least two targets: one that is cell-cycle specific and associated with the observed arrest in the G1 phase and another, unrelated to the cell cycle, which is responsible for triggering apoptosis indiscriminately, regardless of cycle phase I.

Drugs that target lipoxygenases and leukotrienes as emerging therapies for asthma and cancer.

Considerable amount of work has been done in the area of enzymatic and non-enzymatic oxidation of arachidonic acid. This effort resulted in understanding of the functions of lipid mediators--eicosanoids in various aspects of health and disease. A mechanism by which aspirin exerts therapeutic effects puzzled pharmacologists for a long time until John Vane, in 1971, discovered that aspirin and its congeners block formation of prostaglandins, a class of lipids that originate from oxidation of arachidonic acid by cyclooxygenase. Since that discovery the pharmacology of eicosanoids has substantially progressed, which resulted in new drugs available in clinics. In addition to many new inhibitors of cyclooxygenase, two isoforms of which are known, much effort has been given to find inhibitors of synthesis and function of leukotrienes, a class of lipids that are derived from 5-lipoxygenase. These lipids are generated in asthma and their uncontrolled biosynthesis aggravates the symptoms of asthma. A new class of drugs called lukasts, inhibitors of 5-LOX products, has been developed and entered clinics as the first new therapy to treat asthma in nearly 20 years. New discoveries in the field of lipoxygenase show great opportunities for drug development for cancer prevention and treatment as it has been established that lipoxygenases and their products are required for cancer growth. Intense research in this field is likely to produce new drugs in the near future.

Interactions of peroxynitrite and other nitrating substances with human platelets: the role of glutathione and peroxynitrite permeability.

Platelets labeled with 2',7'-dihydrodichlorofluorescein diacetate (DCF-DA) and stimulated with 50-400nM peroxynitrite (ONOO(-)) produced a rapid increase of the fluorescence signal at 523nm with good linearity and reproducibility. Platelet fluorescence was inhibited by 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), suggesting that HCO(3)(-)/Cl(-) transporter mediated ONOO(-) transport into the platelets. Exposure of platelets to potassium superoxide, hydrogen peroxide, and sodium nitroprusside at concentrations of up to 100 microM did not generate a fluorescence signal. We also studied other nitrating compounds to establish the specificity of the DCF-DA-labeled platelet ONOO(-) assay. A rapid increase of fluorescence was observed when sodium hypochlorite (0.15 to 0.75mM) was added to platelets suspended in a buffered nitrite solution. Exposure of platelets to NO(2), nitroglycerin, and tetranitromethane produced a slow sustained increase of fluorescence. Endogenous glutathione appeared to be an essential factor in the generation of fluorescence by ONOO(-) and other nitrating compounds. We further studied other conditions that increased platelet fluorescence. Stimulation of platelets with thrombin (1U/mL) produced a rapid increase in fluorescence that corresponded to the formation of 20.5nmol ONOO(-) per 10(7) cells, whereas stimulation with collagen and arachidonic acid was without effect. Hypoxia of platelets for 20 and 40min followed by 5min of reoxygenation doubled the fluorescence from these platelets compared with control platelets. Thus, thrombin produced an effect that was likely due to the formation of ONOO(-) in platelets, whereas hypoxia-reoxygenation was likely to cause the formation of an active nitroglutathione-like molecule.

Biological nitration of arachidonic acid.

When a spontaneous autoxidation of arachidonic acid to prostaglandin-like products was first described almost 40 years ago, it was thought to be an artifact that interfered with the detection of enzymatically generated prostaglandins. It has now been generally accepted that the autoxidation of arachidonic acid occurs in vivo and leads to formation of isoprostanes and other products. Sensitive methods can detect the isoprostanes as useful biological markers, which help to estimate, non-invasively, the burden of free radicals formed in pathologies resulting from oxidative stress. After the discovery of NO, it has been hypothesized that NO and its active congeners (reactive nitrogen species, RNS), such as nitrogen dioxide radical (NO2), nitrous acid, peroxynitrite, can also participate in lipid peroxidation, either as initiators or modulators of processes initiated by the hydroxyl radical. In biological systems these RNS not only originate from the biosynthesis of NO but also from exogenous sources such as polluted air and dietary nitrite. While the ability of NO2 to induce lipid peroxidation has been long known, more recent studies have discovered novel processes that have been termed lipid nitration. Polyunsaturated fatty acids appear to be readily targeted by RNS. Among the products of arachidonic acid nitration by NO2, interesting lipids have been detected, such as nitroeicosatetraenoic acids, alpha,beta-nitrohydroxyeicosatrienoic acids, and trans-arachidonic acids. The products of fatty acid nitration have the potential to function as biomarkers and/or lipid mediators of mechanisms distinct from fatty acid peroxidation but offering insight into the contribution of specific RNS such as NO2 to the damage of biological membrane resulting from nitrooxidative stress.

Eicosanomics: targeted lipidomics of eicosanoids in biological systems.

Recent advancements in mass spectrometry, especially the development of electrospray tandem mass spectrometry (ESI/LC/MS2) and matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI/TOF), have greatly facilitated analysis of complex biomolecules. It has now become possible to profile, in relatively short periods of time, large multicomponent groups of compounds biosynthesized by biological systems. The efficiency and accuracy of analysis have led to the development of new concepts of mass spectrometric profiling, mapping, and imaging. Profiling of proteins in biological material (proteomics) has become a widely accepted strategy for identification of mechanisms involved in the biochemistry of disease processes, and has become a novel tool for unraveling new drug targets. Evolution of proteomics has relied on ESI/LC/MS2 and MALDI/TOF, techniques that are also useful in the novel area of quantitative proteomics.

Induction of nuclear receptors and drug resistance in the brain microvascular endothelial cells treated with antiepileptic drugs.

Our work contributes to the understanding of the mechanisms of drug resistance in epilepsis. This study aimed to investigate i) the levels of expression of P-glycoprotein (P-gp), and multidrug resistance-associated proteins (MRP)1 and 2, ii) the activation of the pregnane X receptor (PXR) and the constitutive androstane receptor (CAR), and iii) the relationship between increased P-gp and MRPs expression and PXR and CAR activation, in immortalized rat brain microvascular endothelial cell lines, GPNT and RBE4, following treatment with the antiepileptic drugs (AEDs), topiramate, phenobarbital, carbamazepine, tiagabine, levetiracetam, and phenytoin, using Western blotting and immunocytochemistry methods. Carbamazepine, phenobarbital and phenytoin induced the highest levels of P-gp and MPRs expression that was associated with increased activation of PXR and CAR receptors as compared to levetiracetam, tiagabine and topiramate. We conclude that P-gp and MRPs are differently overexpressed in GPNT and RBE4 by various AEDs and both PXR and CAR are involved in the drug-resistant epilepsy induced by carbamazepine, phenobarbital and phenytoin.

Fluoro-edenite fibers induce expression of Hsp70 and inflammatory response.

Many asbestos-like mineral fibers have been detected in the air of mountainous and volcanic areas of Italy and other parts of the world. These fibers have been suspected to be the cause of increased incidences of lung cancer and other lung diseases in these areas. However, the mechanisms of the cellular response and defense following exposure to these microscopic fibers have not been characterized. We continue to study these mechanisms to be able to propose preventive strategies in large populations. The objective of the present study was to determine comparatively biological responses of mesothelial Met-5A and monocyte-macrophage J774 cells following exposure to two types of fluoro-edenite fibers having low and high iron content (labeled 19 and 27, respectively) obtained from Biancavilla (Sicily, Italy). The reference fiber was a non-iron fibrous tremolite from Val di Susa (Piemonte, Italy). The cells were treated with 5, 50, and 100 mug of fibrous matter per 1 ml for 72 hr. We identified several key mechanisms by which cells responded and counteracted the injury induced by these fibers. The fibers caused induction of the heat shock protein 70 (Hsp70), stimulated formation of reactive oxygen species (detected by using DCFH-DA as a fluorescent probe) and NO* (measured as nitrite). Exposure of cells to the fibers induced lactate dehydrogenase activity and decreased viability. The fluoro-endenite type 27 was the most potent fiber tested, which indicated that iron and possibly manganese contribute significantly to this fiber toxicity. The J774 cells were more sensitive to fluoro-edenite than Met-5A cells suggesting that the primary site of the fiber-induced inflammatory response could be the macrophage rather than the pulmonary epithelium. Fluoro-edenite produces more biological alterations with respect to non-iron tremolite. Hsp70 and free radicals could be important factors in the context of mineral fiber-induced acute lung injury leading possibly to mutagenic effects. We anticipate that pharmacological blockade of the fiber-dependent cellular responses could in long term offer preventive approach to combat lung diseases induced by these fibers.