Freeman Lab

The basic investigation and clinical research activities of the Freeman Laboratory focus on the eukaryotic cell production, reactions and signal transduction properties of oxidizing and free radical inflammatory mediators (e.g., superoxide, hydrogen peroxide, nitric oxide (NO), peroxynitrite, nitrogen dioxide, oxidized/nitrated lipids).  In particular, we are interested in the actions of these species as both redox signaling mediators, as pathogenic agents in inflammatory diseases and in the case of nitro-fatty acids as drug candidates.  Our observations regarding O2 and NO-derived reactive species have lent new insight into mechanisms of redox-dependent cell signaling, post-translational protein modification and have revealed new therapeutic strategies for treating acute inflammation, metabolic syndrome, respiratory disorders and cardiovascular diseases.

Below displays the Research Details from the profile of each member of the lab.

Marco Fazzari, PhD

Electrophilic fatty acid nitroalkenes (NO2-FA) are products of nitric oxide and nitrite-mediated unsaturated fatty acid nitration, which mediate pleiotropic signaling actions modulating metabolic and inflammatory responses in cell and animal models. The generation of NO2-FA is promoted by inflammatory responses in cells, such as ischemia-reperfusion injury, and in the gastrointestinal tract, where acidic conditions favor the nitration of unsaturated fatty acids. So far, fatty acid nitration has been predominantly studied using free unsaturated fatty acids because of challenges in the direct mass spectrometric analysis of complex lipids and nitroalkene instability during de-esterification reactions. This is a limitation, as dietary and endogenous free fatty acid levels are proportionately very low when compared to the levels of esterified fatty acids in tissue compartments and foods.

 

My research projects are all directed towards developing a basic understanding of the biological generation and metabolism of electrophilic lipid signaling mediators, which have only been partially studied and still remains a large gap in knowledge regarding the detection, characterization and physiologic actions of NO2-FA-containing complex lipids. Recently I have shown that sources of unsaturated fatty acid-containing triacylglycerides in the diet (e.g. plant or animal lipids) support the formation of NO2-FA upon digestion.

 

 

Furthermore, the direct nitration of complex lipids or the incorporation of free NO2-FA into triglycerides in adipocytes form a reservoir of electrophilic lipids that can react directly with nucleophilic targets or mediate signaling actions upon hydrolysis to free NO2-FA by cellular lipases.

 

 

Finally, I have focused my research interests on the acidic gastric nitration of conjugated linoleic acid (CLA)-containing lipids which generates of unstable nitro-nitrate intermediates. Decomposition of these species, which are spontaneous at physiological pH for free CLA-derived products and catalyzed by lipase-mediated hydrolysis for nitro-nitrate triglycerides, yield nitro-conjugated linoleic acid (NO2-CLA), active nitrogen oxide and nitrosating species with potential beneficial effects.


Bruce A. Freeman, PhD

The basic and clinical research activities of the Freeman Laboratory focus on the eukaryotic cell production, reactions and signal transduction properties of oxidizing and free radical inflammatory mediators (e.g., superoxide, hydrogen peroxide, nitric oxide (NO), peroxynitrite, nitrogen dioxide, oxidized/nitrated lipids). In particular, we are interested in the action of these species as both redox signaling mediators under basal conditions and as pathogenic agents in inflammatory diseases. Our observations regarding O2 and NO-derived reactive species have lent new insight into redox-dependent cell signaling and have revealed new therapeutic strategies for treating acute inflammation, metabolic syndrome, respiratory disorders and cardiovascular diseases.
In the late 1980s, his group studied the cellular and subcellular organelle production of superoxide and hydrogen peroxide. Following the landmark description of endothelial-derived relaxing factor (EDRF) as the free radical NO, the Freeman laboratory pioneered the concept that the inflammatory and signal transduction mediator NO displays unique redox signaling actions following reaction with superoxide, oxidizing fatty acids and heme peroxidases. The “oxidative inactivation” of NO is a kinetically fast reaction, yielding “reactive nitrogen species” as products. This array of reactions of O2-derived species with NO can serve to both impair and transduce NO signaling via non-cGMP dependent mechanisms.
 
There is now a rapidly expanding appreciation that NO-derived reactive species display distinct chemical reactivities and exert cell signaling actions beyond the activation of guanylate cyclase – e.g., via thiol oxidation, electrophilic addition and receptor-dependent reactions. This aspect of redox-related chemical biology is an area that the Freeman laboratory continues to investigate, with the intent of defining the linkages between reactive oxygen species and NO-dependent cell signaling mechanisms. From a translational research perspective, his group is addressing how these interactions impact cell and organ function, with particular directed towards metabolic, cardiovascular and pulmonary diseases.
 
Dr. Freeman's laboratory observed that NO reacts with superoxide (O2-) to yield the potent biological oxidizing and nitrating species peroxynitrite (ONOO-)and its conjugate acid, peroxynitrous acid (ONOOH). Groundbreaking observations were made in this area by Joe Beckman, PhD and Rafael Radi, MD, PhD. Their work showed that peroxynitrite is both a direct oxidant and, after homolytic scission of peroxynitrous acid, yields the potent oxidant hydroxyl radical (OH) and the oxidizing and nitrating species nitrogen dioxide (NO2) (Fig. 1). Also, they identified thiols and carbon dioxide as the principal biological targets of peroxynitrite. It is now known that peroxynitrite accounts for many of the pathogenic actions previously ascribed to its precursors - superoxide (and its products) and NO. Work from many laboratories continues to affirm that peroxynitrite mediates redox cell signaling actions upon the oxidation or nitration of target molecules such as thiols, aromatic amino acids, nucleotides and unsaturated fatty acids – with downstream cell signaling events and reactions of peroxynitrite now appreciated to be a consequence of its potent and unique reactivities.
 

Figure 1. Peroxynitrite is formed from the reaction of NO and superoxide and yields secondary oxidizing and nitrating species

 
 
 
An observation from the Freeman laboratory, related to peroxynitrite biochemistry and pharmacology, has yielded new insight into biochemical and tissue responses to ischemia. Specifically, the CO2 accumulation that occurs during impaired tissue perfusion and oxygen delivery displays potent pro-inflammatory properties. Observations made by Dr. Radi showed that carbon dioxide indirectly affects the reactivity of O2-and NO, via its facile chemical reaction with the superoxide and NO reaction product, peroxynitrite. This reaction yields the potent oxidizing and nitrating species nitrosoperoxocarbonate (ONOOCO2) that in turn yields secondary radical species (Fig. 2). John Lang, MD then discovered in an animal model of sepsis that there is a potent contribution of CO2 to tissue redox signaling and inflammatory responses. For example, clinically-relevant mechanical ventilation strategies performed on anesthetized rabbits reveals that mild hypercapnia amplifies inflammatory lung injury. Of interest, this also causes a CO2-dependent increase in iNOS gene/protein expression and NO/ONOO- production. The discovery that CO2 actively participates in oxidative inflammatory reactions has relevance to ICU-related care and organ transplantation.
 
 

 

Figure 2. The chemical reactivities and cell signaling actions of reactive oxygen species and nitric oxide are intimately linked.

 

Figure3

Figure 3. The PMN-dependent release of MPO results in subendothelial deposition and the generation and reaction of secondary NO-consuming, oxidizing and nitrating species.

 

Studies with wild type and MPO-/- mice undergoing an acute inflammatory response provided another important insight into the actions of MPO during NO signaling. Specifically, reactions catalyzed by MPO directly modulate vascular relaxation and inflammatory responses by regulating NO bioavailability. In addition to directly reacting with NO (a kinetically slow reaction), MPO predominantly alters vascular responsiveness by generating substrate radicals (such as tyrosyl radical and ascorbyl radical) that rapidly consume NO and abrogate its cGMP-dependent signaling capabilities. Thus, multiple reactions of MPO lead to biomolecule nitration and NO consumption.

 


Figure 4. MPO oxidizes nitrite to nitrogen dioxide and catalytically consumes NO.

 

Of important clinical relevance, Drs. Margaret Tarpey and Stephan Baldus have discovered that enzymatic reactions leading to the catalytic consumption of NO impair vascular function and are linked with increased risk for an adverse myocardial event (heart attack or death) in patients. The main perpetrators of oxidative NO consumption in the vasculature appear to be the reactive species derived from xanthine oxidoreductase (XO) and MPO, with both plasma XO and MPO levels elevated in patients with coronary artery disease. The work of others also suggest that a variety of NA(D)PH oxidases act in a similar manner. As for MPO, XO readily binds to and enters the vessel wall, with this occurring to a much greater extent in patients with coronary artery disease (Fig. 5). Work by Dr. Baldus convincingly shows that both coronary blood flow and the risk for adverse myocardial events are strongly linked with plasma MPO levels in patients. More recently, it has been observed that XO also contributes to impaired coronary vasomotion in patients.

 

 

Figure 5. Xanthine oxidase readily binds to and is incorporated by vascular cells.

 

In coronary artery disease patients XO accumulates along the vessels wall and catalytically consumes NO. Dr. Freeman's group is presently actively investigating the pluripotent signaling actions of the NO-derived, nitrated unsaturated fatty acids formed during enzymatic and autocatalytic lipid oxygenation (Fig 6). Homero Rubbo, PhD discovered that NO potently inhibits fatty acid oxidation, via reactions that are >2000 times faster than similar events catalyzed by vitamin E. Dr. Rubbo also observed at the same time that NO-dependent reactions induce fatty acid nitration. Building on this observation, Valerie O’Donnell, PhD lent important structural and functional insight into these endogenously-present species, and how they can be formed biologically.

 

Figure 6. A spectrum of nitrated fatty acids are produced by NO and nitrite-dependent oxidative inflammatory reactions.

 

 

Nitro-fatty acids are present under physiological and pathological conditions in a broad range of species, including insects, fish (e.g., salmon), plants (e.g., olives) and humans. The consumption of diets promoting increased tissue and plasma nitro-fatty acid levels has recently been proposed to account for a significant element of the health benefits linked with Mediterranean and Japanese-like diets.

The use of high performance liquid chromatography techniques coupled with high accuracy mass spectrometry analysis has given precise structural and conformational characterization of these species. As a result, pure preparations of synthetic nitro-fatty acids are structurally identical to those found endogenously in humans at nM to sometimes mM concentrations.

 

Figure 7. High-accuracy mass spectrometry fragmentation analysis of CXA-10

 

 

Figure 8. Endogenous generation and metabolism of nitro-fatty acids

The physiological generation of nitro-fatty acids occurs via two main pathways:

  1. Acid-catalyzed unsaturated fatty acid nitration by dietary nitrite coming from vegetables and cured meats.
     
  2. Free radical-mediated fatty acid nitration reactions during digestion, metabolic stress and inflammatory conditions.

These reaction mechanisms support the formation of nitrogen dioxide (NO2) which rapidly reacts with unsaturated double bonds in fatty acids, giving rise to the formation of nitro-fatty acid derivatives. These products contain a reactive center that interacts with critical protein targets and induces post-translational protein modifications. Somewhat akin to protein phosphorylation or dephosphorylation, nitro-fatty acids transiently alter the function of key target proteins to modulate the activity of critical gene expression programs, enzymatic activities and signaling network activities – thereby inducing characteristic anti-inflammatory, anti-fibrotic and cytoprotective actions.

Nitro-fatty acid metabolism and clearance - The formation of reversible adducts between target proteins and nitro-fatty acids results from the “soft” electrophilic nature of nitro-fatty acids. This chemical property is also the basis for nitro-fatty acid reaction with glutathione (GSH), which helps regulate nitro-fatty acid levels. Products of GSH and nitro-fatty acid reaction are exported from the cell to the circulation, where these adducts are filtered by the kidneys and excreted in urine, where nitro-fatty acid levels can be easily detected by mass spectrometry analysis. Nitro-fatty acids are also irreversibly inactivated by the enzyme prostaglandin reductase-1. Finally, like all fatty acids, nitro-fatty acids are metabolized by the mitochondrial energy-generating process termed beta-oxidation. These pathways all modulate the activity and half-life of both endogenous and exogenous nitro-fatty acids. Importantly, all critical protein adducts, gene expression events and metabolic products can be monitored in accessible tissue compartments, the plasma and urine of humans and animal models, thereby providing real-time insight into the metabolism and actions of nitro-fatty acids.



Figure 9. Metabolism of nitro-fatty acids. Nitro-fatty acids such as nitro-oleic acid or nitro-linoleic acid react with cysteine residues in proteins to modify enzymatic activity and regulate transcription factor function and downstream gene expression. Similarly, the reaction of or transfer of a nitro-fatty acid to GSH reverses protein-nitro-fatty acid adduct formation and facilitates export from the cell to eventual urinary excretion. These species also undergo beta-oxidation reactions and are inactivated by prostaglandin reductase-1.

 

Nitro-fatty acids are adaptive signaling mediators

Biological systems are endowed with an arsenal of sentinel proteins that constantly sense and react to changes in the cellular environment, in order to ensure the continued function of critical life-sustaining processes over a wide range of pathophysiological scenarios. In this regard, the detection of an invading pathogen or a toxin is met by the activation of pro-inflammatory cascades aimed at neutralizing the threat. The excessive or chronic activation of these physiological pathways can, if left unchecked, result in further damage to organs and tissues and the development of disease.

Cells rely on the generation of adaptive signaling mediators that dynamically modulate the extent and duration of inflammatory and metabolic responses to external insults. Nitro-fatty acids were discovered to be a novel family of adaptive signaling mediators that pleiotropically down-regulate inflammation, activate protective cellular responses and induce endogenous production of antioxidant defenses in cells. Specifically, nitro-fatty acids reversibly react with electrophile-susceptible transcription factors and protein thiols to modulate gene expression and enzyme activities via the post-translational modification of functionally-significant proteins.

This ability of nitro-fatty acids to reversibly adduct proteins is at the root of their potent signaling actions and apparent lack of toxicity. For example, the pro-inflammatory actions of bacterial lipopolysaccharides (LPS) are inhibited by nitro-fatty acid adduction of the NFkB p65 subunit that undergoes DNA binding and mediates the stimulation of pro-inflammatory gene expression. Additionally, nitro-fatty acid reaction with critical cysteines in Keap1 results in the nuclear translocation of the transcription factor Nrf2 leading to increased generation of antioxidant and cytoprotective proteins by the cell. Accumulating evidence from multiple international research groups support that these cytoprotective activities of nitro-fatty acids can be harnessed for the development of effective therapies for the treatment of inflammatory and metabolic diseases. Notably, the concentrations of nitro-fatty acids that exert potent protective actions in murine models of disease (obesity-induced diabetes, restenosis, atherosclerosis, inflammatory bowel disease, pulmonary hypertension, acute and chronic renal injury, etc.) are in the 5-25 nM range, well within a pharmacologically attainable and safe range in humans.

Figure 10. Signaling pathways modulated by nitro-fatty acids. This includes the suppression of pro-inflammatory gene expression by NFkB inhibition; induction of the expression of heat shock proteins; and upregulation of antioxidant defenses by the induction of Nrf2-regulated gene expression.

 


Nicholas Khoo, PhD

Link:  Translational Program Project / Vascular Sub-Phenotypes of Lung Disease  http://www.vmi.pitt.edu/tPPG.html 
 
  1. Define the formation and signaling actions of electrophilic nitro-fatty acids. Electrophilic nitroalkene-containing fatty acids (NO2-FA) are products of nitric oxide (NO)/nitrogen dioxide reactions with unsaturated fatty acids that are generated at increased rates during metabolic stress and inflammation.  These endogenously produced mediators display a broad range of adaptive signaling actions. While the nitroalkene moiety confers reversible electrophilic reactivity with nucleophiles such as cysteine and histidine, promoting the post-translational modification of reactive protein thiols, the exact signaling mechanisms underlying the physiological actions of NO2-FA and their conditions of formation and metabolism remain unclear.
  2. Determining the interplay between mechanisms of adipose dysfunction and electrophilic nitro-fatty acid signaling to modulate obesity and insulin resistance. Obesity, a low-grade systemic inflammatory disease, is central to the pathogenesis of insulin resistance and type 2 diabetes. White adipose tissue, a major physiological compartment affected by obesity, has emerged as a signaling nexus that strongly impacts energy balance and glucose homeostasis. While our studies suggest that adipose tissue is a unique environment for NO2-FA signaling, the molecular mechanism(s) by which obesity impacts NO2-FA actions and those accounting for the therapeutic actions of NO2-FA remain unclear. Some of the publications listed below demonstrate the significant impact adipose tissue has on glucose homeostasis, the esterification process of NO2-FAs in adipocytes and that NO2-FAs improve glucose tolerance. 
  3. Define fundamental signaling mechanisms of tissue generation and reactions of reactive oxygen species (ROS). Oxidative stress, which occurs when ROS levels are higher than the antioxidant enzyme activities within a cell, is linked with a plethora of pathologies. However, ROS also have been described as signaling molecules that regulate physiological homeostasis. Assessment of reactive species in tissues and cells is routinely accomplished by measuring the accumulation of more stable secondary byproducts of redox reactions and/or the reduction in concentration of small molecule antioxidants (such as glutathione). Over the last few years we have published techniques to accurately detect reactive species in vivo (DMPO method), determine the source of oxidants from enzymes and the regulation of antioxidant enzymatic activities. These publications are seminal in defining conditions of oxidative stress in tissues and cells.
In summary, these research interests will generate insights into mechanisms leading to obesity and its associated myriad of health problems and/or diseases such as diabetes, atherosclerosis and other cardiovascular complications, which will hopefully elucidate novel preventative and therapeutic strategies.

Dario Vitturi, PhD

Dr. Vitturi's research focuses on the mechanisms by which biological systems harness the generation of free radicals and other reactive species to modulate cell signaling and maintain physiological homeostasis. He is particularly interested in nitrite as a physiologically relevant precursor for nitric oxide (NO) formation that is activated under conditions of acidosis or through metalloprotein-dependent reduction in hypoxic environments. Nitrite has been shown to be a promising therapeutic intervention for many conditions associated with uncontrolled inflammation, decreased NO availability and ischemia-reperfusion events. Importantly, NO generation is just one aspect of the biological reactivity of nitrite, with this molecule also acting as a precursor for the formation of secondary species such as S-nitrosothiols, N-nitrosamines, and nitrated fatty acids, all of which are capable of mediating specific cellular responses. He is also interested in elucidating pathways of formation and signaling actions of endogenous electrophilic molecules. Electrophiles react covalently with nucleophilic residues in proteins, thus modulating enzyme activity and eliciting concerted cell signaling responses which ultimately have the potential to modulate antioxidant responses, inflammation and tissue injury under pathological conditions. Thus, his research encompasses the study of biochemical pathways leading to the formation of reactive species, the elucidation of their allied biological actions and ultimately the development of clinically viable interventions for their modulation under pathological conditions.
 

Stacy Gelhaus Wendell, PhD

Link:  Translational Program Project / Vascular Sub-Phenotypes of Lung Disease  http://www.vmi.pitt.edu/tPPG.html 


Nitrated and oxidized metabolites of omega-3 and omega-6 fatty acids, such as docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA) arachidonic acid (AA) and linoleic acid (LA), are potent signaling mediators involved in inflammation and resolution, amongst a variety of other regulatory pathways.  These oxidized metabolites are produced by reactive oxygen and nitrogen species as well as through enzymatic pathways including cyclooxygenase (COX), lipoxygenase (LO), and cytochrome P450s (CYP450).  Dr. Gelhaus’ research is specifically focused on understanding the anti-inflammatory mechanisms of a specific subgroup of these metabolites; the electrophilic fatty acids.  Examples of electrophilic fatty acids include nitrated fatty acids such as nitro-oleic, nitro-linoleic, and nitro-conjugated linoleic acid or oxidized lipids that contain an a-,ß-unsaturated ketone or epoxide moiety.  Electrophilic fatty acid metabolites can exert their effects through the modulation of transcriptional regulatory proteins.  Many transcriptional regulatory proteins contain a nucleophilic amino acid residue, such as a cysteine or histidine, to which the electrophilic moiety of the fatty acid can form a reversible Michael addition.  Many of these interactions have been described with the nitrated fatty acids, particularly nitro-oleic acid.  In terms of cellular signaling, the Freeman lab has described several protein targets of posttranslational nitroalkylation modification, resulting in activation of pro-MMP9, PPARϒ, heat shock factors, Keap1/Nrf2 and the inhibition of the pro-inflammatory transcription factor NF-κB.

 

The current focus of Dr. Wendell's research is on the mechanism of these electrophilic fatty acids in asthma.  Asthma is a complicated disease that much like cancer is comprised of numerous disease states and phenotypes.  In many ways it is an umbrella of respiratory diseases that share some similar phenotypes such as airway hyper-responsiveness, increased mucus secretion, increased smooth muscle contraction and decreased airflow.  In the most severe of cases, airway remodeling and corticosteroid resistance are not uncommon.  Asthma affects over 30 million worldwide and is an economic burden with therapeutic costs upwards of $19 billion dollars per year.  While the number of asthmatics in Westernized countries seems to be plateauing, the world-wide number of asthmatics is projected to hit over 100 million by 2025, mostly in low/middle economically developing countries. 

 

Dr. Wendell is looking at the signaling of electrophilic fatty acids in transformed stable cell lines while collaborating with clinicians to study the formation and signaling of electrophilic fatty acids in healthy controls, mild to moderate, and severe asthmatic subjects.  In this study, subjects undergo a baseline bronchoscopy after which they are placed randomly in one of three groups—control, aspirin, or indomethacin.  The thought here is that indomethacin will completely inhibit cyclooxygenase activity; therefore, shunting metabolism to other pathways including lipoxygenase or CYP450.  Aspirin will inactivate COX-1, but acetylate COX-2 at S516, thus altering enzymatic activity and the stereochemistry of product formation.  Following 5 days of treatment, subjects return for a second bronchoscopy.  At each bronchoscopy, blood, urine, bronchoalveolar lavage, and bronchial brushings are taken.   The epithelial cells from the brushings can be cultured at the air liquid interface for mechanistic studies and identification of key electrophilic fatty acids.  To reach these end goals, the lab utilizes molecular biology and analytical techniques, primarily mass spectrometry, (triple quadrupole and ion trap) to elucidate the structures of novel electrophilic species, accurately detect and quantify key electrophilic fatty acid oxidation products in biological matrices, and establish mechanisms of action in asthma and other lung and airway diseases.  Furthermore, the implications of electrophilic fatty acid formation and signaling under inflammatory conditions and the ability of electrophilic fatty acids to decrease airway hyperresponsiveness are being investigated in a house dust mite allergen murine model of asthma.


Marco Fazzari, PhD
Research Assistant Professor


Bruce A. Freeman, PhD
Irwin Fridovich Distinguished Professor and Chair


Nicholas Khoo, PhD
Research Assistant Professor


Lihua Li
Research III


Crystal Uvalle
Research Specialist IV


Dario Vitturi, PhD
Research Assistant Professor


Stacy Gelhaus Wendell, PhD
Research Associate Professor

Marco Fazzari, PhD

Journal Articles

M Fazzari, N Khoo, SR Woodcock, DK Jorkasky, L Li, FJ Schopfer and BA Freeman. Nitro-fatty acid pharmacokinetics in the adipose tissue compartment. Journal of Lipid Research 58:375-385, 2017. 
SR Salvatore, DA Vitturi, M Fazzari, DK Jorkasky and FJ Schopfer. Evaluation of 10-nitro oleic acid bio-elimination in rats and humans. Scientific Reports 7: 39900, 2017.
M Fazzari, N Khoo, SR Woodcock, L Li, BA Freeman and FJ Schopfer. Generation and esterification of electrophilic fatty acid nitroalkenes in triacylglycerides. Free Radical Biology & Medicine 87:113-124, 2015. 
DA Vitturi, L Minarrieta, SR Salvatore, EM Postlethwait, M Fazzari, G Ferrer-Sueta, JR Lancaster, BA Freeman and FJ Schopfer. Convergence of biological nitration and nitrosation reactions via symmetrical nitrous anhydride. Nature Chemical Biology 11:504-510, 2015.
M Fazzari, A Trostchansky, F.J Schopfer, SR Salvatore, B Sanchez-Calvo, D Vitturi, R Valderrama, JB Barroso, R Radi, BA Freeman and H Rubbo. Olives and olive oil are sources of electrophilic Fatty Acid nitroalkenes. PLoS One 9:e84884, 2014.

Bruce A. Freeman, PhD

Journal Articles

Vitturi DA, L Minarrieta, SR Salvatore, EM Postlethwait, M Fazzari, G Ferrer-Sueta, JR Lancaster, BA Freeman* and FJ Schopfer*.       Convergence of biological nitration and nitrosation via symmetrical nitrous anhydride. Nature Chem Biol  doi: 10.1038/nchembio.1814, 2015. *Corresponding authors
Wendell SG, F Golin-Bisello, S Wenzel, RW Sobol, F Holguin and BA Freeman. 15-hydroxy-prostaglandin dehydrogenase generation of electrophilic lipid signaling mediators from hydroxy Ω-3 fatty acids. J Biol Chem 290:5868-5880, 2015.
Woodcock SR, SR Salvatore, G Bonacci, FJ Schopfer and BA Freeman. Biomimetic nitration of conjugated linoleic acid: Formation, characterization and synthesis of naturally occurring conjugated nitrodienes. J Org Chem 79:25-33, 2014.
Charles RL, O Rudyk, O Prysyazhna, A Kamyina, J Yang, C Morisseau, BD Hammock, BA Freeman and P Eaton. Protection from hypertension in mice by the Mediterranean diet is mediated by nitro fatty acid inhibition of soluble epoxide hydrolase.       Proc Nat Acad Sci 111:8167-8172, 2014.
Rudolph V, TK Rudolph, FJ Schopfer, G Bonacci, SR Woodcock, MP Cole, PRS Baker, R Ramani and BA Freeman.  Endogenous generation and protective effects of nitro-fatty acids in a murine model of focal cardiac ischaemia and reperfusion.  Cardiovascular Research 85:155-166, 2010.
Groeger AL, C Cipollina, MP Cole, SR Woodcock, G Bonacci, TK Rudolph, V Rudolph, BA Freeman and FJ Schopfer.  Cyclooxygenase-2 generates anti-inflammatory mediators from omega-3 fatty acids.  Nature Chemical Biology 6:433-441, 2010.
Batthyany C, FJ Schopfer, PR Baker, R Duran, LM Baker, Y Huang, C Cervenansky, BP Branchaud and BA Freeman. Reversible post-translational modification of proteins by nitrated fatty acids in vivo. J Biol Chem 281: 20450-20463, 2006.
Schopfer F, Y Lin, P Baker, T Cui, M Garcia-Barrio, J Zhang, K Chen, Y Chen and BA Freeman.       Nitrolinoleic acid – an endogenous PPARγ ligand. Proc Nat Acad Sci 102:2340-2345, 2005.
Eiserich JP, S Baldus, M-L Brennan, W Ma, C Zhang, A Tousson, L Castro, AJ Lusis, CR White and BA Freeman. Myeloperoxidase: A leukocyte-derived vascular ·NO oxidase. Science 296:2391-2394, 2002.
Eiserich JP*, M Hristova, CE Cross, AD Jones, BA Freeman, B Halliwell and A van der Vliet*. Formation of nitric oxide-derived inflammatory oxidants by myeloperoxidase in neutrophils. Nature 391:393-397, 1998. *PhD students.
Rubbo H, R Radi, M Trujillo, R Telleri, B Kalyanaraman, S Barnes, M Kirk and BA Freeman. Nitric oxide regulation of superoxide and peroxynitrite-dependent lipid peroxidation: Formation of novel nitrogen-containing oxidized lipid derivatives. J Biol Chem 265:26066-26075, 1994.
Radi R, JS Beckman and BA Freeman. Peroxynitrite oxidation of sulfhydryls: The cytotoxic potential of endothelial-derived superoxide and nitric oxide. J Biol Chem 266:4244-4250, 1991.
Beckman JS, TW Beckman, J Chen, PA Marshall and BA Freeman. Apparent hydroxyl radical production by peroxynitrite: Implications for endothelial injury from nitric oxide and superoxide. Proc Natl Acad Sci 87:1620-1624, 1990.
Rosen GR and BA Freeman. Detection of superoxide in endothelial cells. Proc Natl Acad Sci 81:7269-7273, 1984.

Complete List of Published Work in MyBibliography - ~225 research articles, 65 reviews, H index 91

http://www.ncbi.nlm.nih.gov/sites/myncbi/collections/public/16gVumapi7L0c0xgqU9Vuej5m/?sort=date&direction=ascending

Nicholas Khoo, PhD

Journal Articles

Fazzari M, NK Khoo, SR Woodcock, L Li, BA Freeman and FJ Schopfer.  Generation and esterification of electrophilic fatty acid nitroalkenes in triacylglycerides.  Free Radic Biol Med 87:113-124, 2015.
Chartoumpekis DV, DL Palliyaguru, N Wakabayashi, NK Khoo, G Schoiswohl, RM O'Doherty and TW Kensler. Notch intracellular domain overexpression in adipocytes confers lipodystrophy in mice. Molecular Metabolism 4:543-550, 2015.
Kelley EE, J Baust, G Bonacci, F Golin-Bisello, JE Devlin, CM St Croix, SC Watkins, S Gor, N Cantu-Medellin, ER Weidert, JC Frisbee, MT Gladwin, HC Champion, BA Freeman and NK Khoo. Fatty acid nitroalkenes ameliorate glucose intolerance and pulmonary hypertension in high-fat diet-induced obesity. Cardiovasc Res 101:352-363, 2014.
Khoo NK, L  Mo, S Zharikov, C Kamga-Pride, K Quesnelle, F Golin-Bisello, L Li, Y Wang and S Shiva. Nitrite augments glucose uptake in adipocytes through the protein kinase A-dependent stimulation of mitochondrial fusion. Free Radic Biol Med 70:45-53, 2014.
Khoo NK, S Hebbar, W Zhao, SA Moore, FE Domann and ME Robbins. Differential activation of catalase expression and activity by PPAR agonists: Implications for astrocyte protection in anti-glioma therapy. Redox Biol 1:70-79, 2013. 
Khoo NKH, N Cantu-Medellin, JE Devlin, CM St Croix, SC Watkins, AM Fleming, HC Champion, RP Mason, BA Freeman and EE Kelley.  Obesity-induced tissue free radical generation:  An in vivo immuno-spin trapping study.  Free Radical Biol Med 52:2312-2319, 2012.
Khoo NKH, V Rudolph, MP Cole, F Golin-Bisello, SR Woodcock, C Batthyany and BA Freeman.  Activation of vascular endothelial nitric oxide synthase and heme oxygenase-1 by electrophilic nitro-fatty acids.  Free Radical Biology and Medicine 48:230-239, 2010.
Khoo NKH, CR White, L Pozzo-Miller, F Zhou, C Constance, T Inoue, RP Patel and DA Parks.  Dietary flavonoid quercetin stimulates vasorelaxation in aortic vessels.  Free Radical Biology and Medicine 49:339-447, 2010.

Dario Vitturi, PhD

Journal Articles

Villacorta L, L Minarrieta, SR Salvatore, NK Khoo, O Rom, Z Gao, RC Berman, S Jobbagy, L Li, SR  Woodcock, YE Chen, BA Freeman, AM Ferreira, FJ Schopfer and DA Vitturi. In situ generation, metabolism and immunomodulatory signaling actions of nitro-conjugated linoleic acid in a murine model of inflammation. Redox Biol 5:522-531, 2018.
 
Turell L, DA Vitturi, EL Coitino, L Lebrato, M Möller, C Sagasti, SR Salvatore, SR Woodcock, B Alvarez and FJ Schopfer. The chemical basis of thiol addition to nitro-conjugated linoleic acid, a protective cell-signaling lipid. J Biol Chem 292:1145-1159, 2017. 
Vitturi DA, L Minarrieta, SR Salvatore, EM Postlethwait, M Fazzari, G Ferrer-Sueta, JR Lancaster Jr., BA Freeman and Schopfer FJ. Convergence of biological nitration and nitrosation via symmetrical nitrous anhydride. Nat Chem Biol 11:504-510, 2015.
 
Vitturi DA, CS Chen, SR Woodcock, SR Salvatore, G Bonacci, JR Koenitzer, NA Stewart, N Wakabayashi, TW Kensler, BA Freeman and FJ Schopfer.  Modulation of nitro-fatty acid signaling:  Prostaglandin reductase-1 is a nitroalkene reductase. J Biol Chem 288:25626-25637, 2013.
Salvatore SR, DA Vitturi, PR Baker, G Bonacci, JR Koenitzer, SR Woodcock, BA Freeman and FJ Schopfer.  Characterization and quantification of endogenous fatty acid nitroalkene metabolites in human urine.  J Lipid Res 54:1998-2009, 2013.
Vitturi DA, CW Sun, VM Harper, B Thrash-Williams, N Cantu-Medellin, BK Chacko, N Peng, Y Dai, JM Wyss, T Townes and RP Patel.  Antioxidant functions for the hemoglobin ß93 cysteine residue in erythrocytes and in the vascular compartment in vivo.  Free Radic Biol Med 55:119-129, 2013.
Rodriguez C, DA Vitturi, J He, M Vandromme, A Brandon, A Hutchings, LW Rue, JD Kerby and RP Patel.  Sodium nitrite therapy attenuates the hypertensive effects of HBOC-201 via nitrite reduction.  Biochem J 422:423-432, 2009.
Vitturi DA, X Teng, JC Toledo, S Matalon, JR Lancaster and RP Patel.  Regulation of nitrite transport in red blood cells by hemoglobin oxygen fractional saturation.  Am J Physiol Heart Circ Physiol 296:H1394-H1407, 2009.
Isbell TS, CW Sun, LC Wu, X Teng, DA Vitturi, BG Branch, CG Kevil, N Peng, JM Wyss, N Ambalavanan, L Schwiebert, J Ren, KM Pawlik, MB Renfrow, RP Patel and TM Townes.  SNO-hemoglobin is not essential for red blood cell-dependent hypoxic vasodilation.  Nat Med 14:773-777, 2008.
Ferrer-Sueta G, D Vitturi, I Batinic-Haberle, I Fridovich, S Goldstein,G Czapski and R Radi. Reactions of manganese porphyrins with peroxynitrite and carbonate radical anion. J Biol Chem 278:27432-27438, 2003.
 

Stacy Gelhaus Wendell, PhD

Journal Articles

Delmastro-Greenwood M, BA Freeman and SG Wendell.  Redox-dependent anti-inflammatory signaling actions of unsaturated fatty acids.  Annual Review of Physiology 76:79-105, 2014.
Woodcock S, G Bonacci, SL Gelhaus and F Schopfer.  Nitrated fatty acids:  Synthesis and measurement.  Free Radical Biology and Medicine 59:14-26, 2013.
Fajt ML, SL Gelhaus, B Freeman, CE Uvalle, JB Trudeau, F Holguin and SE Wenzel.  Prostaglandin D2 pathway upregulation:  Relation to asthma severity, control, and Th2 inflammation.  J Allergy and Clin Immunol 131:1504-1512, 2013.
Gelhaus SL, O Gilad, TM Penning and IA Blair.  Multidrug resistance protein (MRP) 4 attenuates benzo[a]pyrene-mediated DNA-adduct formation in human bronchoalveolar H358 cells.  Toxicology Letters 209:58-66, 2012.
Gelhaus SL, RG Harvey, TM Penning and IA Blair.  Regulation of benzo[a]pyrene-mediated DNA- and glutathione-adduct formation by 2,3,7,8-tetrachlorodibenzo-p-dioxin in human lung cells.  Chemical Research in Toxicology 24:89-98, 2011.
Bhat S, SL Gelhaus, C Mesaros, A Vachani and IA Blair.  A new liquid chromatography-mass spectrometry method for analysis of urinary 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL).  Rapid Communications in Mass Spectrometry 25:115-121, 2011.
Basu S, C Mesaros, SL Gelhaus and IA Blair.  Stable isotope labeling by essential nutrients in cell culture (SILEC) for preparation of labeled coenzyme A and its thioesters.  Analytical Chemistry 83:1363-1369, 2011.
Park J-H, SL Gelhaus, S Vendantam, AL Oliva, A Batra, IA Blair, AB Troxel, J Field and TM Penning.  The pattern of p53 mutations caused by PAH o-quinones is driven by 8-oxo-dGuo formation while the spectrum of mutations is determined by biological selection for dominance.  Chemical Research in Toxicology 21:1039-1049, 2008.

Steven R. Woodcock, PhD

Journal Articles

Woodcock SR, SR Salvatore, G Bonacci, BA Freeman and FJ Schopfer.  Biomimetic nitration of conjugated linoleic acid:  Characterization and synthesis of naturally-occurring conjugated dienes.  J Org Chem 79:25-33, 2014.
Vitturi DA, CS Chen, SR Woodcock, N Stewart, N Wakabayashi, SR Salvatore, G Bonacci, JR Koenitzer, TW Kensler, BA Freeman and FJ Schopfer.  Modulation of nitro-fatty acid signaling:  Prostaglandin reductase-1 is a nitroalkene reductase.  J Bio Chem 288:25626-25637, 2013.
Woodcock SR, G Bonacci, SL Gelhaus and FJ Schopfer.  Nitrated fatty acids:  Synthesis and measurement.  Free Rad Biol Med 59:14-26, 2013.
Bonacci G, PR Baker, SR Salvatore, D Shores, NK Khoo, JR Koenitzer, DA Vitturi, SR Woodcock, F Golin-Bisello, MP Cole, S Watkins, C St. Croix, CI Batthyany, BA Freeman and FJ Schopfer.  Conjugated linoleic acid is a preferential substrate for fatty acid nitration.  J Biol Chem 287:44071-44082, 2012.
Rudolph V, FJ Schopfer, NK Khoo, TK Rudolph, MP Cole, SR Woodcock, G Bonacci, AL Groeger, F Golin-Bisello, CS Chen, PR Baker and BA Freeman.  Nitro-fatty acid metabolome:  Saturation, desaturation, beta-oxidation, and protein adduction.  J Biol Chem 284:1461-1473, 2009.
Woodcock SR, AJV Marwitz, P Bruno and BP Branchaud.  Synthesis of nitrolipids:  All four possible diastereomers of nitro-oleic acids:  (E)- and (Z)-, 9- and 10-Nitro-octadec-9-enoic acids.  Org Lett 8:3931-3833, 2006.