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 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.
Figure 7. PPAR activation by nitro-linoleic acid.
The Freeman group is now appreciating that nitrated fatty acid derivatives are abundant bioactive oxides of nitrogen in blood and tissues that can be formed by oxidative inflammatory reactions. Nitrated fatty acids are potent PPAR ligands that exert potent receptor-dependent cell signaling and gene expression regulatory actions.
Figure 8. Activation of vascular heme oxygenase-1 expression by nitro-linoleic acid.
Nitro fatty acid-protein adducts are clinically detectable, with this adduction reaction affecting protein structure, function and cellular distribution. In aggregate, nitrated fatty acids are a new class of cell signaling mediators that represent a convergence of NO and oxygenated lipid redox signaling pathways. The endogenous generation and cell signaling actions of NO2-FA and other electrophilic lipids reveal that mammals and other species have evolved networks of lipid receptors, transcription factors and signaling pathways that sense changes in tissue levels of oxidative inflammatory mediators. These stimulus-response mechanisms confer to organisms the ability to sense and rapidly adapt to changes in metabolic, redox and inflammatory status via an immediate impact on protein function and by altering patterns of gene expression. The formation and signaling actions of nitrated unsaturated fatty acids thus expand the array of molecular targets regulated by NO and lipid mediators, and represent a redox-sensitive signaling mechanism that responds adaptively to multiple facets of cell metabolism.
Figure 9. Signaling actions of nitro-fatty acids.
In summary, Dr. Freeman's investigation of NO reactions with intermediates of lipid peroxidation and eicosanoid synthesis, as well as with oxidant/free radical products of oxidases and peroxidases (e.g., xanthine oxidase and myeloperoxidase) are revealing a) clinically-significant mechanisms of pathogenic NO scavenging that occurs during inflammation and b) novel strategies for preserving NO-dependent signaling and treating inflammatory injury in diverse disease processes.
Discoveries made by Jason Eiserich, PhD and Stephan Baldus, MD shed important light on the roles of neutrophil-derived myeloperoxidase (MPO) as both a marker and mediator of vascular dysfunction. Dr. Eiserich made the seminal observation that MPO readily oxidizes nitrite (NO2-) to the free radical product NO2, a species capable of oxidizing and nitrating biomolecules. With Dr. Baldus, Dr. Freeman then observed that upon neutrophil degranulation, MPO concentrates in the subendothelial matrix of vascular tissues by a transcytotic mechanism, acting there to catalyze cellular and extracellular matrix protein tyrosine nitration. This transcytosis and interstitial matrix deposition of MPO occurs independent of leukocyte emigration and confers specificity to nitration of functionally-significant matrix proteins. In patients with coronary artery disease, there is an abundant deposition of MPO in coronary vessel walls that remains redox reactive, and a strong co-distribution between MPO and nitrotyrosine adducts of vessel wall proteins.
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