Ruba S. Deeb

Maintaining Equilibrium by Selective Targeting of Cyclooxygenase Pathways Promising Offensives Against Vascular Injury

Cardiovascular disease is the leading cause of death in most Western countries. Vascular abnormalities associated with cardiovascular disease are attributable to a variety of risk factors, including hypercholesterolemia, hypertension, hyperglycemia, lipotoxicity, obesity, aging, and tobacco smoke.1,2 Importantly, altered ·NO bioavailability is a major underlying mechanism linking each of these risk factors. Indeed, the endothelium is recognized as a pivotal regulator of vascular function by maintaining homeostatic levels of ·NO and prostanoids derived from arachidonic acid (AA) metabolism.3 Eicosanoids, synthesized from unsaturated fatty acids, are biologically active molecules that play a wide range of regulatory roles in the cardiovascular, renal, immune, and gastrointestinal systems.4 Alterations in their biosyntheses can promote hypertension, diabetes, and, in particular, atherosclerosis, an inflammatory disease characterized by the accretion of fat-laden plaques in the arterial wall that can lead to vasoocclusive events. During atherogenesis, eicosanoid production (from the cyclooxygenases [COXs], lipoxygenases [LOXs], and cytochrome P450s pathways) is altered by mechanisms that are not yet well understood. The dichotomous nature of eicosanoids requires that their balance is maintained, and, as such, these pathways are a relevant therapeutic target against cardiovascular disease.5 Thus, it is now appreciated that cardiovascular disease can be triggered by an absolute deficiency of ·NO and/or an imbalance between “beneficial” and “harmful” eicosanoids in the vasculature and that these pathways are mutually interactive. In this review we consider pharmacological therapies possessing the potential for greater safety and efficacy than nonsteroidal antiinflammatory drugs (NSAIDs) in the treatment of chronic vasoinflammatory conditions. Fundamental physiological distinctions are now recognized for the structurally similar enzymes, COX-1 and COX-2. Constitutively expressed COX-1 is found in almost all of the tissues where it subserves diverse physiological functions. Basal expression of COX-2 is limited to the kidney, brain, and reproductive system4 but can be …

Tyrosine nitration in prostaglandin H2 synthase

In this study, we investigated the effects of various nitrogen oxide (NOx) species on the extent of prostaglandin H2 synthase-1 (PGHS-1) nitration in purified protein and in vascular smooth muscle cells. We also examined PGHS-1 activity under these conditions and found the degree of nitration to correlate inversely with enzyme activity. In addition, since NOx species are thought to invoke damage during the pathogenesis of atherosclerosis, we examined human atheromatous tissue for PGHS-1 nitration. Both peroxynitrite and tetranitromethane induced Tyr nitration of purified PGHS-1, whereas 1-hydroxy-2-oxo-3-(N-methyl-aminopropyl)-3-methyl-1-triazene (NOC-7; a nitric oxide-releasing compound) did not. Smooth muscle cells treated with peroxynitrite showed PGHS-1 nitration. The extent of nitration by specific NOx species was determined by electrospray ionization mass spectrometry. Tetranitromethane was more effective than peroxynitrite, NOC-7, and nitrogen dioxide at nitrating a tyrosine-containing peptide (12%, 5%, 1%, and <1% nitration, respectively). Nitrogen dioxide and, to a lesser extent, peroxynitrite, induced dityrosine formation. Using UV/Vis spectroscopy, it was estimated that the reaction of PGHS-1 with excess peroxynitrite yielded two nitrated tyrosines/PGHS-1 subunit. Finally, atherosclerotic tissue obtained from endarterectomy patients was shown to contain nitrated PGHS-1. Thus, prolonged exposure to elevated levels of peroxynitrite may cause oxidative damage through tyrosine nitration.

Protein 3-Nitrotyrosine in Complex Biological Samples: Quantification by High-Pressure Liquid Chromatography/ Electrochemical Detection and Emergence of Proteomic Approaches for Unbiased Identification of Modification Sites

Nitration of tyrosine residues by nitric oxide (NO)-derived species results in the accumulation of 3-nitrotyrosine in proteins, a hallmark of nitrosative stress in cells and tissues. Tyrosine nitration is recognized as one of the multiple signaling modalities used by NO-derived species for the regulation of protein structure and function in health and disease. Various methods have been described for the quantification of protein 3-nitrotyrosine residues, and several strategies have been presented toward the goal of proteome-wide identification of protein tyrosine modification sites. This chapter details a useful protocol for the quantification of 3-nitrotyrosine in cells and tissues using high-pressure liquid chromatography with electrochemical detection. Additionally, this chapter describes a novel biotin-tagging strategy for specific enrichment of 3-nitrotyrosine-containing peptides. Application of this strategy, in conjunction with high-throughput MS/MS-based peptide sequencing, is anticipated to fuel efforts in developing comprehensive inventories of nitrosative stress-induced protein-tyrosine modification sites in cells and tissues.

Heme catalyzes tyrosine 385 nitration and inactivation of prostaglandin H2 synthase-1 by peroxynitrite

The mechanism by which the inflammatory enzyme prostaglandin H2 synthase-1 (PGHS-1) deactivates remains undefined. This study aimed to determine the stabilizing parameters of PGHS-1 and identify factors leading to deactivation by nitric oxide species (NOx). Purified PGHS-1 was stabilized when solubilized in β-octylglucoside (rather than Tween-20 or CHAPS) and when reconstituted with hemin chloride (rather than hematin). Peroxynitrite (ONOO−) activated the peroxidase site of PGHS-1 independently of the cyclooxygenase site. After ONOO− exposure, holoPGHS-1 could not metabolize arachidonic acid and was structurally compromised, whereas apoPGHS-1 retained full activity once reconstituted with heme. After incubation of holoPGHS-1 with ONOO−, heme absorbance was diminished but to a lesser extent than the loss in enzymatic function, suggesting the contribution of more than one process to enzyme inactivation. Hydroperoxide scavengers improved enzyme activity, whereas hydroxyl radical scavengers provided no protection from the effects of ONOO−. Mass spectral analyses revealed that tyrosine 385 (Tyr 385) is a target for nitration by ONOO− only when heme is present. Multimer formation was also observed and required heme but could be attenuated by arachidonic acid substrate. We conclude that the heme plays a role in catalyzing Tyr 385 nitration by ONOO− and the demise of PGHS-1.

Characterization of a cellular denitrase activity that reverses nitration of cyclooxygenase

Protein 3-nitrotyrosine (3-NT) formation is frequently regarded as a simple biomarker of disease, an irreversible posttranslational modification that can disrupt protein structure and function. Nevertheless, evidence that protein 3-NT modifications may be site selective and reversible, thus allowing for physiological regulation of protein activity, has begun to emerge. We have previously reported that cyclooxygenase (COX)-1 undergoes heme-dependent nitration of Tyr(385), an internal and catalytically essential residue. In the present study, we demonstrate that nitrated COX-1 undergoes a rapid reversal of nitration by substrate-selective and biologically regulated denitrase activity. Using nitrated COX-1 as a substrate, denitrase activity was validated and quantified by analytic HPLC with electrochemical detection and determined to be constitutively active in murine and human endothelial cells, macrophages, and a variety of tissue samples. Smooth muscle cells, however, contained little denitrase activity. Further characterizing this denitrase activity, we found that it was inhibited by free 3-NT and may be enhanced by endogenous nitric oxide and exogenously administered carbon monoxide. Finally, we describe a purification protocol that results in significant enrichment of a discrete denitrase-containing fraction, which maintains activity throughout the purification process. These findings reveal that nitrated COX-1 is a substrate for a denitrase in cells and tissues, implying that the reciprocal processes of nitration and denitration may modulate bioactive lipid synthesis in the setting of inflammation. In addition, our data reveal that denitration is a controlled process that may have broad importance for regulating cell signaling events in nitric oxide-generating systems during oxidative/nitrosative stress.

Inducible nitric oxide synthase gene deletion exaggerates MAPK-mediated cyclooxygenase-2 induction by inflammatory stimuli

Cyclooxygenase (COX)-2 and inducible nitric oxide (NO) synthase (iNOS) are responsive to a wide array of inflammatory stimuli, have been localized to vascular smooth muscle cells (SMCs), and are intimately linked to the progression of vascular disease, including atherosclerotic lesion formation. We and others have shown that the production and subsequent impact of COX products appear to be correlative with the status of NO synthesis. This study examined the impact of inflammation-driven NO production on COX-2 expression in SMCs. Concurrent stimulation of quiescent rat aortic SMCs with lipopolysaccharide (LPS) and interferon (IFN)-gamma increased COX-2, iNOS, and nitrite production. Pharmacological inhibition of NO synthase (N(G)-monomethyl-l-arginine) concentration- and time-dependently magnified LPS + IFN-gamma-mediated COX-2 mRNA and protein induction in a cGMP-independent manner. COX-2 induction was associated with activation of the ERK, p38, and JNK mitogen-activated protein kinase (MAPK) pathways. Interestingly, NO synthase inhibition enhanced ERK, p38, and to a lesser extent JNK phosphorylation but suppressed MAPK phosphatase (MKP)-1 induction in response to LPS + IFN-gamma. Similarly, the exposure of SMCs from iNOS(-/-) mice to LPS + IFN-gamma produced an enhancement of COX-2 induction, p38, and JNK phosphorylation and an attenuated upregulation of MKP-1 versus their wild-type counterparts. Taken together, our data indicate that NO, in part derived from iNOS, negatively regulates the immediate early induction of COX-2 in response to inflammatory stimuli.

Decreased Neurotrophin TrkB Receptor Expression Reduces Lesion Size in the Apolipoprotein E–Null Mutant Mouse

Background— Accumulation of macrophages and smooth muscle cells in the vascular wall is critical for the development of atherosclerotic lesions. Although much is known about the factors that regulate macrophage recruitment to the vascular wall, the ability of growth factors to regulate smooth muscle cell recruitment in lesion development in vivo is unclear. Our previous studies demonstrated that neurotrophins and their receptors, the Trk receptor tyrosine kinases, are potent chemotactic factors for smooth muscle cells, and the expression of brain-derived neurotrophic factor (BDNF) and its cognate receptor, TrkB, is upregulated in human atherosclerotic lesions. Methods and Results— TrkB+/− mice on a 129/B6 background were backcrossed to apolipoprotein E (ApoE)–null (ApoE−/−) mice on the C57Bl/6 background for 6 to 8 generations. Immunohistochemical analysis demonstrated BDNF immunoreactivity in areas of macrophage and smooth muscle cell infiltration, whereas TrkB immunoreactivity was predominant in areas of neointimal smooth muscle cells. Moreover, haplodeficient expression of TrkB in ApoE−/− mice was associated with a 30% to 40% reduction in lesion size compared with ApoE−/− mice with normal expression of TrkB and a 45% decrease in smooth muscle cell accumulation in the lesions. Finally, reconstitution with bone marrow from ApoE−/− mice with normal TrkB expression did not restore lesion development in TrKB+/−/ApoE−/− mice. Conclusions— These results suggest that TrkB expression on smooth muscle cells contributes to lesion development in the cholesterol-fed ApoE–null mutant mouse. These data demonstrate, for the first time, a role for the neurotrophin TrkB receptor in atherosclerotic lesion development.

Smoking-Associated Disordering of the Airway Basal Stem/Progenitor Cell Metabotype

The airway epithelium is a complex pseudostratified multicellular layer lining the tracheobronchial tree, functioning as the primary defense against inhaled environmental contaminants. The major cell types of the airway epithelium include basal, intermediate columnar, ciliated, and secretory. Basal cells (BCs) are the proliferating stem/progenitor population that differentiate into the other specialized cell types of the airway epithelium during normal turnover and repair. Given that cigarette smoke delivers thousands of xenobiotics and high levels of reactive molecules to the lung epithelial surface, we hypothesized that cigarette smoke broadly perturbs BC metabolism. To test this hypothesis, primary airway BCs were isolated from healthy nonsmokers (n = 11) and healthy smokers (n = 7) and assessed by global metabolic profiling by liquid chromatography-mass spectrometry. The analysis identified 52 significant metabolites in BCs differentially expressed between smokers and nonsmokers (P < 0.05). These changes included metabolites associated with redox pathways, energy production, and inflammatory processes. Notably, BCs from smokers exhibited altered levels of the key enzyme cofactors/substrates nicotinamide adenine dinucleotide, flavin adenine dinucleotide, acetyl coenzyme A, and membrane phospholipid levels. Consistent with the high burden of oxidants in cigarette smoke, glutathione levels were diminished, whereas 3-nitrotyrosine levels were increased, suggesting that protection of airway epithelial cells against oxidative and nitrosative stress is significantly compromised in smoker BCs. It is likely that this altered metabotype is a reflection of, and likely contributes to, the disordered biology of airway BCs consequent to the stress cigarette smoking puts on the airway epithelium.

Physical evidence for substrate binding in preventing cyclooxygenase inactivation under nitrative stress

Prostaglandin biosynthesis is catalyzed by two spatially and functionally distinct active sites in cyclooxygenase (COX) enzymes. Despite the crucial role of COXs in biology, molecular details regarding the function and regulation of these enzymes are incompletely defined. Reactive nitrogen species, formed during oxidative stress, produce modifications that alter COX functionalities and prostaglandin biosynthesis. We previously established that COX-1 undergoes selective nitration on Tyr385 via a mechanism that requires the presence of bound heme cofactor. As this is a critical residue for COX-1 catalysis, nitration at this site results in enzyme inactivation. We now show that occupancy of the COX-1 active site with substrate protects against Tyr385 nitration and redirects nitration to alternative Tyr residues on COX-1, preserving catalytic activity. This study reveals a novel role for the substrate in protecting COX-1 from inactivation by nitration in pathophysiological settings.

Serum Metabolite Biomarkers Discriminate Healthy Smokers from COPD Smokers

COPD (chronic obstructive pulmonary disease) is defined by a fixed expiratory airflow obstruction associated with disordered airways and alveolar destruction. COPD is caused by cigarette smoking and is the third greatest cause of mortality in the US. Forced expiratory volume in 1 second (FEV1) is the only validated clinical marker of COPD, but it correlates poorly with clinical features and is not sensitive enough to predict the early onset of disease. Using LC/MS global untargeted metabolite profiling of serum samples from a well-defined cohort of healthy smokers (n = 37), COPD smokers (n = 41) and non-smokers (n = 37), we sought to discover serum metabolic markers with known and/or unknown molecular identities that are associated with early-onset COPD. A total of 1,181 distinct molecular ions were detected in 95% of sera from all study subjects and 23 were found to be differentially-expressed in COPD-smokers vs. healthy-smokers. These 23 putative biomarkers were differentially-correlated with lung function parameters and used to generate a COPD prediction model possessing 87.8% sensitivity and 86.5% specificity. In an independent validation set, this model correctly predicted COPD in 8/10 individuals. These serum biomarkers included myoinositol, glycerophopshoinositol, fumarate, cysteinesulfonic acid, a modified version of fibrinogen peptide B (mFBP), and three doubly-charged peptides with undefined sequence that significantly and positively correlate with mFBP levels. Together, elevated levels of serum mFBP and additional disease-associated biomarkers point to a role for chronic inflammation, thrombosis, and oxidative stress in remodeling of the COPD airways. Serum metabolite biomarkers offer a promising and accessible window for recognition of early-stage COPD.