Marie Luise Brennan

Apolipoprotein A-I is a selective target for myeloperoxidase-catalyzed oxidation and functional impairment in subjects with cardiovascular disease

In recent studies we demonstrated that systemic levels of protein-bound nitrotyrosine (NO(2)Tyr) and myeloperoxidase (MPO), a protein that catalyzes generation of nitrating oxidants, serve as independent predictors of atherosclerotic risk, burden, and incident cardiac events. We now show both that apolipoprotein A-I (apoA-I), the primary protein constituent of HDL, is a selective target for MPO-catalyzed nitration and chlorination in vivo and that MPO-catalyzed oxidation of HDL and apoA-I results in selective inhibition in ABCA1-dependent cholesterol efflux from macrophages. Dramatic selective enrichment in NO(2)Tyr and chlorotyrosine (ClTyr) content within apoA-I recovered from serum and human atherosclerotic lesions is noted, and analysis of serum from sequential subjects demonstrates that the NO(2)Tyr and ClTyr contents of apoA-I are markedly higher in individuals with cardiovascular disease (CVD). Analysis of circulating HDL further reveals that higher NO(2)Tyr and ClTyr contents of the lipoprotein are each significantly associated with diminished ABCA1-dependent cholesterol efflux capacity of the lipoprotein. MPO as a likely mechanism for oxidative modification of apoA-I in vivo is apparently facilitated by MPO binding to apoA-I, as revealed by cross-immunoprecipitation studies in plasma, recovery of MPO within HDL-like particles isolated from human atheroma, and identification of a probable contact site between the apoA-I moiety of HDL and MPO. To our knowledge, the present results provide the first direct evidence for apoA-I as a selective target for MPO-catalyzed oxidative modification in human atheroma. They also suggest a potential mechanism for MPO-dependent generation of a proatherogenic dysfunctional form of HDL in vivo.

Relationship of Paraoxonase 1 (PON1) Gene Polymorphisms and Functional Activity With Systemic Oxidative Stress and Cardiovascular Risk

CONTEXT Paraoxonase 1 (PON1) is reported to have antioxidant and cardioprotective properties. The relationship between PON1 genotypes and functional activity with systemic measures of oxidative stress and cardiovascular disease (CVD) risk in humans has not been systematically investigated. OBJECTIVE To investigate the relationship of genetic and biochemical determinants of PON1 activity with systemic measures of oxidative stress and CVD risk in humans. DESIGN, SETTING, AND PARTICIPANTS The association between systemic PON1 activity measures and a functional polymorphism (Q192R) resulting in high PON1 activity with prevalent CVD and future major adverse cardiac events (myocardial infarction, stroke, or death) was evaluated in 1399 sequential consenting patients undergoing diagnostic coronary angiography between September 2002 and November 2003 at the Cleveland Clinic. Patients were followed up until December 2006. Systemic levels of multiple structurally defined fatty acid oxidation products were also measured by mass spectrometry in 150 age-, sex-, and race-matched patients and compared with regard to PON1 genotype and activity. MAIN OUTCOME MEASURES Relationship between a functional PON1 polymorphism and PON1 activity with global indices of systemic oxidative stress and risk of CVD. RESULTS The PON1 genotype demonstrated significant dose-dependent associations (QQ192 > QR192 > RR192) with decreased levels of serum PON1 activity and with increased levels of systemic indices of oxidative stress. Compared with participants with either the PON1 RR192 or QR192 genotype, participants with the QQ192 genotype demonstrated an increased risk of all-cause mortality (43/681 deaths [6.75%] in RR192 and QR192 and 62/584 deaths [11.1%] in QQ192; adjusted hazard ratio, 2.05; 95% confidence interval [CI], 1.32-3.18) and of major adverse cardiac events (88/681 events [13.6%] in RR192 and QR192 and 102/584 events [18.0%] in QQ192; adjusted hazard ratio, 1.48; 95% CI, 1.09-2.03; P = .01). The incidence of major adverse cardiac events was significantly lower in participants in the highest PON1 activity quartile (23/315 [7.3%]) and 235/324 [7.7%] for paraoxonase and arylesterase, respectively) compared with those in the lowest activity quartile (78/311 [25.1%] and 75/319 [23.5%]; P < .001 for paraoxonase and arylesterase, respectively). The adjusted hazard ratios for major adverse cardiac events between the highest and lowest PON1 activity quartiles were, for paraoxonase, 3.4 (95% CI, 2.1-5.5; P < .001) and for arylesterase, 2.9 (95% CI, 1.8-4.7; P < .001) and remained independent in multivariate analysis. CONCLUSION This study provides direct evidence for a mechanistic link between genetic determinants and activity of PON1 with systemic oxidative stress and prospective cardiovascular risk, indicating a potential mechanism for the atheroprotective function of PON1.

Statins Promote Potent Systemic Antioxidant Effects Through Specific Inflammatory Pathways

Background—The pleiotropic actions of hydroxymethylglutaryl CoA reductase inhibitors (statins) include antiinflammatory and antioxidant actions. We recently reported that statins induce reductions in plasma protein levels of nitrotyrosine (NO2Tyr), a modification generated by nitric oxide–derived oxidants. Whether alternative oxidative pathways are suppressed in vivo after statin administration has not yet been reported. Methods and Results—As an extension of our prior study, hypercholesterolemic subjects with no known coronary artery disease were evaluated at baseline and after 12 weeks of atorvastatin therapy (10 mg/d). Plasma levels of protein-bound chlorotyrosine, NO2Tyr, dityrosine, and orthotyrosine, specific molecular fingerprints for distinct oxidative pathways upregulated in atheroma, were determined by mass spectrometry. In parallel, alterations in lipoproteins and C-reactive protein were determined. Statin therapy caused significant reductions in chlorotyrosine, NO2Tyr, and dityrosine (30%, 25%, and 32%, respectively; P <0.02 each) that were similar in magnitude to reductions in total cholesterol and apolipoprotein B-100 (25% and 29%, P <0.001 each). Nonsignificant decreases in orthotyrosine and C-reactive protein levels were observed (9% and 11%, respectively; P >0.10 each). Statin-induced reductions in oxidation markers were independent of decreases in lipids and lipoproteins. Conclusions—Statins promote potent systemic antioxidant effects through suppression of distinct oxidation pathways. The major pathways inhibited include formation of myeloperoxidase-derived and nitric oxide–derived oxidants, species implicated in atherogenesis. The present results suggest potential mechanisms that may contribute to the beneficial actions of statins. They also have important implications for monitoring the antiinflammatory and antioxidant actions of these agents.

Serum Myeloperoxidase Levels Independently Predict Endothelial Dysfunction in Humans

Background—In vitro and animal studies demonstrate that myeloperoxidase catalytically consumes nitric oxide as a substrate, limiting its bioavailability and function. We therefore hypothesized that circulating levels of myeloperoxidase would predict risk of endothelial dysfunction in human subjects. Methods and Results—Serum myeloperoxidase was measured by enzyme-linked immunoassay, and brachial artery flow–mediated dilation and nitroglycerin-mediated dilation were determined by ultrasound in a hospital-based population of 298 subjects participating in an ongoing study of the clinical correlates of endothelial dysfunction (age, 51±16; 61% men, 51% with cardiovascular disease). A strong inverse relation between brachial artery flow–mediated dilation and increasing quartile of serum myeloperoxidase level was observed (11.0±6.0%, 9.4±5.3%, 8.6±5.8%, and 6.4±4.5% for quartiles 1 through 4, respectively; P<0.001 for trend). Using the median as a cut point to define endothelial dysfunction, increasing quartile of myeloperoxidase predicted endothelial dysfunction after adjustment for classic cardiovascular disease risk factors, C-reactive protein levels, prevalence of cardiovascular disease, and ongoing treatment with cardiovascular medications (OR, 6.4; 95% CI, 2.6 to 16; P=0.001 for highest versus lowest quartile). Conclusions—Serum myeloperoxidase levels serve as a strong and independent predictor of endothelial dysfunction in human subjects. Myeloperoxidase-mediated endothelial dysfunction may be an important mechanistic link between oxidation, inflammation, and cardiovascular disease.

Endothelial transcytosis of myeloperoxidase confers specificity to vascular ECM proteins as targets of tyrosine nitration

Nitrotyrosine formation is a hallmark of vascular inflammation, with polymorphonuclear neutrophil-derived (PMN-derived) and monocyte-derived myeloperoxidase (MPO) being shown to catalyze this posttranslational protein modification via oxidation of nitrite (NO(2)(-)) to nitrogen dioxide (NO(2)(*)). Herein, we show that MPO concentrates in the subendothelial matrix of vascular tissues by a transcytotic mechanism and serves as a catalyst of ECM protein tyrosine nitration. Purified MPO and MPO released by intraluminal degranulation of activated human PMNs avidly bound to aortic endothelial cell glycosaminoglycans in both cell monolayer and isolated vessel models. Cell-bound MPO rapidly transcytosed intact endothelium and colocalized abluminally with the ECM protein fibronectin. In the presence of the substrates hydrogen peroxide (H(2)O(2)) and NO(2)(-), cell and vessel wall-associated MPO catalyzed nitration of ECM protein tyrosine residues, with fibronectin identified as a major target protein. Both heparin and the low-molecular weight heparin enoxaparin significantly inhibited MPO binding and protein nitrotyrosine (NO(2)Tyr) formation in both cultured endothelial cells and rat aortic tissues. MPO(-/-) mice treated with intraperitoneal zymosan had lower hepatic NO(2)Tyr/tyrosine ratios than did zymosan-treated wild-type mice. These data indicate that MPO significantly contributes to NO(2)Tyr formation in vivo. Moreover, transcytosis of MPO, occurring independently of leukocyte emigration, confers specificity to nitration of vascular matrix proteins.

Myeloperoxidase and Plasminogen Activator Inhibitor 1 Play a Central Role in Ventricular Remodeling after Myocardial Infarction

Left ventricular (LV) remodeling after myocardial infarction (MI) results in LV dilation, a major cause of congestive heart failure and sudden cardiac death. Ischemic injury and the ensuing inflammatory response participate in LV remodeling, leading to myocardial rupture and LV dilation. Myeloperoxidase (MPO), which accumulates in the infarct zone, is released from neutrophils and monocytes leading to the formation of reactive chlorinating species capable of oxidizing proteins and altering biological function. We studied acute myocardial infarction (AMI) in a chronic coronary artery ligation model in MPO null mice (MPO−/−). MPO−/− demonstrated decreased leukocyte infiltration, significant reduction in LV dilation, and marked preservation of LV function. The mechanism appears to be due to decreased oxidative inactivation of plasminogen activator inhibitor 1 (PAI-1) in the MPO−/−, leading to decreased tissue plasmin activity. MPO and PAI-1 are shown to have a critical role in the LV response immediately after MI, as demonstrated by markedly delayed myocardial rupture in the MPO−/− and accelerated rupture in the PAI-1−/−. These data offer a mechanistic link between inflammation and LV remodeling by demonstrating a heretofore unrecognized role for MPO and PAI-1 in orchestrating the myocardial response to AMI.

p53 is a suppressor of inflammatory response in mice

Chronic inflammation is known to promote cancer, suggesting that negative regulation of inflammation is likely to be tumor suppressive. We found that p53 is a general inhibitor of inflammation that acts as an antagonist of nuclear factor κB (NFκB). We first observed striking similarities in global gene expression profiles in human prostate cancer cells LNCaP transduced with p53 inhibitory genetic element or treated with TNF, suggesting that p53 inhibits transcription of TNF‐inducible genes that are largely regulated by NFκB. Consistently, ectopically expressed p53 acts as an inhibitor of transcription of NFκB‐dependent promoters. Furthermore, suppression of inflammatory response by p53 was observed in vivo in mice by comparing wild‐type and p53 null animals at molecular (inhibition of transcription of genes encoding cytokines and chemokines, reducing accumulation of reactive oxygen species and protein oxidation products), cellular (activation of macrophages and neutrophil clearance) and organismal (high levels of metabolic markers of inflammation in tissues of p53‐deficient mice and their hypersensitivity to LPS) levels. These observations indicate that p53, acting through suppression of NFκB, plays the role of a general “buffer” of innate immune response in vivo that is well consistent with its tumor suppressor function and frequent constitutive activation of NFκB in tumors.

Identification of α-Chloro Fatty Aldehydes and Unsaturated Lysophosphatidylcholine Molecular Species in Human Atherosclerotic Lesions

Background—A role for myeloperoxidase (MPO) as a mediator of coronary artery disease and acute coronary syndromes has recently received considerable attention. Although active MPO and hypochlorite-modified proteins and peptides have been detected in human atherosclerotic lesions, detection of novel chlorinated oxidized lipid species with proatherogenic properties in vivo has not yet been reported. In this study we show that MPO-generated reactive chlorinating species promote selective oxidative cleavage of plasmalogens, liberating &agr;-chloro fatty aldehydes and unsaturated lysophosphatidylcholine in human atherosclerotic lesions. Methods and Results—Stable isotope dilution gas chromatography–mass spectrometry methods were used to identify and quantitate the &agr;-chloro fatty aldehyde, 2-chlorohexadecanal, in atherosclerotic versus normal human aorta. Compared with normal aorta, 2-chlorohexadecanal levels were elevated more than 1400-fold in atherosclerotic tissues. Parallel electrospray ionization mass spectrometry studies confirmed 34- and 20-fold increases in the plasmalogen cooxidation products, unsaturated lysophosphatidylcholine molecular species containing linoleic and arachidonic acid, respectively, within atherosclerotic compared with normal aorta. Unsaturated lysophosphatidylcholine containing docosahexaenoic acid was also detected in atherosclerotic but not in normal aorta. Exposure of primary human coronary artery endothelial cells to plasmalogen-derived lysophosphatidylcholine molecular species produced marked increases in P-selectin surface expression. Conclusions—The present studies demonstrate that plasmalogens are attacked by MPO-derived reactive chlorinating species within human atheroma. The resultant species formed, &agr;-chloro fatty aldehydes and unsaturated lysophospholipids, possess proatherogenic properties, as shown by induction of P-selectin surface expression in primary human coronary artery endothelial cells.

Concurrent evaluation of novel cardiac biomarkers in acute coronary syndrome: myeloperoxidase and soluble CD40 ligand and the risk of recurrent ischaemic events in TACTICS-TIMI 18

AIMS We investigated the prognostic performance of myeloperoxidase (MPO), and soluble CD40 ligand (sCD40L) along with B-type natriuretic peptide (BNP), high-sensitivity C-reactive protein (hsCRP), and cardiac troponin I (cTnI) for non-fatal recurrent ischaemic events in non-ST elevation acute coronary syndrome (ACS). METHODS AND RESULTS We measured plasma MPO and sCD40L in 1524 patients with ACS treated with tirofiban and randomized to early invasive vs. conservative management in the TACTICS-TIMI 18 trial who survived to 180 days. Patients with elevated baseline MPO (>884 pM) were at higher risk of non-fatal myocardial infarction or rehospitalization for ACS at 30 days (9.3 vs. 4.6%, P < 0.001). In contrast, no difference was observed with higher sCD40L (>989 pg/mL, 7.6 vs. 6.3%, P = 0.31). MPO remained associated with recurrent ischaemic events after adjustment for age, ST-deviation, diabetes, prior coronary artery disease, heart failure, cTnI, hsCRP, and sCD40L (OR 2.10; 95% CI 1.36-3.23, P = 0.001). This association was attenuated by 180 days (OR 1.26; 0.95-1.68). Stratification using baseline MPO, BNP, and cTnI identified a >3-fold gradient of risk. CONCLUSION MPO adds to BNP and cTnI for short-term risk assessment for recurrent ischaemic events in non-ST elevation ACS. sCD40L was not associated with risk in this population treated with a platelet GPIIb/IIIa receptor antagonist.

Levels of soluble endothelium-derived adhesion molecules in patients with sickle cell disease are associated with pulmonary hypertension, organ dysfunction, and mortality

Endothelial cell adhesion molecules orchestrate the recruitment and binding of inflammatory cells to vascular endothelium. With endothelial dysfunction and vascular injury, the levels of endothelial bound and soluble adhesion molecules increase. Such expression is modulated by nitric oxide (NO), and in patients with sickle cell disease (SCD), these levels are inversely associated with measures of NO bioavailability. To further evaluate the role of endothelial dysfunction in a population study of SCD, we have measured the levels of soluble endothelium‐derived adhesion molecules in the plasma specimens of 160 adult patients with SCD during steady state. Consistent with a link between endothelial dysfunction and end‐organ disease, we found that higher levels of soluble vascular cell adhesion molecule‐1 (sVCAM‐1) were associated with markers indicating renal dysfunction and hepatic impairment. Analysis of soluble intercellular cell adhesion molecule‐1 (sICAM‐1), sE‐selectin and sP‐selectin levels indicated partially overlapping associations with sVCAM‐1, with an additional association with inflammatory stress and triglyceride levels. Importantly, increased soluble adhesion molecule expression correlated with severity of pulmonary hypertension, a clinical manifestation of endothelial dysfunction. Soluble VCAM‐1, ICAM‐1, and E‐selectin were independently associated with the risk of mortality in this cohort. Our data are consistent with steady state levels of soluble adhesion molecules as markers of pulmonary hypertension and risk of death.