David Schmitt

Myeloperoxidase-generated reactive nitrogen species convert LDL into an atherogenic form in vitro

Oxidized LDL is implicated in atherosclerosis; however, the pathways that convert LDL into an atherogenic form in vivo are not established. Production of reactive nitrogen species may be one important pathway, since LDL recovered from human atherosclerotic aorta is enriched in nitrotyrosine. We now report that reactive nitrogen species generated by the MPO-H2O2-NO2- system of monocytes convert LDL into a form (NO2-LDL) that is avidly taken up and degraded by macrophages, leading to massive cholesterol deposition and foam cell formation, essential steps in lesion development. Incubation of LDL with isolated MPO, an H2O2-generating system, and nitrite (NO2-)-- a major end-product of NO metabolism--resulted in nitration of apolipoprotein B 100 tyrosyl residues and initiation of LDL lipid peroxidation. The time course of LDL protein nitration and lipid peroxidation paralleled the acquisition of high-affinity, concentration-dependent, and saturable binding of NO2-LDL to human monocyte-derived macrophages and mouse peritoneal macrophages. LDL modification and conversion into a high-uptake form occurred in the absence of free metal ions, required NO2-, occurred at physiological levels of Cl-, and was inhibited by heme poisons, catalase, and BHT. Macrophage binding of NO2-LDL was specific and mediated by neither the LDL receptor nor the scavenger receptor class A type I. Exposure of macrophages to NO2-LDL promoted cholesteryl ester synthesis, intracellular cholesterol and cholesteryl ester accumulation, and foam cell formation. Collectively, these results identify MPO-generated reactive nitrogen species as a physiologically plausible pathway for converting LDL into an atherogenic form.

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.

Macrophage scavenger receptor CD36 is the major receptor for LDL modified by monocyte-generated reactive nitrogen species

The oxidative conversion of LDL into an atherogenic form is considered a pivotal event in the development of cardiovascular disease. Recent studies have identified reactive nitrogen species generated by monocytes by way of the myeloperoxidase-hydrogen peroxide-nitrite (MPO-H(2)O(2)-NO(2)(-)) system as a novel mechanism for converting LDL into a high-uptake form (NO(2)-LDL) for macrophages. We now identify the scavenger receptor CD36 as the major receptor responsible for high-affinity and saturable cellular recognition of NO(2)-LDL by murine and human macrophages. Using cells stably transfected with CD36, CD36-specific blocking mAbs, and CD36-null macrophages, we demonstrated CD36-dependent binding, cholesterol loading, and macrophage foam cell formation after exposure to NO(2)-LDL. Modification of LDL by the MPO-H(2)O(2)-NO(2)(-) system in the presence of up to 80% lipoprotein-deficient serum (LPDS) still resulted in the conversion of the lipoprotein into a high-uptake form for macrophages, whereas addition of less than 5% LPDS totally blocked Cu(2+)-catalyzed LDL oxidation and conversion into a ligand for CD36. Competition studies demonstrated that lipid oxidation products derived from 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphocholine can serve as essential moieties on NO(2)-LDL recognized by CD36. Collectively, these results suggest that MPO-dependent conversion of LDL into a ligand for CD36 is a likely pathway for generating foam cells in vivo. MPO secreted from activated phagocytes may also tag phospholipid-containing targets for removal by CD36-positive cells.

Formation of Nitric Oxide–Derived Oxidants by Myeloperoxidase in Monocytes: Pathways for Monocyte-Mediated Protein Nitration and Lipid Peroxidation In Vivo

Protein nitration and lipid peroxidation are implicated in the pathogenesis of atherosclerosis; however, neither the cellular mediators nor the reaction pathways for these events in vivo are established. In the present study, we examined the chemical pathways available to monocytes for generating reactive nitrogen species and explored their potential contribution to the protein nitration and lipid peroxidation of biological targets. Isolated human monocytes activated in media containing physiologically relevant levels of nitrite (NO(2)(-)), a major end product of nitric oxide ((*)NO) metabolism, nitrate apolipoprotein B-100 tyrosine residues and initiate LDL lipid peroxidation. LDL nitration (assessed by gas chromatography-mass spectrometry quantification of nitrotyrosine) and lipid peroxidation (assessed by high-performance liquid chromatography with online tandem mass spectrometric quantification of distinct products) required cell activation and NO(2)(-); occurred in the presence of metal chelators, superoxide dismutase (SOD), and scavengers of hypohalous acids; and was blocked by myeloperoxidase (MPO) inhibitors and catalase. Monocytes activated in the presence of the exogenous (*)NO generator PAPA NONOate (Z-[N-(3-aminopropyl)-N-(n-propyl)amino]diazen-1-ium-1,2- diolate) promoted LDL protein nitration and lipid peroxidation by a combination of pathways. At low rates of (*)NO flux, both protein nitration and lipid peroxidation were inhibited by catalase and peroxidase inhibitors but not SOD, suggesting a role for MPO. As rates of (*)NO flux increased, both nitrotyrosine formation and 9-hydroxy-10,12-octadecadienoate/9-hydroperoxy-10,12-octadecadieno ic acid production by monocytes became insensitive to the presence of catalase or peroxidase inhibitors, but they were increasingly inhibited by SOD and methionine, suggesting a role for peroxynitrite. Collectively, these results demonstrate that monocytes use distinct mechanisms for generating (*)NO-derived oxidants, and they identify MPO as a source of nitrating intermediates in monocytes.

Paradoxical Association of Enhanced Cholesterol Efflux With Increased Incident Cardiovascular Risks

Objective—Diminished cholesterol efflux activity of apolipoprotein B (apoB)–depleted serum is associated with prevalent coronary artery disease, but its prognostic value for incident cardiovascular events is unclear. We investigated the relationship of cholesterol efflux activity with both prevalent coronary artery disease and incident development of major adverse cardiovascular events (death, myocardial infarction, or stroke). Approach and Results—Cholesterol efflux activity from free cholesterol–enriched macrophages was measured in 2 case–control cohorts: (1) an angiographic cohort (n=1150) comprising stable subjects undergoing elective diagnostic coronary angiography and (2) an outpatient cohort (n=577). Analysis of media from cholesterol efflux assays revealed that the high-density lipoprotein fraction (1.063<d<1.21) contained only a minority (≈40%) of [14C]cholesterol released, with the majority found within the lipoprotein particle–depleted fraction, where ≈60% was recovered after apolipoprotein A1 immunoprecipitation. Albumin immunoprecipitation recovered another ≈30% of radiolabeled cholesterol within this fraction. Enhanced cholesterol efflux activity from ATP-binding cassette transporter A1–stimulated macrophages was associated with reduced risk of prevalent coronary artery disease in unadjusted models within both cohorts; however, the inverse risk relationship remained significant after adjustment for traditional coronary artery disease risk factors only within the outpatient cohort. Surprisingly, higher cholesterol efflux activity was associated with increase in prospective (3 years) risk of myocardial infarction/stroke (adjusted hazard ratio, 2.19; 95% confidence interval, 1.02–4.74) and major adverse cardiovascular events (adjusted hazard ratio, 1.85; 95% confidence interval, 1.11–3.06). Conclusions—Heightened cholesterol efflux to apoB-depleted serum was paradoxically associated with increased prospective risk for myocardial infarction, stroke, and death. The majority of released radiolabeled cholesterol from macrophages in cholesterol efflux activity assays does not reside within a high-density lipoprotein particle.

Cancer Stem Cell-Specific Scavenger Receptor CD36 Drives Glioblastoma Progression

Glioblastoma (GBM) contains a self‐renewing, tumorigenic cancer stem cell (CSC) population which contributes to tumor propagation and therapeutic resistance. While the tumor microenvironment is essential to CSC self‐renewal, the mechanisms by which CSCs sense and respond to microenvironmental conditions are poorly understood. Scavenger receptors are a broad class of membrane receptors well characterized on immune cells and instrumental in sensing apoptotic cellular debris and modified lipids. Here, we provide evidence that CSCs selectively use the scavenger receptor CD36 to promote their maintenance using patient‐derived CSCs and in vivo xenograft models. CD36 expression was observed in GBM cells in addition to previously described cell types including endothelial cells, macrophages, and microglia. CD36 was enriched in CSCs and was able to functionally distinguish self‐renewing cells. CD36 was coexpressed with integrin alpha 6 and CD133, previously described CSC markers, and CD36 reduction resulted in concomitant loss of integrin alpha 6 expression, self‐renewal, and tumor initiation capacity. We confirmed oxidized phospholipids, ligands of CD36, were present in GBM and found that the proliferation of CSCs, but not non‐CSCs, increased with exposure to oxidized low‐density lipoprotein. CD36 was an informative biomarker of malignancy and negatively correlated to patient prognosis. These results provide a paradigm for CSCs to thrive by the selective enhanced expression of scavenger receptors, providing survival, and metabolic advantages. Stem Cells 2014;32:1746–1758

MyD88-dependent interplay between myeloid and endothelial cells in the initiation and progression of obesity-associated inflammatory diseases.

MyD88-dependent GM-CSF production by endothelial cells plays a role in the initiation of obesity-associated inflammation by promoting adipose macrophage recruitment and M1-like polarization.

Cholesteryl Hydroperoxyoctadecadienoate from Oxidized Low Density Lipoprotein Inactivates Platelet-derived Growth Factor

Both oxidized low density lipoprotein (ox-LDL) and platelet-derived growth factor (PDGF) have been implicated in the genesis of various inflammatory responses, including atherosclerosis. We demonstrate here a novel interaction between specific oxidized lipids derived from ox-LDL and PDGF. The lipid moieties of ox-LDL caused concentration-dependent inactivation of PDGF as measured by loss of its mitogenic activity and its binding to high affinity receptors. Reverse-phase and normal-phase HPLC were used to purify the inactivating component in the lipid mixture. By fast atom bombardment mass spectrometry and infrared spectroscopy, we identified the inactivating lipids as the 9- and 13-hydroperoxy derivatives of cholesteryl linoleate, cholesteryl hydroperoxyoctadecadienoate. When a series of cholesteryl esters were subjected to oxidizing conditions, only those containing two or more double bonds caused inactivation of PDGF; the extent of inactivation increased with increased levels of oxidation. Exposing PDGF to cumene hydroperoxide, t-butyl hydroperoxide, or hydrogen peroxide did not affect the activity of the mitogen. The oxidized lipid had no effect on the mitogenic activity of epidermal growth factor but did abolish the mitogenic activity of basic fibroblast growth factor and the antiproliferative activity of transforming growth factor β1. The inactivation of PDGF and other cytokines by lipid hydroperoxides may occur in such processes as vascular disease, inflammation, and wound healing.