Gut Microbial Metabolite TMAO Enhances Platelet Hyperreactivity and Thrombosis Risk.

Normal platelet function is critical to blood hemostasis and maintenance of a closed circulatory system. Heightened platelet reactivity, however, is associated with cardiometabolic diseases and enhanced potential for thrombotic events. We now show gut microbes, through generation of trimethylamine N-oxide (TMAO), directly contribute to platelet hyperreactivity and enhanced thrombosis potential. Plasma TMAO levels in subjects (n > 4,000) independently predicted incident (3 years) thrombosis (heart attack, stroke) risk. Direct exposure of platelets to TMAO enhanced sub-maximal stimulus-dependent platelet activation from multiple agonists through augmented Ca(2+) release from intracellular stores. Animal model studies employing dietary choline or TMAO, germ-free mice, and microbial transplantation collectively confirm a role for gut microbiota and TMAO in modulating platelet hyperresponsiveness and thrombosis potential and identify microbial taxa associated with plasma TMAO and thrombosis potential. Collectively, the present results reveal a previously unrecognized mechanistic link between specific dietary nutrients, gut microbes, platelet function, and thrombosis risk.

D-2-Hydroxyglutarate Inhibits Calcineurin Phosphatase Activity to Abolish NF-AT Activation and IL-2 Induction in Stimulated Lymphocytes.

Gliomas expressing mutant isocitrate dehydrogenases excessively synthesize d-2-hydroxyglutarate (D2HG), suppressing immune surveillance. A portion of this D2HG is released from these tumor cells, but the way environmental D2HG inhibits lymphocyte function is undefined. We incubated human PBLs or Jurkat T cells with D2HG at concentrations present within and surrounding gliomas or its obverse l-2-hydroxyglutarate (L2HG) stereoisomer. We quantified each 2HG stereoisomer within washed cells by N-(p-toluenesulfonyl)-l-phenylalanyl chloride derivatization with stable isotope-labeled D2HG and L2HG internal standards, HPLC separation, and mass spectrometry. D2HG was present in quiescent cells and was twice as abundant as L2HG. Extracellular 2HG rapidly increased intracellular levels of the provided stereoisomer by a stereoselective, concentration-dependent process. IL-2 expression, even when elicited by A23187 and PMA, was abolished by D2HG in a concentration-dependent manner, with significant reduction at just twice its basal level. In contrast, L2HG was only moderately inhibitory. IL-2 expression is regulated by increased intracellular Ca2+ that stimulates calcineurin to dephosphorylate cytoplasmic phospho-NF-AT, enabling its nuclear translocation. D2HG abolished stimulated expression of a stably integrated NF-AT-driven luciferase reporter that precisely paralleled its concentration-dependent inhibition of IL-2. D2HG did not affect intracellular Ca2+. Rather, surface plasmon resonance showed D2HG, but not L2HG, bound calcineurin, and D2HG, but not L2HG, inhibited Ca2+-dependent calcineurin phosphatase activity in stimulated Jurkat extracts. Thus, D2HG is a stereoselective calcineurin phosphatase inhibitor that prevents NF-AT dephosphorylation and so abolishes IL-2 transcription in stimulated lymphocytes. This occurs at D2HG concentrations found within and adjacent to gliomas independent of its metabolic or epigenetic transcriptional regulation.

Dysregulated platelet function in patients with postacute sequelae of COVID-19.

BACKGROUND: Postacute sequelae of COVID-19 (PASC), also referred to as "Long COVID", sometimes follows COVID-19, a disease caused by SARS-CoV-2. Although SARS-CoV-2 is well known to promote a prothrombotic state, less is known about the thrombosis risk in PASC. Our objective was to evaluate platelet function and thrombotic potential in patients following recovery from SARS-CoV-2, but with clear symptoms of patients with PASC. METHODS: patients with PASC and matched healthy controls were enrolled in the study on average 15 months after documented SARS-CoV-2 infection. Platelet activation was evaluated by light transmission aggregometry (LTA) and flow cytometry in response to platelet surface receptor agonists. Thrombosis in platelet-deplete plasma was evaluated by Factor Xa activity. A microfluidics system assessed thrombosis in whole blood under shear stress conditions. RESULTS: A mild increase in platelet aggregation in patients with PASC through the thromboxane receptor was observed, and platelet activation through the glycoprotein VI (GPVI) receptor was decreased in patients with PASC compared to age- and sex-matched healthy controls. Thrombosis under shear conditions as well as Factor Xa activity were reduced in patients with PASC. Plasma from patients with PASC was an extremely potent activator of washed, healthy platelets - a phenomenon not observed when stimulating healthy platelets after incubation with plasma from healthy individuals. CONCLUSIONS: patients with PASC show dysregulated responses in platelets and coagulation in plasma, likely caused by a circulating molecule that promotes thrombosis. A hitherto undescribed protective response appears to exist in patients with PASC to counterbalance ongoing thrombosis that is common to SARS-CoV-2 infection.

CD36 Enhances Vascular Smooth Muscle Cell Proliferation and Development of Neointimal Hyperplasia.

Objective- Dysregulated proliferation of vascular smooth muscle cells (VSMC) plays an essential role in neointimal hyperplasia. CD36 functions critically in atherogenesis and thrombosis. We hypothesize that CD36 regulates VSMC proliferation and contributes to the development of obstructive vascular diseases. Approach and Results- We found by immunofluorescent staining that CD36 was highly expressed in human vessels with obstructive diseases. Using guidewire-induced carotid artery injury and shear stress-induced intima thickening models, we compared neointimal hyperplasia in Apoe-/-, Cd36-/- /Apoe-/-, and CD36 specifically deleted in VSMC (VSMC cd36-/-) mice. CD36 deficiency, either global or VSMC-specific, dramatically reduced injury-induced neointimal thickening. Correspondingly, carotid artery blood flow was significantly increased in Cd36-/- /Apoe-/- compared with Apoe-/- mice. In cultured VSMCs from thoracic aorta of wild-type and Cd36-/- mice, we found that loss of CD36 significantly decreased serum-stimulated proliferation and increased cell populations in S phase, suggesting that CD36 is necessary for VSMC S/G2-M-phase transition. Treatment of VSMCs with a TSR (thrombospondin type 1 repeat) peptide significantly increased wild-type, but not Cd36-/- VSMC proliferation. TSR or serum treatment significantly increased cyclin A expression in wild-type, but not in Cd36-/- VSMCs. STAT3 (signal transducer and activator of transcription), which reportedly enhances both VSMC differentiation and maturation, was higher in Cd36-/- VSMCs. CD36 deficiency significantly decreased expression of Col1A1 (type 1 collagen A1 chain) and TGF-ß1 (transforming growth factor beta 1), and increased expression of contractile proteins, including calponin 1 and smooth muscle α actin, and dramatically increased cell contraction. Conclusions- CD36 promotes VSMC proliferation via upregulation of cyclin A expression that contributes to the development of neointimal hyperplasia, collagen deposition, and obstructive vascular diseases.

Epidermal Growth Factor (EGF) Autocrine Activation of Human Platelets Promotes EGF Receptor-Dependent Oral Squamous Cell Carcinoma Invasion, Migration, and Epithelial Mesenchymal Transition.

Activated platelets release functional, high m.w. epidermal growth factor (HMW-EGF). In this study, we show platelets also express epidermal growth factor (EGF) receptor (EGFR) protein, but not ErbB2 or ErbB4 coreceptors, and so might respond to HMW-EGF. We found HMW-EGF stimulated platelet EGFR autophosphorylation, PI3 kinase-dependent AKT phosphorylation, and a Ca2+ transient that were blocked by EGFR tyrosine kinase inhibition. Strong (thrombin) and weak (ADP, platelet-activating factor) G protein-coupled receptor agonists and non-G protein-coupled receptor collagen recruited EGFR tyrosine kinase activity that contributed to platelet activation because EGFR kinase inhibition reduced signal transduction and aggregation induced by each agonist. EGF stimulated ex vivo adhesion of platelets to collagen-coated microfluidic channels, whereas systemic EGF injection increased initial platelet deposition in FeCl3-damaged murine carotid arteries. EGFR signaling contributes to oral squamous cell carcinoma (OSCC) tumorigenesis, but the source of its ligand is not established. We find individual platelets were intercalated within OSCC tumors. A portion of these platelets expressed stimulation-dependent Bcl-3 and IL-1ß and so had been activated. Stimulated platelets bound OSCC cells, and material released from stimulated platelets induced OSCC epithelial-mesenchymal transition and stimulated their migration and invasion through Matrigel barriers. Anti-EGF Ab or EGFR inhibitors abolished platelet-induced tumor cell phenotype transition, migration, and invasion; so the only factor released from activated platelets necessary for OSCC metastatic activity was HMW-EGF. These results establish HMW-EGF in platelet function and elucidate a previously unsuspected connection between activated platelets and tumorigenesis through rapid, and prolonged, autocrine-stimulated release of HMW-EGF by tumor-associated platelets.

Modulation of inflammatory platelet-activating factor (PAF) receptor by the acyl analogue of PAF.

Platelet-activating factor (PAF) is a potent inflammatory mediator that exerts its actions via the single PAF receptor (PAF-R). Cells that biosynthesize alkyl-PAF also make abundant amounts of the less potent PAF analogue acyl-PAF, which competes for PAF-R. Both PAF species are degraded by the plasma form of PAF acetylhydrolase (PAF-AH). We examined whether cogenerated acyl-PAF protects alkyl-PAF from systemic degradation by acting as a sacrificial substrate to enhance inflammatory stimulation or as an inhibitor to dampen PAF-R signaling. In ex vivo experiments both PAF species are prothrombotic in isolation, but acyl-PAF reduced the alkyl-PAF-induced stimulation of human platelets that express canonical PAF-R. In Swiss albino mice, alkyl-PAF causes sudden death, but this effect can also be suppressed by simultaneously administering boluses of acyl-PAF. When PAF-AH levels were incrementally elevated, the protective effect of acyl-PAF on alkyl-PAF-induced death was serially decreased. We conclude that, although acyl-PAF in isolation is mildly proinflammatory, in a pathophysiological setting abundant acyl-PAF suppresses the action of alkyl-PAF. These studies provide evidence for a previously unrecognized role for acyl-PAF as an inflammatory set-point modulator that regulates both PAF-R signaling and hydrolysis.

The soluble protease ADAMDEC1 released from activated platelets hydrolyzes platelet membrane pro-epidermal growth factor (EGF) to active high-molecular-weight EGF.

Platelets are the sole source of EGF in circulation, yet how EGF is stored or released from stimulated cells is undefined. In fact, we found platelets did not store EGF, synthesized as a single 6-kDa domain in pro-EGF, but rather expressed intact pro-EGF precursor on granular and plasma membranes. Activated platelets released high-molecular-weight (HMW)-EGF, produced by a single cleavage between the EGF and the transmembrane domains of pro-EGF. We synthesized a fluorogenic peptide encompassing residues surrounding the putative sessile arginyl residue and found stimulated platelets released soluble activity that cleaved this pro-EGF1020-1027 peptide. High throughput screening identified chymostatins, bacterial peptides with a central cyclic arginyl structure, as inhibitors of this activity. In contrast, the matrix metalloproteinase/TACE (tumor necrosis factor-α-converting enzyme) inhibitor GM6001 was ineffective. Stimulated platelets released the soluble protease ADAMDEC1, recombinant ADAMDEC1 hydrolyzed pro-EGF1020-1027, and this activity was inhibited by chymostatin and not GM6001. Biotinylating platelet surface proteins showed ADAMDEC1 hydrolyzed surface pro-EGF to HMW-EGF that stimulated HeLa EGF receptor (EGFR) reporter cells and EGFR-dependent tumor cell migration. This proteolysis was inhibited by chymostatin and not GM6001. Metabolizing pro-EGF Arg1023 to citrulline with recombinant polypeptide arginine deiminase 4 (PAD4) abolished ADAMDEC1-catalyzed pro-EGF1020-1027 peptidolysis, while pretreating intact platelets with PAD4 suppressed ADAMDEC1-, thrombin-, or collagen-induced release of HMW-EGF. We conclude that activated platelets release ADAMDEC1, which hydrolyzes pro-EGF to soluble HMW-EGF, that HMW-EGF is active, that proteolytic cleavage of pro-EGF first occurs at the C-terminal arginyl residue of the EGF domain, and that proteolysis is the regulated and rate-limiting step in generating soluble EGF bioactivity from activated platelets.

The Role of Platelet Activation and Inflammation in Early Brain Injury Following Subarachnoid Hemorrhage.

BACKGROUND: Early brain injury (EBI) following aneurysmal subarachnoid hemorrhage (SAH) is an important predictor of poor functional outcome, yet the underlying mechanism is not well understood. Animal studies suggest that platelet activation and inflammation with subsequent microthrombosis and ischemia may be a mechanism of EBI. METHODS: A prospective, hypothesis-driven study of spontaneous, SAH patients and controls was conducted. Platelet activation [thromboelastography maximum amplitude (MA)] and inflammation [C-reactive protein (CRP)] were measured serially over time during the first 72 h following SAH onset. Platelet activation and inflammatory markers were compared between controls and SAH patients with mild [Hunt-Hess (HH) 1-3] versus severe (HH 4-5) EBI. The association of these biomarkers with 3-month functional outcomes was evaluated. RESULTS: We enrolled 127 patients (106 SAH; 21 controls). Platelet activation and CRP increased incrementally with worse EBI/HH grade, and both increased over 72 h (all P < 0.01). Both were higher in severe versus mild EBI (MA 68.9 vs. 64.8 mm, P = 0.001; CRP 12.5 vs. 1.5 mg/L, P = 0.003) and compared to controls (both P < 0.003). Patients with delayed cerebral ischemia (DCI) had more platelet activation (66.6 vs. 64.9 in those without DCI, P = 0.02) within 72 h of ictus. At 3 months, death or severe disability was more likely with higher levels of platelet activation (mRS4-6 OR 1.18, 95 % CI 1.05-1.32, P = 0.007) and CRP (mRS4-6 OR 1.02, 95 % CI 1.00-1.03, P = 0.041). CONCLUSIONS: Platelet activation and inflammation occur acutely after SAH and are associated with worse EBI, DCI and poor 3-month functional outcomes. These markers may provide insight into the mechanism of EBI following SAH.

Thymidine phosphorylase participates in platelet signaling and promotes thrombosis.

RATIONALE: Platelets contain abundant thymidine phosphorylase (TYMP), which is highly expressed in diseases with high risk of thrombosis, such as atherosclerosis and type II diabetes mellitus. OBJECTIVE: To test the hypothesis that TYMP participates in platelet signaling and promotes thrombosis. METHODS AND RESULTS: By using a ferric chloride (FeCl3)-induced carotid artery injury thrombosis model, we found time to blood flow cessation was significantly prolonged in Tymp(-/-) and Tymp(+/-) mice compared with wild-type mice. Bone marrow transplantation and platelet transfusion studies demonstrated that platelet TYMP was responsible for the antithrombotic phenomenon in the TYMP-deficient mice. Collagen-, collagen-related peptide-, adenosine diphosphate-, or thrombin-induced platelet aggregation were significantly attenuated in Tymp(+/-) and Tymp(-/-) platelets, and in wild type or human platelets pretreated with TYMP inhibitor KIN59. Tymp deficiency also significantly decreased agonist-induced P-selectin expression. TYMP contains an N-terminal SH3 domain-binding proline-rich motif and forms a complex with the tyrosine kinases Lyn, Fyn, and Yes in platelets. TYMP-associated Lyn was inactive in resting platelets, and TYMP trapped and diminished active Lyn after collagen stimulation. Tymp/Lyn double haploinsufficiency diminished the antithrombotic phenotype of Tymp(+/-) mice. TYMP deletion or inhibition of TYMP with KIN59 dramatically increased platelet-endothelial cell adhesion molecule 1 tyrosine phosphorylation and diminished collagen-related peptide- or collagen-induced AKT phosphorylation. In vivo administration of KIN59 significantly inhibited FeCl3-induced carotid artery thrombosis without affecting hemostasis. CONCLUSIONS: TYMP participates in multiple platelet signaling pathways and regulates platelet activation and thrombosis. Targeting TYMP might be a novel antiplatelet and antithrombosis therapy.

Deubiquitinases Modulate Platelet Proteome Ubiquitination, Aggregation, and Thrombosis.

OBJECTIVE: Platelets express a functional ubiquitin-proteasome system. Mass spectrometry shows that platelets contain several deubiquitinases, but whether these are functional, modulate the proteome, or affect platelet reactivity are unknown. APPROACH AND RESULTS: Platelet lysates contained ubiquitin-protein deubiquitinase activity hydrolyzing both Lys48 and Lys63 polyubiquitin conjugates that was suppressed by the chemically unrelated deubiquitinase inhibitors PYR41 and PR619. These inhibitors acutely and markedly increased monoubiquitination and polyubiquitination of the proteome of resting platelets. PYR41 (intravenous, 15 minutes) significantly impaired occlusive thrombosis in FeCl3-damaged carotid arteries, and deubiquitinase inhibition reduced platelet adhesion and retention during high shear flow of whole blood through microfluidic chambers coated with collagen. Total internal reflection microscopy showed that adhesion and spreading in the absence of flow were strongly curtailed by these inhibitors with failure of stable process extension and reduced the retraction of formed clots. Deubiquitinase inhibition also sharply reduced homotypic platelet aggregation in response to not only the incomplete agonists ADP and collagen acting through glycoprotein VI but also to the complete agonist thrombin. Suppressed aggregation was accompanied by curtailed procaspase activating compound-1 binding to activated IIb/IIIa and inhibition of P-selectin translocation to the platelet surface. Deubiquitinase inhibition abolished the agonist-induced spike in intracellular calcium, suppressed Akt phosphorylation, and reduced agonist-stimulated phosphatase and tensin homolog phosphatase phosphorylation. Platelets express the proteasome-associated deubiquitinases USP14 and UCHL5, and selective inhibition of these enzymes by b-AP15 reproduced the inhibitory effect of the general deubiquitinase inhibitors on ex vivo platelet function. CONCLUSIONS: Remodeling of the ubiquitinated platelet proteome by deubiquitinases promotes agonist-stimulated intracellular signal transduction and platelet responsiveness.

Proteasome proteolysis supports stimulated platelet function and thrombosis.

OBJECTIVE: Proteasome inhibitors used in the treatment of hematologic cancers also reduce thrombosis. Whether the proteasome participates in platelet activation or function is unclear because little is known of the proteasome in these terminally differentiated cells. APPROACH AND RESULTS: Platelets displayed all 3 primary proteasome protease activities, which MG132 and bortezomib (Velcade) inhibited. Proteasome substrates are marked by ubiquitin, and platelets contained a functional ubiquitination system that modified the proteome by monoubiquitination and polyubiquitination. Systemic MG132 strongly suppressed the formation of occlusive, platelet-rich thrombi in FeCl3-damaged carotid arteries. Transfusion of platelets treated ex vivo with MG132 and washed before transfusion into thrombocytopenic mice also reduced carotid artery thrombosis. Proteasome inhibition reduced platelet aggregation by low thrombin concentrations and ristocetin-stimulated agglutination through the glycoprotein Ib-IX-V complex. This receptor was not appropriately internalized after proteasome inhibition in stimulated platelets, and spreading and clot retraction after MG132 exposure also were decreased. The effects of proteasome inhibitors were not confined to a single receptor as MG132 suppressed thrombin-stimulated, ADP-stimulated, and lipopolysaccharide-stimulated microparticle shedding. Proteasome inhibition increased ubiquitin decoration of cytoplasmic proteins, including the cytoskeletal proteins Filamin A and Talin-1. Mass spectrometry revealed a single MG132-sensitive tryptic cleavage after R1745 in an extended Filamin A loop, which would separate its actin-binding domain from its carboxy terminal glycoprotein Ibα-binding domain. CONCLUSIONS: Platelets contain a ubiquitin/proteasome system that marks cytoskeletal proteins for proteolytic modification to promote productive platelet-platelet and platelet-wall interactions.

Lipopolysaccharide stimulates platelets through an IL-1ß autocrine loop.

LPS activates platelets through TLR4, aiding productive sepsis, with stimulated splicing and translation of stored heteronuclear pro-IL-1ß RNA. Although the IL-1R type 1 (IL-1R1) receptor for IL-1 shares downstream components with the TLR4 receptor, platelets are not known to express IL-1R1, nor are they known to respond to this cytokine. We show by flow cytometry and Western blotting that platelets express IL-1R1, and that IL-1ß and IL-1α stimulate heteronuclear I-1ß splicing and translation of the newly made mRNA in platelets. Platelets also respond to the IL-1ß they make, which is exclusively associated with shed microparticles. Specific blockade of IL-1R1 with IL-1R antagonist suppressed platelet stimulation by IL-1, so IL-1ß stimulates its own synthesis in an autocrine signaling loop. Strikingly, IL-1R antagonist inhibition, pharmacologic or genetic suppression of pro-IL-1ß processing to active cytokine by caspase-1, or blockade of de novo protein synthesis also blocked LPS-induced IL-1ß mRNA production. Robust stimulation of platelets by LPS therefore also required IL-1ß amplification. Activated platelets made IL-1ß in vivo as IL-1ß rapidly accumulated in occluded murine carotid arteries by posttranscriptional RNA splicing unique to platelets. We conclude that IL-1ß is a platelet agonist, that IL-1ß acts through an autocrine stimulatory loop, that an IL-1ß autocrine loop is required to amplify platelet activation by LPS, and that platelets immobilized in occlusive thrombi are activated over time to produce IL-1ß. IL-1 is a new platelet agonist that promotes its own synthesis, connecting thrombosis with immunity.

Aspirin hydrolysis in plasma is a variable function of butyrylcholinesterase and platelet-activating factor acetylhydrolase 1b2 (PAFAH1b2).

Aspirin is rapidly hydrolyzed within erythrocytes by a heterodimer of PAFAH1b2/PAFAH1b3 but also in plasma by an unidentified activity. Hydrolysis in both compartments was variable, with a 12-fold variation in plasma among 2226 Cleveland Clinic GeneBank patients. Platelet inhibition by aspirin was suppressed in plasma that rapidly hydrolyzed aspirin. Plasma aspirin hydrolysis was significantly higher in patients with coronary artery disease compared with control subjects (16.5 ± 4.4 versus 15.1 ± 3.7 nmol/ml/min; p = 3.4 × 10(-8)). A genome-wide association study of 2054 GeneBank subjects identified a single locus immediately adjacent to the BCHE (butyrylcholinesterase) gene associated with plasma aspirin hydrolytic activity (lead SNP, rs6445035; p = 9.1 × 10(-17)). However, its penetrance was low, and plasma from an individual with an inactivating mutation in BCHE still effectively hydrolyzed aspirin. A second aspirin hydrolase was identified in plasma, the purification of which showed it to be homomeric PAFAH1b2. This is distinct from the erythrocyte PAFAH1b2/PAFAH1b3 heterodimer. Inhibitors showed that both butyrylcholinesterase (BChE) and PAFAH1b2 contribute to aspirin hydrolysis in plasma, with variation primarily reflecting non-genetic variation of BChE activity. Therefore, aspirin is hydrolyzed in plasma by two enzymes, BChE and a new extracellular form of platelet-activating factor acetylhydrolase, PAFAH1b2. Hydrolytic effectiveness varies widely primarily from non-genetic variation of BChE activity that affects aspirin bioavailability in blood and the ability of aspirin to inhibit platelet aggregation.

Apolipoprotein A1 regulates coenzyme Q10 absorption, mitochondrial function, and infarct size in a mouse model of myocardial infarction.

HDL and apolipoprotein A1 (apoA1) concentrations inversely correlate with risk of death from ischemic heart disease; however, the role of apoA1 in the myocardial response to ischemia has not been well defined. To test whether apoA1, the primary HDL apolipoprotein, has an acute anti-inflammatory role in ischemic heart disease, we induced myocardial infarction via direct left anterior descending coronary artery ligation in apoA1 null (apoA1(-/-)) and apoA1 heterozygous (apoA1(+/-)) mice. We observed that apoA1(+/-) and apoA1(-/-) mice had a 52% and 125% increase in infarct size as a percentage of area at risk, respectively, compared with wild-type (WT) C57BL/6 mice. Mitochondrial oxidation contributes to tissue damage in ischemia-reperfusion injury. A substantial defect was present at baseline in the electron transport chain of cardiac myocytes from apoA1(-/-) mice localized to the coenzyme Q (CoQ) pool with impaired electron transfer (67% decrease) from complex II to complex III. Administration of coenzyme Q10 (CoQ10) to apoA1 null mice normalized the cardiac mitochondrial CoQ pool and reduced infarct size to that observed in WT mice. CoQ10 administration did not significantly alter infarct size in WT mice. These data identify CoQ pool content leading to impaired mitochondrial function as major contributors to infarct size in the setting of low HDL/apoA1. These data suggest a previously unappreciated mechanism for myocardial stunning, cardiac dysfunction, and muscle pain associated with low HDL and low apoA1 concentrations that can be corrected by CoQ10 supplementation and suggest populations of patients that may benefit particularly from CoQ10 supplementation.

OxNASH score correlates with histologic features and severity of nonalcoholic fatty liver disease.

BACKGROUND AND AIM: Oxidative stress is a core abnormality responsible for disease progression in nonalcoholic fatty liver disease (NAFLD). By employing a highly sensitive liquid chromatography-tandem mass spectrometry (LC/MS/MS) approach we recently were able to define the circulating profile of bioactive lipid peroxidation products characteristic of patients with nonalcoholic steatohepatitis (NASH) and developed the OxNASH score for NASH diagnosis. The aims of this study were to assess the utility of OxNASH as a predictor of NASH and study the association between OxNASH and specific histologic features of NAFLD. METHODS: Our cohort consisted of 122 patients undergoing liver biopsy for clinical suspicion of NAFLD. The NAFLD activity score (NAS) was calculated for each patient. Levels of fatty acid oxidation products were quantified using stable isotope dilution LC/MS/MS, and OxNASH was calculated. RESULTS: The mean age of our patients was 49.3 (±11.6) years, and the mean body mass index was 31.5 (±4.8) kg/m(2). The majority of patients were Caucasian (82 %) and 48 % were female. OxNASH correlated with NAS and with the individual histologic features of NAFLD, namely, steatosis, inflammation, and ballooning (P < 0.05), with the strongest association being with inflammation [rho (ρ) 0.40, 95 % confidence interval 0.23, 0.57, P < 0.001]. There was also a correlation between the stage of fibrosis and OxNASH (P = 0.001). These associations remained statistically significant after adjustment for multiple confounders. CONCLUSIONS: Based on our results, in adult patients with NAFLD, OxNASH correlates with histologic features of NASH and appears to be a promising noninvasive marker.

Oxidatively truncated phospholipids are required agents of tumor necrosis factor α (TNFα)-induced apoptosis.

TNFα generates reactive oxygen species (ROS) at the cell surface that induce cell death, but how ROS communicate to mitochondria and their specific apoptotic action(s) are both undefined. ROS oxidize phospholipids to hydroperoxides that are friable and fragment adjacent to the (hydro)peroxide function, forming truncated phospholipids, such as azelaoyl phosphatidylcholine (Az-PC). Az-PC is relatively soluble, and exogenous Az-PC rapidly enters cells to damage mitochondrial integrity and initiate intrinsic apoptosis. We determined whether this toxic phospholipid is formed within cells during TNFα stimulation in sufficient quantities to induce apoptosis and if they are essential in TNFα-induced cytotoxicity. We found that TNFα induced ROS formation and phospholipid peroxidation in Jurkat cells, and either chemical interference with NADPH oxidase activity or siRNA suppression of the NADPH oxidase-4 subunit blocked ROS accumulation and phospholipid peroxidation. Mass spectrometry showed that phospholipid peroxides and then Az-PC increased after TNFα exposure, whereas ROS inhibition abolished Az-PC accumulation and TNFα-induced cell death. Glutathione peroxidase-4 (GPx4), which specifically metabolizes lipid hydroperoxides, fell in TNFα-stimulated cells prior to death. Ectopic GPx4 overcame this, reduced peroxidized phospholipid accumulation, blocked Az-PC accumulation, and prevented death. Conversely, GPx4 siRNA knockdown enhanced phospholipid peroxidation, increasing TNFα-stimulated Az-PC formation and apoptosis. Truncated phospholipids were essential elements of TNFα-induced apoptosis because overexpression of PAFAH2 (a phospholipase A(2) that selectively hydrolyzes truncated phospholipids) blocked TNFα-induced Az-PC accumulation without affecting phospholipid peroxidation. PAFAH2 also abolished apoptosis. Thus, phospholipid oxidation and truncation to apoptotic phospholipids comprise an essential element connecting TNFα receptor signaling to mitochondrial damage and apoptotic death.

Bioactive oxidatively truncated phospholipids in inflammation and apoptosis: formation, targets, and inactivation.

This report reviews structurally related phospholipid oxidation products that are biologically active where molecular mechanisms have been defined. Phospholipids containing polyunsaturated fatty acyl residues are chemically or enzymatically oxidized to phospholipid hydroperoxides, which may fragment on either side of the newly introduced peroxy function to form phospholipids with a truncated sn-2 residue. These truncated phospholipids not subject to biologic control of their production and, depending on the sn-2 residue length and structure, can stimulate the plasma membrane receptor for PAF. Alternatively, these chemically formed products can be internalized by a transport system to either stimulate the lipid activated nuclear transcription factor PPARγ or at higher levels interact with mitochondria to initiate the intrinsic apoptotic cascade. Intracellular PAF acetylhydrolases specifically hydrolyze truncated phospholipids, and not undamaged, biosynthetic phospholipids, to protect cells from oxidative death. Truncated phospholipids are also formed within cells where they couple cytokine stimulation to mitochondrial damage and apoptosis. The relevance of intracellular truncated phospholipids is shown by the complete protection from cytokine induced apoptosis by PAF acetylhydrolase expression. This protection shows truncated phospholipids are the actual effectors of cytokine mediated toxicity. This article is part of a Special Issue entitled: Oxidized phospholipids-their properties and interactions with proteins.

Translational control of JunB, an AP-1 transcription factor, in activated human endothelial cells.

Stimulated endothelial cells (EC) assume an activated phenotype with pro-inflammatory and prothrombotic features, requiring new gene and protein expression. New protein synthesis in activated EC is largely regulated by transcriptional events controlled by a variety of transcription factors. However, post-transcriptional control of gene expression also influences phenotype and allows the cell to alter protein expression in a faster and more direct way than is typically possible with transcriptional mechanisms. We sought to demonstrate that post-transcriptional control of gene expression occurs during EC activation. Using thrombin-activated EC and a high-throughput, microarray-based approach, we identified a number of gene products that may be regulated through post-transcriptional mechanisms, including the AP-1 transcription factor JunB. Using polysome profiling, cytoplasts and other standard cell biologic techniques, JunB is shown to be regulated at a post-transcriptional level during EC activation. In activated EC, the AP-1 transcription factor JunB, is regulated on a post-transcriptional level. Signal-dependent control of translation may regulate transcription factor expression and therefore, subsequent transcriptional events in stimulated EC.

Circulating platelet-activating factor is primarily cleared by transport, not intravascular hydrolysis by lipoprotein-associated phospholipase A2/ PAF acetylhydrolase.

RATIONALE: The phospholipid platelet-activating factor (PAF) stimulates all cells of the innate immune system and numerous cardiovascular cells. A single enzyme (plasma PAF acetylhydrolase [PAF-AH] or lipoprotein-associated phospholipase [Lp-PL]A(2)) in plasma hydrolyzes PAF, but significant controversy exists whether its action is pro- or antiinflammatory and accordingly whether its inhibition will slow cardiovascular disease. OBJECTIVE: We sought to define how PAF and related short-chain oxidized phospholipids turnover in vivo and the role of PAF acetylhydrolase/Lp-PLA(2) in this process. METHODS AND RESULTS: [(3)H-acetyl]PAF was hydrolyzed by murine or human plasma (t(1/2), 3 and 7 minutes, respectively), but injected [(3)H-acetyl]PAF disappeared from murine circulation more quickly (t(1/2), <30 seconds). [(3)H]PAF clearance was unchanged in PAF receptor(-/-) animals, or over the first 2 half-lives in PAF-AH(-/-) animals. [(3)H]PAF turnover was reduced by coinjecting excess unlabeled PAF or an oxidatively truncated phospholipid, and [(3)H]PAF clearance was slowed in hyperlipidemic apolipoprotein (apo)E(-/-) mice with excess circulating oxidatively truncated phospholipids. [(3)H]PAF, fluorescent NBD-PAF, or fluorescent oxidatively truncated phospholipid were primarily accumulated by liver and lung, and were transported into endothelium as intact phospholipids through a common mechanism involving TMEM30a. CONCLUSIONS: Circulating PAF and oxidized phospholipids are continually and rapidly cleared, and hence continually and rapidly produced. Saturable PAF receptor-independent transport, rather than just intravascular hydrolysis, controls circulating inflammatory and proapoptotic oxidized phospholipid mediators. Intravascular PAF has access to intracellular compartments. Inflammatory and proapoptotic phospholipids may accumulate in the circulation as transport is overwhelmed by substrates in hyperlipidemia.

Lipopolysaccharide signaling without a nucleus: kinase cascades stimulate platelet shedding of proinflammatory IL-1ß-rich microparticles.

Platelets contain unspliced heteronuclear IL-1ß RNA, which is rapidly spliced and translated upon activation. LPS is a superior agonist for this atypical platelet response, but how LPS induces proinflammatory cytokine production in anucleate cells lacking NF-κB is unknown. Platelets express functional TLR4, and stimulation by LPS induced rapid splicing, translation, and secretion of mature IL-1ß after caspase-1 processing. LPS stimulated microparticle shedding, and secreted IL-1ß was exclusively present in these particles. Microparticles from LPS-stimulated platelets induced VCAM-1 production by cultured human endothelial cells, and blockade of endothelial IL-1ß receptor with IL-1 receptor antagonist completely suppressed endothelial activation. Splicing was posttranscriptional as the SR kinase inhibitor TG003 blocked IL-1ß RNA production by platelets, but not by monocytes, and was dependent on exogenous CD14--a property of platelets. We used a combination of small-molecule inhibitors, cell-penetrating chimeric peptide inhibitors, and gene-targeted animals to show splicing required MyD88 and TIRAP, and IRAK1/4, Akt, and JNK phosphorylation and activation. Traf6 couples MyD88 to the Akt pathway and, remarkably, a Traf6 interacting peptide-antennapedia chimera was more effective than LPS in stimulating IL-1ß splicing. The Traf6 chimera did not, however, stimulate microparticle shedding, nor was IL-1ß released. We conclude LPS-induced kinase cascades are sufficient to alter cellular responses, that three signals emanate from platelet TLR4, and that Akt and JNK activation are sufficient to initiate posttranscriptional splicing while another event couples microparticle shedding to TLR4 activation. Platelets contribute to the inflammatory response to LPS through production of microparticles that promote endothelial cell activation.