Search

Objective: Adiposity is associated with oxidative stress, inflammation, and glucose intolerance. Previous data suggest that platelet gene expression is associated with key cardiometabolic phenotypes, including body mass index but stable in healthy individuals over time. However, modulation of gene expression in platelets in response to metabolic shifts (eg, weight reduction) is unknown and may be important to defining mechanism. Approach and Results: Platelet RNA sequencing and aggregation were performed from 21 individuals with massive weight loss (>45 kg) following bariatric surgery. Based on RNA sequencing data, we measured the expression of 67 genes from isolated platelet RNA using high-throughput quantitative reverse transcription quantitative PCR in 1864 FHS (Framingham Heart Study) participants. Many transcripts not previously studied in platelets were differentially expressed with bariatric surgical weight loss, appeared specific to platelets (eg, not differentially expressed in leukocytes), and were enriched for a nonalcoholic fatty liver disease pathway. Platelet aggregation studies did not detect alteration in platelet function after significant weight loss. Linear regression models demonstrated several platelet genes modestly associated with cross-sectional cardiometabolic phenotypes, including body mass index. There were no associations between studied transcripts and incident diabetes or cardiovascular end points. Conclusions: In summary, while there is no change in platelet aggregation function after significant weight loss, the human platelet experiences a dramatic transcriptional shift that implicates pathways potentially relevant to improved cardiometabolic risk postweight loss (eg, nonalcoholic fatty liver disease). Further studies are needed to determine the mechanistic importance of these observations.

Summary The presence of nonhuman RNAs in man has been questioned and it is unclear if food-derived miRNAs cross into the circulation. In a large population study, we found nonhuman miRNAs in plasma by RNA sequencing and validated a small number of pine-pollen miRNAs by RT-qPCR in 2,776 people. The presence of these pine-pollen miRNAs associated with hay fever and not with overt cardiovascular or pulmonary disease. Using in vivo and in vitro models, we found that transmission of pollen-miRNAs into the circulation occurs via pulmonary transfer and this transfer was mediated by platelet-pulmonary vascular cell interactions and platelet pollen-DNA uptake. These data demonstrate that pollen-derived plant miRNAs can be horizontally transferred into the circulation via the pulmonary system in humans. Although these data suggest mechanistic plausibility for pulmonary-mediated plant-derived miRNA transfer into the human circulation, our large observational cohort data do not implicate major disease or risk factor association., Graphical Abstract, Highlights • Pollen-derived miRNAs can be found in the human circulation via pulmonary transfer • This process is partially mediated by platelets' ability to uptake and digest pollen DNA • The presence of circulating pollen miRNAs has modest clinical implications, Biological Sciences; Molecular Biology; Omics; Transcriptomics

Influenza infection increases the incidence of myocardial infarction but the reason is unknown. Platelets mediate vascular occlusion through thrombotic functions but are also recognized to have immunomodulatory activity. To determine if platelet processes are activated during influenza infection, we collected blood from 18 patients with acute influenza infection. Microscopy reveals activated platelets, many containing viral particles and extracellular-DNA associated with platelets. To understand the mechanism, we isolate human platelets and treat them with influenza A virus. Viral-engulfment leads to C3 release from platelets as a function of TLR7 and C3 leads to neutrophil-DNA release and aggregation. TLR7 specificity is confirmed in murine models lacking the receptor, and platelet depletion models support platelet-mediated C3 and neutrophil-DNA release post-influenza infection. These findings demonstrate that the initial intrinsic defense against influenza is mediated by platelet–neutrophil cross-communication that tightly regulates host immune and complement responses but can also lead to thrombotic vascular occlusion., Influenza viremia is rare in human blood and not well studied. Here, the authors show that influenza can be found in human platelets and that platelet engulfment of influenza A results in TLR7-dependent C3 release, which in turn promotes neutrophil-DNA release and formation of platelet-DNA aggregates.

Rationale: Coronavirus disease 2019 (COVID-19) is characterized by increased incidence of microthrombosis with hyperactive platelets sporadically containing viral RNA. It is unclear if SARS-CoV-2 (severe acute respiratory syndrome, corona virus-2) directly alters platelet activation or if these changes are a reaction to infection-mediated global inflammatory alterations. Importantly, the direct effect of SARS-CoV-2 on platelets has yet to be studied. Objective: To characterize the direct SARS-CoV-2–platelet interactions using in vitro studies with purified infectious virions and samples from infected patients. Methods and Results: Platelet RNA analyzed by ARTIC v3 sequencing for SARS-CoV-2 showed presence of fragmented viral genome in all patients with COVID-19. Immunofluorescent imaging of platelets from patients with COVID-19 confirmed presence of SARS-CoV-2 proteins, whereas there was no detection of viral RNA by real-time quantitative polymerase chain reaction. Transmission electron microscopy of platelets incubated with purified SARS-CoV-2 virions demonstrated rapid internalization and digestion leading to distinct morphological changes and resulted in a release of extracellular vesicles. Interactions between SARS-CoV-2 and platelets occurred with or without ACE (angiotensin-converting enzyme) 2 presence as measured by immunofluorescence. Transmission electron microscopy showed that SARS-CoV-2 virions became internalized when they were attached to microparticles, bypassing the need for ACE2. Enrichment analysis of platelet transcriptome from patients with acute COVID-19, compared with those with clinical thrombosis, suggested upregulation of pathways related to virally mediated cell death, specifically necroptosis and apoptosis. Platelets incubated with infectious virus appeared to undergo cell death in 30 minutes postincubation as assessed by transmission electron microscopy and platelets from patients with COVID-19 showed evidence of increased markers of apoptosis and necroptosis by Western blot. Immunofluorescence confirmed colocalization of SARS-CoV-2 with phospho-MLKL (mixed lineage kinase domain-like pseudokinase) and caspase 3 on nonpermeabilized platelets in vitro and in COVID-19 platelets. Conclusions: Platelets internalize SARS-CoV-2 virions, directly or attached to microparticles, and viral internalization leads to rapid digestion, programmed cell death, and extracellular vesicle release. During COVID-19, platelets mediate a rapid response to SARS-CoV-2 and this response can contribute to dysregulated immunity and thrombosis.

Objective— Mechanisms of early and late improvements in cardiovascular risk after bariatric surgery and applicability to larger, at-risk populations remain unclear. We aimed to identify proteins altered after bariatric surgery and their relations to metabolic syndrome and diabetes mellitus. Approach and Results— We identified 19 proteins altered in 32 nonfasting plasma samples from a study of patients undergoing bariatric surgery who were evaluated preoperatively (visit 1) versus both early (visit 2; ≈3 months) and late (visit 3; ≈12 months) postoperative follow-up using predefined protein panels (Olink). Using in silico methods and publicly available gene expression repositories, we found that genes encoding 8 out of 19 proteins had highest expression in liver relative to other assayed tissues, with the top biological and disease processes, including major obesity-related vascular diseases. Of 19 candidate proteins in the surgical cohort, 6 were previously measured in >3000 FHS (Framingham Heart Study) participants (IGFBP [insulin-like growth factor binding protein]-1, IGFBP-2, P-selectin, CD163, LDL (low-density lipoprotein)-receptor, and PAI [plasminogen activator inhibitor]-1). A higher concentration of IGFBP-2 at baseline was associated with a lower risk of incident metabolic syndrome (odds ratio per log-normal unit, 0.45; 95% CI, 0.32–0.64; P =7.7×10 -6 ) and diabetes mellitus (odds ratio, 0.63; 95% CI, 0.49–0.79; P =0.0001) after multivariable adjustment. Conclusions— Using a directed protein quantification platform (Olink), we identified known and novel proteins altered after surgical weight loss, including IGFBP-2. Future efforts in well-defined obesity intervention settings may further define and validate novel targets for the prevention of vascular disease in obesity.

Background Given high on-treatment mortality in heart failure (HF), identifying molecular pathways that underlie adverse cardiac remodeling may offer novel biomarkers and therapeutic avenues. Circulating extracellular RNAs (ex-RNAs) regulate important biological processes and are emerging as biomarkers of disease, but less is known about their role in the acute setting, particularly in the setting of HF. Methods We examined the ex-RNA profiles of 296 acute coronary syndrome (ACS) survivors enrolled in the Transitions, Risks, and Actions in Coronary Events Center for Outcomes Research and Education Cohort. We measured 374 ex-RNAs selected a priori, based on previous findings from a large population study. We employed a two-step, mechanism-driven approach to identify ex-RNAs associated with echocardiographic phenotypes (left ventricular [LV] ejection fraction, LV mass, LV end-diastolic volume, left atrial [LA] dimension, and LA volume index) then tested relations of these ex-RNAs with prevalent HF (N=31, 10.5%). We performed further bioinformatics analysis of microRNA (miRNAs) predicted targets' genes ontology categories and molecular pathways. Results We identified 44 ex-RNAs associated with at least one echocardiographic phenotype associated with HF. Of these 44 exRNAs, miR-29-3p, miR-584-5p, and miR-1247-5p were also associated with prevalent HF. The three microRNAs were implicated in the regulation p53 and transforming growth factor-β signaling pathways and predicted to be involved in cardiac fibrosis and cell death; miRNA predicted targets were enriched in gene ontology categories including several involving the extracellular matrix and cellular differentiation. Conclusions Among ACS survivors, we observed that miR-29-3p, miR-584-5p, and miR-1247-5p were associated with both echocardiographic markers of cardiac remodeling and prevalent HF. Relevance for patients miR-29c-3p, miR-584-5p, and miR-1247-5p were associated with echocardiographic phenotypes and prevalent HF and are potential biomarkers for adverse cardiac remodeling in HF.

Transcripts in platelets are largely produced in precursor megakaryocytes but remain physiologically active as platelets translate RNAs and regulate protein/RNA levels. Recent studies using transcriptome sequencing (RNA-seq) characterized the platelet transcriptome in limited number of non-diseased individuals. Here, we expand upon these RNA-seq studies by completing RNA-seq in platelets from 32 patients with acute myocardial infarction (MI). Our goals were to characterize the platelet transcriptome using a population of patients with acute MI and relate gene expression to platelet aggregation measures and ST-segment elevation MI (STEMI) (n = 16) vs. non-STEMI (NSTEMI) (n = 16) subtypes. Similar to other studies, we detected 9565 expressed transcripts, including several known platelet-enriched markers (e.g. PPBP, OST4). Our RNA-seq data strongly correlated with independently ascertained platelet expression data and showed enrichment for platelet-related pathways (e.g. wound response, hemostasis, and platelet activation), as well as actin-related and post-transcriptional processes. Several transcripts displayed suggestively higher (FBXL4, ECHDC3, KCNE1, TAOK2, AURKB, ERG, and FKBP5) and lower (MIAT, PVRL3, and PZP) expression in STEMI platelets compared to NSTEMI. We also identified transcripts correlated with platelet aggregation to TRAP (ATP6V1G2, SLC2A3), collagen (CEACAM1, ITGA2), and ADP (PDGFB, PDGFC, ST3GAL6). Our study adds to current platelet gene expression resources by providing transcriptome-wide analyses in platelets isolated from patients with acute MI. In concert with prior studies, we identify various genes for further study in regards to platelet function and acute MI. Future platelet RNA-seq studies examining more diverse sets of healthy and diseased samples will add to our understanding of platelet thrombotic and non-thrombotic functions.

Cells of the innate immune system use Toll-like receptors (TLRs) to initiate the proinflammatory response to microbial infection. Recent studies have shown acute infections are associated with a transient increase in the risk of vascular thrombotic events. Although platelets play a central role in acute thrombosis and accumulating evidence demonstrates their role in inflammation and innate immunity, investigations into the expression and functionality of platelet TLRs have been limited. In the present study, we demonstrate that human platelets express TLR2, TLR1, and TLR6. Incubation of isolated platelets with Pam3CSK4, a synthetic TLR2/TLR1 agonist, directly induced platelet aggregation and adhesion to collagen. These functional responses were inhibited in TLR2-deficient mice and, in human platelets, by pretreatment with TLR2-blocking antibody. Stimulation of platelet TLR2 also increased P-selectin surface expression, activation of integrin αIIbβ3, generation of reactive oxygen species, and, in human whole blood, formation of platelet–neutrophil heterotypic aggregates. TLR2 stimulation also activated the phosphoinositide 3-kinase (PI3-K)/Akt signaling pathway in platelets, and inhibition of PI3-K significantly reduced Pam3CSK4-induced platelet responses. In vivo challenge with livePorphyromonas gingivalis, a Gram-negative pathogenic bacterium that uses TLR2 for innate immune signaling, also induced significant formation of platelet–neutrophil aggregates in wild-type but not TLR2-deficient mice. Together, these data provide the first demonstration that human platelets express functional TLR2 capable of recognizing bacterial components and activating the platelet thrombotic and/or inflammatory pathways. This work substantiates the role of platelets in the immune and inflammatory response and suggests a mechanism by which bacteria could directly activate platelets.

SummaryIn addition to haemostasis, platelets mediate inflammation and clearance of bacteria from the bloodstream. As with platelet-platelet interactions, platelet-bacteria interactions involve cytoskeletal rearrangements and release of granular content. Stimulation of the immune Toll-like receptor 2 (TLR2) on the platelet surface, activates phosphoinositide-3-kinase (PI3K) and causes platelet activation and plateletdependent thrombosis. It remains unknown if platelet activation by immune versus thrombotic pathways leads to the differential regulation of signal transduction, protein-protein interactions, and α-granule release, and the physiological relevance of these potential differences. We investigated these processes after immune versus thrombotic platelet stimulation. We examined selected signalling pathways and found that phosphorylation kinetics of Akt, ERK1/2 and p38 differed dramatically between agonists. Next, we investigated platelet protein-protein interactions by mass spectrometry (MS)-based proteomics specifically targeting cytosolic factor XIIIa (FXIIIa) because of its function as a cytoskeleton-crosslinking protein whose binding partners have limited characterisation. Four FXIIIa-binding proteins were identified, two of which are novel interactions: FXIIIa-focal adhesion kinase (FAK) and FXIIIa-gelsolin. The binding of FAK to FXIIIa was found to be altered differentially by immune versus thrombotic stimulation. Lastly, we studied the effect of thrombin versus Pam3CSK4 stimulation on α-granule release and observed differential release patterns for selected granule proteins and decreased fibrin clot formation compared with thrombin. The inhibition of PI3K caused a decrease in protein release after Pam3CSK4 -but not after thrombin-stimulation. In summary, stimulation of platelets by either thrombotic or immune receptors leads to markedly different signalling responses and granular protein release consistent with differential contribution to coagulation and thrombosis.

SummaryMultiple studies have shown the effects of long-term exposure to high-fat or western diets on the vascular system. There is limited knowledge on the acute effects of high circulating fat levels, specifically on platelets, which have a role in many processes, including thrombosis and inflammation. This study investigated the effects of acute, high-fat exposure on platelet function and transcript profile. Twenty healthy participants were given an intravenous infusion of 20% Intralipid emulsion and heparin over 6 hours. Blood samples were taken prior to and the day after infusion to measure platelet function and transcript expression levels. Platelet aggregation was not significantly affected by Intralipid infusion, but, when mitochondria function was inhibited by carbonyl cyanide 3-chlorophenylhydrazone (CCCP) or oligomycin, platelet aggregation was higher in the post-infusion state compared to baseline. Through RNA sequencing, and verified by RT-qPCR, 902 miRNAs and 617 mRNAs were affected by Intralipid infusion. MicroRNAs increased include miR-4259 and miR-346, while miR-517b and miR-517c are both decreased. Pathway analysis identified two clusters significantly enriched, including cell motility. In conclusion, acute exposure to high fat affects mitochondrial-dependent platelet function, as well as the transcript profile.

Objective— Soluble CD40 ligand (sCD40L) has been recently implicated in the pathogenesis of atherosclerosis. Elevated levels of sCD40L in acute coronary syndrome patients suggests enhanced platelet function; however, the exact mechanism by which this occurs is unknown. In this study, we examined the effect of sCD40L on platelet function and reactive oxygen and nitrogen species (RONS) generation. Methods and Results— Platelet stimulation in the presence of recombinant sCD40L (rsCD40L) led to enhanced generation of RONS as measured by DCFHDA oxidation and confocal microscopy. Incubation with rsCD40L led to enhanced platelet P-selectin expression, aggregation, and platelet-leukocyte conjugation. Platelets isolated from CD40L-deficient mice had decreased agonist-induced NO release as compared with wild-type mice. Incubation of platelets with rsCD40L enhanced stimulation-induced p38 MAP kinase and Akt phosphorylation. Conclusion— Soluble CD40L enhances platelet activation, aggregation, and platelet-leukocyte conjugation, as well as increases stimulation-induced platelet release of RONS through activation of Akt and p38 MAP kinase signaling pathways. These data suggest that sCD40L regulates platelet-dependent inflammatory and thrombotic responses that contribute to the pathogenesis of atherothrombosis.

Red wine and purple grape juice contain polymeric flavonoids with antioxidant properties believed to be protective against cardiovascular events but the alcohol and sugar content of these beverages has curtailed their medicinal use. Acute cardiac events are also associated with enhanced inflammation and thrombosis. In this study, the extracts from grape skins or seeds were examined for their anti-inflammatory properties and effect on platelet release of reactive oxygen intermediates. Incubation of platelets with seed or skin extract led to a decrease in platelet aggregation from 68.8+/-19.8% to 45+/-3.6% for seeds and to 27+/-7.2% for skin, respectively (P

Aminoacyl-tRNA synthetases are essential enzymes that catalyze attachment of amino acids to tRNAs for decoding of genetic information. In higher eukaryotes, several synthetases associate with non-synthetase proteins to form a high-molecular mass complex that may improve the efficiency of protein synthesis. This multi-synthetase complex is not found in bacteria. Here we describe the isolation of a non-synthetase protein from the archaeon Methanocaldococcus jannaschii that was copurified with prolyl-tRNA synthetase (ProRS). This protein, Mj1338, also interacts with several other tRNA synthetases and has an affinity for general tRNA, suggesting the possibility of forming a multi-synthetase complex. However, unlike the non-synthetase proteins in the eukaryotic complex, the protein Mj1338 is predicted to be a metabolic protein, related to members of the family of H(2)-forming N(5),N(10)-methylene tetrahydromethanopterin (5,10-CH(2)-H(4)MP) dehydrogenases that are involved in the one-carbon metabolism of the archaeon. The association of Mj1338 with ProRS, and with other components of the protein synthesis machinery, thus suggests the possibility of a closer link between metabolism and decoding in archaea than in eukarya or bacteria.

Viral infections have been associated with reduced platelet counts, the biological significance of which has remained elusive. Here, we show that infection with encephalomyocarditis virus (EMCV) rapidly reduces platelet count, and this response is attributed to platelet Toll-like receptor 7 (TLR7). Platelet-TLR7 stimulation mediates formation of large platelet-neutrophil aggregates, both in mouse and human blood. Intriguingly, this process results in internalization of platelet CD41-fragments by neutrophils, as assessed biochemically and visualized by microscopy, with no influence on platelet prothrombotic properties. The mechanism includes TLR7-mediated platelet granule release, translocation of P-selectin to the cell surface, and a consequent increase in platelet-neutrophil adhesion. Viral infection of platelet-depleted mice also led to increased mortality. Transfusion of wild-type, TLR7-expressing platelets into TLR7-deficient mice caused a drop in platelet count and increased survival post EMCV infection. Thus, this study identifies a new link between platelets and their response to single-stranded RNA viruses that involves activation of TLR7. Finally, platelet-TLR7 stimulation is independent of thrombosis and has implications to the host immune response and survival.

Valyl-tRNA synthetase (ValRS) has difficulty discriminating between its cognate amino acid, valine, and structurally similar amino acids. To minimize translational errors, the enzyme catalyzes a tRNA-dependent editing reaction that prevents accumulation of misacylated tRNA(Val). Editing occurs with threonine, alanine, serine, and cysteine, as well as with several nonprotein amino acids. The 3'-end of tRNA plays a vital role in promoting the tRNA-dependent editing reaction. Valine tRNA having the universally conserved 3'-terminal adenosine replaced by any other nucleoside does not stimulate the editing activity of ValRS. As a result 3'-end tRNA(Val) mutants, particularly those with 3'-terminal pyrimidines, are stably misacylated with threonine, alanine, serine, and cysteine. Valyl-tRNA synthetase is unable to hydrolytically deacylate misacylated tRNA(Val) terminating in 3'-pyrimidines but does deacylate mischarged tRNA(Val) terminating in adenosine or guanosine. Evidently, a purine at position 76 of tRNA(Val) is essential for translational editing by ValRS. We also observe misacylation of wild-type and 3'-end mutants of tRNA(Val) with isoleucine. Valyl-tRNA synthetase does not edit wild-type tRNA(Val)(A76) mischarged with isoleucine, presumably because isoleucine is only poorly accommodated at the editing site of the enzyme. Misacylated mutant tRNAs as well as 3'-end-truncated tRNA(Val) are mixed noncompetitive inhibitors of the aminoacylation reaction, suggesting that ValRS, a monomeric enzyme, may bind more than one tRNA(Val) molecule. Gel-mobility-shift experiments to characterize the interaction of tRNA(Val) with the enzyme provide evidence for two tRNA binding sites on ValRS.

The role of platelets in hemostasis and thrombosis is clearly established; however, the mechanisms by which platelets mediate inflammatory and immune pathways are less well understood. Platelets interact and modulate the function of blood and vascular cells by releasing bioactive molecules. Although the platelet is anucleate, it contains transcripts that may mirror disease. Platelet mRNA is only associated with low-level protein translation; however, platelets have a unique membrane structure allowing for the passage of small molecules, leading to the possibility that its cytoplasmic RNA may be passed to nucleated cells. To examine this question, platelet-like particles with labeled RNA were cocultured with vascular cells. Coculture of platelet-like particles with activated THP-1, monocytic, and endothelial cells led to visual and functional RNA transfer. Posttransfer microarray gene expression analysis of THP-1 cells showed an increase in HBG1/HBG2 and HBA1/HBA2 expression that was directly related to the transfer. Infusion of wild-type platelets into a TLR2-deficient mouse model established in vivo confirmation of select platelet RNA transfer to leukocytes. By specifically transferring green fluorescent protein, we also observed external RNA was functional in the recipient cells. The observation that platelets possess the capacity to transfer cytosolic RNA suggests a new function for platelets in the regulation of vascular homeostasis.

Introduction: Cells require energy provided by glycolytic and mitochondrial sources. Obesity, a health issue worldwide, affects mitochondrial function. Platelets possess mitochondria although their role in platelet function is unclear. We assessed the hypothesis that platelet mitochondria contribute to cellular processes and the mitochondrial disorder in obesity may also affect platelet functions. Methods: Platelet aggregation and adhesion were assessed to estimate the effects of selective metabolic inhibitors on platelet functions. Mitochondrial ultrastructure was analyzed by transmission electron microscopy in the presence of these inhibitors. C57BL/6J mice were used to investigate the mitochondrial contribution to the effects of high fat diet (HFD) on platelet adhesion. mRNA expression in mouse platelets was measured by RT-PCR. Results: The disruption of the mitochondrial function by complementary inhibitors resulted in a reduction of platelet aggregation to 80.9 ± 7.7% (antimycin A), 77.5 ± 9.5% (oligomycin), 59.1 ± 3.2% (carbonyl cyanide 3-chlorophenylhydrazone, CCCP), and 37.2 ± 4.3% (potassium cyanide); n=3, p p

Cerebrovascular disease is a major cause of death and disability, with a poorer outcome in patients having select risk factors including diabetes and hypertension. Risk factors and the state of cerebral ischemia-reperfusion associated with cerebrovascular occlusion are known to cause inflammatory changes. These events and the inflammatory state are reflected by transcript changes in various components of the blood and can be specifically measured. By defining these changes, new insight into cerebrovascular disease and its therapeutics is being achieved.

Objective: Inflammatory activity in fat tissue has recently been implicated in mechanisms of insulin resistance and obesity-related metabolic dysfunction. Toll-like receptors (TLRs) play a key role in innate immune responses and recent studies implicate the TLR pathway in mechanisms of inflammation and atherosclerosis. The aim of this study was to examine differential TLR expression and function in human adipose tissue. Methods and Procedures: We biopsied subcutaneous abdominal fat from 16 obese subjects (age 39 ± 11 years, BMI 49 ± 14 kg/m2) and characterized TLR expression using quantitative real-time PCR and confocal immunofluorescence imaging. In tissue culture, we stimulated isolated human adipocytes with Pam3CSK4 and lipopolysaccharide (LPS) (TLR2 and TLR4 agonists, respectively) and quantified TLR activity, interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) production, and nuclear factor-κB (NF-κB) p65 nuclear activation using real-time PCR, enzyme-linked immunosorbent assay (ELISA), and immunofluorescence. Results: TLR1, 2, and 4 protein colocalized with adiponectin in human adipocytes with TLR4 exhibiting the highest immunohistochemical expression. Using real-time PCR, we confirmed higher level of gene expression for TLR4 as compared to other members of the TLR family (TLR1, 2, 7, 8) in human adipose depots (P < 0.001). In tissue culture, adipocyte TLR2/TLR4 mRNA expression and protein increased significantly following Pam3CSK4 and LPS (P < 0.001). TLR2/TLR4 stimulation was associated with NF-κB p65 nuclear translocation and proinflammatory cytokine production. Discussion: The findings demonstrate that TLRs are inducible in adipose tissue and linked with downstream NF-κB activation and cytokine release. Adipose stores may play a dynamic role in the regulation of inflammation and innate immunity in human subjects via modulation of the TLR/NF-κB regulatory pathway.

The angiotensin-converting enzyme (ACE) is a membrane-bound peptidyl dipeptidase known to act on a variety of peptide substrates in the extracellular space. Its most notable functions are the formation of angiotensin II and the degradation of bradykinin. In the current experiments, we found that exogenous ACE added to vascular smooth muscle cell culture strongly induces and upregulates the genes of bradykinin receptors B1and B2. This transcriptional regulatory property of ACE was shown to be unrelated to its known enzymatic properties. Indeed, ACE at 3.75 μg/ml added in the culture medium of vascular smooth muscle cells was found to cause marked upregulation of the mRNA expression of the genes for the B1and B2receptors of bradykinin by 22- and 11-fold, respectively. This phenomenon was not altered by the addition of specific angiotensin II antagonists for the AT1or AT2receptors. Moreover, the ACE inhibitor captopril, which inhibited ACE enzymatic activity, did not block its effect at the bradykinin receptor gene transcription level. Expression of both receptor genes was completely abolished by actinomycin D. Furthermore, transcriptional upregulation was inhibited by curcumin, suggesting involvement of different transcriptional factors in this phenomenon. Electrophoretic mobility shift assay revealed increase in NF-κB and activator protein-1 protein binding for consensus sequences, between ACE-treated cells versus untreated cells. The data indicate a novel biological function of the ACE unrelated to its well-known enzymatic function as a peptidyl dipeptidase.

Platelet and vascular stimulation leads to release of reactive oxygen species (ROS) that are known to influence vascular reactivity and thrombosis. Dipyridamole is a vasodilator and platelet inhibitor that has previously been shown to have direct antioxidant properties. The antioxidant effects of dipyridamole on vascular cell-derived ROS are not known; therefore, dipyridamole was incubated with endothelial cells and platelets and cellular redox status and release of endogenous ROS were assessed. Dipyridamole decreased intracellular basal ROS generation from endothelial cells as measured by DCFDA (2',7'-dichlorodihydrofluorescein diacetate) oxidation. Incubation of endothelial cells with dipyridamole also attenuated t-butylhydroperoxide-induced oxidative stress. Using a redox-sensitive fluorescent dye, dipyridamole improved cellular activity after treatment with t-butylhydroperoxide. Incubation with dipyridamole did not alter platelet release of nitric oxide or hydrogen peroxide but significantly attenuated superoxide release. Using flow cytometry and confocal microscopy, dipyridamole decreased platelet ROS generation. Dipyridamole also suppressed platelet-soluble CD40 ligand release. In summary, at therapeutically relevant concentrations, dipyridamole suppresses the formation of ROS in platelets and endothelial cells and improves cellular redox status. These data suggest that the redox-dependent properties of dipyridamole have a direct effect on vascular cells.

The application of soluble CD40 ligand (sCD40L) as a biomarker has garnered great scientific and clinical interest. However, there are many uncertainties with regard to the biology of sCD40L. Although presumed to be a marker of platelet activation, relative levels in plasma, serum, and platelet expression are unknown, as is the optimal method for its measurement. We measured CD40L from serum, platelet-poor plasma, and platelet surface in adults who had stable cardiovascular disease (CVD) and those who had unstable CVD (n = 40). Plasma sCD40L did not differ significantly between groups. Serum sCD40L was significantly lower (1.4 +/- 1.3 vs 5.2 +/- 3.7 ng/ml, p0.001) and platelet membrane CD40L expression was higher (1.4 +/- 0.7% vs 0.9 +/- 0.6%, p = 0.03) in unstable compared with stable CVD. When the 2 groups were considered together, there was a significant correlation between plasma and serum sCD40L levels (rho = 0.4, p = 0.02) and negative correlations between plasma (rho = -0.3, p = 0.04) and serum (rho = -0.4, p = 0.01) sCD40L levels with platelet membrane CD40L expression. In unstable CVD, the correlation between sCD40L measurements was poor. Consistent with enhanced platelet activation, there was a positive correlation between platelet aggregation and surface CD40L expression (rho = 0.5, p = 0.02) and between platelet expression of CD40L and P-selectin (rho = 0.4, p = 0.05) in unstable CVD. There was no correlation between CD40L and platelet count or C-reactive protein. Only surface expression of CD40L compared with platelet-derived (plasma) or total (serum) CD40L level proved a reliable marker of platelet function in patients who had stable CVD and those who had unstable CVD. In conclusion, our data demonstrate the complex nature of CD40L and highlight the distinct processes of expression, shedding, and clearance of this ligand in patient populations.

The balance between thrombosis and hemorrhage is carefully regulated. Nitric oxide (NO) is an important mediator of these processes, as it prevents platelet adhesion to the endothelium and inhibits platelet recruitment. Although endothelial NO synthase (eNOS)-deficient mice have decreased vascular reactivity and mild hypertension, enhanced thrombosis in vivo has not been demonstrated. To determine the role of endogenous NO in hemostasis, a model of carotid arterial injury and thrombosis was performed using eNOS-deficient and wild-type mice. Paradoxically, the eNOS-deficient animals had a prolongation of time to occlusion compared with the wild-type mice ( P < 0.001). Consistent with this finding, plasma markers suggesting enhanced fibrinolysis [tissue plasminogen activator (t-PA) activity and antigen and D-dimer levels] were significantly elevated in eNOS-deficient animals. Vascular tissue expression of t-PA and platelet activity levels were not altered. In endothelial cells, t-PA is stored in Weibel-Palade bodies, and exocytosis of these storage granules is inhibited by NO. Thus in the absence of NO, release of Weibel-Palade body contents (and t-PA) could be enhanced; this observation is also supported by increased von Willebrand factor levels observed in eNOS-deficient animals. In summary, although eNOS deficiency attenuates vascular reactivity and increases platelet recruitment, it is also associated with enhanced fibrinolysis due to lack of NO-dependent inhibition of Weibel-Palade body release. These processes highlight the complexity of NO-dependent regulation of vascular homeostasis. Such compensatory mechanisms may partially explain the lack of spontaneous thrombosis, minimally elevated baseline blood pressure, and normal life span that are seen in animals deficient in a pivotal regulator of vascular patency.

To the Editor: Moderate consumption of red wine is associated with a reduction in cardiovascular disease,1–4 and some of the cardioprotective effects have been attributed to the distinct polyphenolic compounds present in red wine and purple grape products. These polyphenols possess multiple biological effects, including antioxidant and free radical–scavenging properties5,6 as well as inhibition of platelet activation.7 The growing interest in the presumed benefits of wine in protecting against coronary heart disease coupled with the inherent limitations in promoting alcohol consumption8,9 has led to the examination of the potential beneficial vascular properties of alternative purple grape products. The effect of these flavonoid-rich substances in subjects with cardiovascular disease taking aspirin is unknown. In addition, their impact on the inflammatory properties of platelets has not been studied. A double blind crossover study was conducted, in which 20 subjects with previously diagnosed coronary disease on standard medications were randomly assigned to drink either placebo or purple grape juice …

Tamoxifen is effective in the prevention and treatment of breast cancer, but its use is associated with an increased risk of thrombosis. The mechanism for this effect is unknown. Reactive oxygen intermediates enhance platelet-dependent thrombosis, and in oncological studies, tamoxifen has been shown to increase production of reactive oxygen species. Therefore, the effects of tamoxifen and its bioactive metabolites on platelet activity and platelet reactive oxygen species were determined. Platelets were incubated with tamoxifen or the metabolites 4-hydroxy-tamoxifen (4-OH), N-desmethyl tamoxifen, or 4-hydroxy-N-desmethyl tamoxifen (endoxifen). Tamoxifen metabolites have been previously shown to possess enhanced bioactivity, and consistent with this observation, tamoxifen metabolites but not tamoxifen modestly increased platelet aggregation. These effects were similar with platelets isolated from male or female subjects. Platelet nitric oxide release or cGMP levels were not altered by incubation with tamoxifen or any of its metabolites. Incubation with tamoxifen metabolites increased stimulation-dependent platelet superoxide release [8.1 +/- 1.6 arbitrary units (a.u.) for control versus 15.2 +/- 3.5 a.u. for 4-OH; P < 0.01]. Coincubation with a superoxide dismutase mimetic eliminated the tamoxifen metabolite-induced enhancement of platelet aggregation. Corresponding to increased superoxide release, incubation with tamoxifen metabolites enhanced the functional activation of NADPH oxidase as determined by phosphorylation of its subunits p47(phox) and p67(phox). In summary, incubation of platelets with the active metabolites of tamoxifen increases stimulation-dependent superoxide release through a NADPH oxidase-dependent mechanism. This results in modest changes in platelet function and seems to be consistent with previous oncological studies demonstrating tamoxifen-dependent increase in reactive oxygen species generation.

Cysteinyl-tRNA synthetase catalyzes the addition of cysteine to its cognate tRNA. The available eukaryotic sequences for this enzyme contain several insertions that are absent from bacterial sequences. To gain insights into the differences between the bacterial and eukaryotic forms, we previously studied the E. coli cysteinyl-tRNA synthetase. In this study, we sought to clone and express the full-length gene for the human cytoplasmic cysteinyl-tRNA synthetase. Although a gene encoding the human enzyme has been described, the predicted protein sequence, consisting of 638 amino acids, lacks homology with other eukaryotic enzymes in the carboxyl-terminus. This suggested that a further investigation was necessary to obtain the definitive sequence for the human enzyme. Here we report the isolation of a full-length cDNA that encodes a protein of 748 amino acids. The predicted protein sequence shows considerable similarity to other eukaryotic cysteinyl-tRNA synthetases in the carboxyl-terminus. We also found that approximately 20% of the mRNA encoding the cytoplasmic cysteinyl-tRNA synthetase contained an insertion of 8 bases in the 3' coding region of the mRNA. This insertion arises from an alternative splicing between the last two exons of the gene. The alternative splicing alters the reading frame and results in the replacement of the carboxy-terminal 44 amino acids with a novel sequence of 22 amino acids. Expression of the full-length and alternative forms of the enzyme in E. coli generated functional proteins that were active in aminoacylation of human cytoplasmic tRNA(Cys) with cysteine.

Background Platelet stimulation leads to release of reactive oxygen species (ROS) that are known to influence platelet function and thrombosis. Dipyridamole is a vasodilator and platelet inhibitor that decreases adenosine uptake and may be a highly efficient chain breaking antioxidant. The antioxidant effects of dipyridamole on platelet-derived ROS and the potential (anti)inflammatory effects are not known. Methods Human platelets were incubated with dipyridamole (0–100 μM) and specific antioxidant and antiflammatory mediators were measured. Results Incubation with dipyridamole did not alter platelet release of nitric oxide but significantly attenuated superoxide release (17±3.7 a.u. for control vs. 7.4±1.1 a.u. for 20μM; n=5, P

OR-15 Key Words: Vascular Smooth Muscle Cells, Cellular Uptake, Transcriptional Regulation