RESUMO
Exercise improves cardiometabolic and vascular function, although the mechanisms remain unclear. Our objective was to demonstrate the diversity of circulating extracellular RNA (ex-RNA) release during acute exercise in humans and its relevance to exercise-mediated benefits on vascular inflammation. We performed plasma small RNA sequencing in 26 individuals undergoing symptom-limited maximal treadmill exercise, with replication of our top candidate miRNA in a separate cohort of 59 individuals undergoing bicycle ergometry. We found changes in miRNAs and other ex-RNAs with exercise (e.g., Y RNAs and tRNAs) implicated in cardiovascular disease. In two independent cohorts of acute maximal exercise, we identified miR-181b-5p as a key ex-RNA increased in plasma after exercise, with validation in a separate cohort. In a mouse model of acute exercise, we found significant increases in miR-181b-5p expression in skeletal muscle after acute exercise in young (but not older) mice. Previous work revealed a strong role for miR-181b-5p in vascular inflammation in obesity, insulin resistance, sepsis, and cardiovascular disease. We conclude that circulating ex-RNAs were altered in plasma after acute exercise target pathways involved in inflammation, including miR-181b-5p. Further investigation into the role of known (e.g., miRNA) and novel (e.g., Y RNAs) RNAs is warranted to uncover new mechanisms of vascular inflammation on exercise-mediated benefits on health.NEW & NOTEWORTHY How exercise provides benefits to cardiometabolic health remains unclear. We performed RNA sequencing in plasma during exercise to identify the landscape of small noncoding circulating transcriptional changes. Our results suggest a link between inflammation and exercise, providing rich data on circulating noncoding RNAs for future studies by the scientific community.
Assuntos
MicroRNA Circulante/sangue , Exercício Físico , Inflamação/sangue , Síndrome Metabólica/sangue , RNA de Transferência/sangue , Adulto , Idoso , Animais , Ciclismo , MicroRNA Circulante/genética , Teste de Esforço/métodos , Feminino , Marcadores Genéticos , Nível de Saúde , Humanos , Inflamação/diagnóstico , Inflamação/genética , Masculino , Síndrome Metabólica/diagnóstico , Síndrome Metabólica/genética , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/sangue , MicroRNAs/genética , MicroRNAs/metabolismo , Pessoa de Meia-Idade , Músculo Esquelético/metabolismo , RNA de Transferência/genética , Fatores de TempoRESUMO
AIMS: Rho-associated coiled-coil containing kinase (ROCK)-2 is an important mediator of the actin cytoskeleton. Because changes in the actin cytoskeleton are critical for platelet function, we hypothesized that ROCK2 in platelets will play important role in thrombosis and can be potentially a target for therapeutic intervention in thromboembolic stroke. METHODS AND RESULTS: We generated platelet-specific ROCK2-deficient mice (ROCK2Plt-/-) from conditional ROCK2fl°x/fl°x and platelet factor (PF)-4-Cre transgenic mice. Platelets from ROCK2Plt-/- mice were less responsive to thrombin stimulation in terms of pseudopodia formation, collagen adhesion, and in the formation of homotypic and heterotypic aggregates. This corresponded to prolonged bleeding time and delayed vascular occlusion following vessel injury. To determine whether these changes in platelet function could affect thrombotic disease, we utilized a clot-embolic model of ischaemic stroke. When pre-formed clots from ROCK2Plt-/- mice were injected into the middle cerebral artery of control mice, cerebral blood flow recovery occurred more rapidly, leading to decreased cerebral injury and neurological deficits, compared to pre-formed clots from control mice. Interestingly, pre-formed clots from control mice produced similar degree of cerebral injury when injected into control or ROCK2Plt-/- mice, suggesting that platelet ROCK2 deficiency affects clot formation but not propagation. Indeed, in a non-thrombotic intra-filament MCA occlusion model of stroke, platelet ROCK2 deletion was not protective. Furthermore, ROCK2Plt-/- mice exhibit similar atherosclerosis severity and vascular remodeling as control mice. CONCLUSION: These findings indicate that platelet ROCK2 plays important role in platelet function and thrombosis, but does not contribute to the pathogenesis of atherosclerosis and vascular remodeling.
Assuntos
Acidente Vascular Cerebral/metabolismo , Remodelação Vascular/genética , Quinases Associadas a rho/deficiência , Animais , Aterosclerose/genética , Aterosclerose/metabolismo , Plaquetas/metabolismo , Modelos Animais de Doenças , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Ativação Plaquetária/genética , Agregação Plaquetária , Acidente Vascular Cerebral/genética , Trombina/metabolismo , Trombose/genética , Trombose/patologiaRESUMO
The role of platelets in regulating vascular homeostasis has expanded beyond mediation of haemostasis and thrombosis. The discovery of platelet RNA and the presence of subpopulations of platelets containing varying amounts of RNA suggest a role for platelet transcripts in vascular function. As the RNA in anucleated platelets is biologically functional and may transfer to other vascular cells, we hypothesised that platelet RNA diminishes over the lifespan of the platelet with diminishing platelet size due to horizontal cellular transfer. The purpose of this study is to determine if platelet RNA variance is the result of horizontal cellular transfer between platelets and other vascular cells. Utilising platelet sorting and RNA sequencing, we found that smaller platelets contained a more diverse set of transcripts than larger platelets. Further investigation using fluorescence imaging, gene expression analyses and in vitro and in vivo modelling revealed that platelets take up RNA from other vascular cells in a complex manner, revealing a dynamic role for platelets in modulating vascular homeostasis through bidirectional RNA transfer. The resultant RNA profile heterogeneity suggests unique functional roles for platelets dependent on size and complexity. This study expands our basic understanding of platelet function and heterogeneity and is the first to evaluate endogenous vascular RNA uptake and its relation to platelet processes. Our findings describe a novel endogenous phenomenon that can help elucidate the platelet's role in these non-thrombotic and haemostatic fields, as well as present potential for diagnostic and therapeutic development.
Assuntos
Plaquetas/metabolismo , Transporte de RNA , RNA Mensageiro/sangue , Animais , Apoptose , Plaquetas/patologia , Tamanho Celular , Regulação da Expressão Gênica , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Leucócitos Mononucleares/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Estabilidade de RNA , RNA Mensageiro/genética , Receptor 2 Toll-Like/sangue , Receptor 2 Toll-Like/genética , Transcrição GênicaRESUMO
Platelets, responsible for clot formation and blood vessel repair, are produced by megakaryocytes in the bone marrow. Platelets are critical for hemostasis and wound healing, and are often provided following surgery, chemotherapy, and major trauma. Despite their importance, platelets today are derived exclusively from human volunteer donors. They have a shelf life of just five days, making platelet shortages common during long weekends, civic holidays, bad weather, and during major emergencies when platelets are needed most. Megakaryocytes in the bone marrow generate platelets by extruding long cytoplasmic extensions called proplatelets through gaps/fenestrations in blood vessels. Proplatelets serve as assembly lines for platelet production by sequentially releasing platelets and large discoid-shaped platelet intermediates called preplatelets into the circulation. Recent advances in platelet bioreactor development have aimed to mimic the key physiological characteristics of bone marrow, including extracellular matrix composition/stiffness, blood vessel architecture comprising tissue-specific microvascular endothelium, and shear stress. Nevertheless, how complex interactions within three-dimensional (3D) microenvironments regulate thrombopoiesis remains poorly understood, and the technical challenges associated with designing and manufacturing biomimetic microfluidic devices are often under-appreciated and under-reported. We have previously reviewed the major cell culture, platelet quality assessment, and regulatory roadblocks that must be overcome to make human platelet production possible for clinical use [1]. This review builds on our previous manuscript by: (1) detailing the historical evolution of platelet bioreactor design to recapitulate native platelet production ex vivo, and (2) identifying the associated challenges that still need to be addressed to further scale and validate these devices for commercial application. While platelets are among the first cells whose ex vivo production is spearheading major engineering advancements in microfluidic design, the resulting discoveries will undoubtedly extend to the production of other human tissues. This work is critical to identify the physiological characteristics of relevant 3D tissue-specific microenvironments that drive cell differentiation and elaborate upon how these are disrupted in disease. This is a burgeoning field whose future will define not only the ex vivo production of platelets and development of targeted therapies for thrombocytopenia, but the promise of regenerative medicine for the next century.
Assuntos
Reatores Biológicos , Plaquetas , Técnicas de Cultura de Células , Megacariócitos , Animais , Plaquetas/citologia , Plaquetas/metabolismo , Técnicas de Cultura de Células/instrumentação , Técnicas de Cultura de Células/métodos , Humanos , Megacariócitos/citologia , Megacariócitos/metabolismoRESUMO
Multiple 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.
Assuntos
Plaquetas/metabolismo , Fosfolipídeos/administração & dosagem , Óleo de Soja/administração & dosagem , Adulto , Idoso , Dieta Hiperlipídica/efeitos adversos , Emulsões/administração & dosagem , Feminino , Voluntários Saudáveis , Humanos , Infusões Intravenosas , Masculino , MicroRNAs/sangue , MicroRNAs/genética , Pessoa de Meia-Idade , Agregação Plaquetária , RNA Mensageiro/sangue , RNA Mensageiro/genética , RNA Nucleolar Pequeno/sangue , RNA Nucleolar Pequeno/genética , Transcrição Gênica , Adulto JovemRESUMO
INTRODUCTION: Diverse and multi-factorial processes contribute to the progression of cardiovascular disease. These processes affect cells involved in the development of this disease in varying ways, ultimately leading to atherothrombosis. The goal of our study was to compare the differential effects of specific stimuli--two bacterial infections and a Western diet--on platelet responses in ApoE-/- mice, specifically examining inflammatory function and gene expression. Results from murine studies were verified using platelets from participants of the Framingham Heart Study (FHS; n = 1819 participants). METHODS: Blood and spleen samples were collected at weeks 1 and 9 from ApoE-/- mice infected with Porphyromonas gingivalis or Chlamydia pneumoniae and from mice fed a Western diet for 9 weeks. Transcripts based on data from a Western diet in ApoE-/- mice were measured in platelet samples from FHS using high throughput qRT-PCR. RESULTS: At week 1, both bacterial infections increased circulating platelet-neutrophil aggregates. At week 9, these cells individually localized to the spleen, while Western diet resulted in increased platelet-neutrophil aggregates in the spleen only. Microarray analysis of platelet RNA from infected or Western diet-fed mice at week 1 and 9 showed differential profiles. Genes, such as Serpina1a, Ttr, Fgg, Rpl21, and Alb, were uniquely affected by infection and diet. Results were reinforced in platelets obtained from participants of the FHS. CONCLUSION: Using both human studies and animal models, results demonstrate that variable sources of inflammatory stimuli have the ability to influence the platelet phenotype in distinct ways, indicative of the diverse function of platelets in thrombosis, hemostasis, and immunity.
Assuntos
Plaquetas/patologia , Dieta Ocidental/efeitos adversos , Inflamação/patologia , Agregação Plaquetária/fisiologia , Animais , Apolipoproteínas E/metabolismo , Aterosclerose/metabolismo , Aterosclerose/patologia , Plaquetas/metabolismo , Plaquetas/microbiologia , Chlamydophila pneumoniae/patogenicidade , Modelos Animais de Doenças , Humanos , Inflamação/metabolismo , Inflamação/microbiologia , Masculino , Camundongos , Neutrófilos/microbiologia , Neutrófilos/patologia , Neutrófilos/fisiologia , Porphyromonas gingivalis/patogenicidade , Trombose/metabolismo , Trombose/patologiaRESUMO
BACKGROUND: Atherosclerosis is a progressive disease characterized by inflammation and accumulation of lipids in vascular tissue. Porphyromonas gingivalis (Pg) and Chlamydia pneumoniae (Cp) are associated with inflammatory atherosclerosis in humans. Similar to endogenous mediators arising from excessive dietary lipids, these Gram-negative pathogens are pro-atherogenic in animal models, although the specific inflammatory/atherogenic pathways induced by these stimuli are not well defined. In this study, we identified gene expression profiles that characterize P. gingivalis, C. pneumoniae, and Western diet (WD) at acute and chronic time points in aortas of Apolipoprotein E (ApoE-/-) mice. RESULTS: At the chronic time point, we observed that P. gingivalis was associated with a high number of unique differentially expressed genes compared to C. pneumoniae or WD. For the top 500 differentially expressed genes unique to each group, we observed a high percentage (76%) that exhibited decreased expression in P. gingivalis-treated mice in contrast to a high percentage (96%) that exhibited increased expression in WD mice. C. pneumoniae treatment resulted in approximately equal numbers of genes that exhibited increased and decreased expression. Gene Set Enrichment Analysis (GSEA) revealed distinct stimuli-associated phenotypes, including decreased expression of mitochondrion, glucose metabolism, and PPAR pathways in response to P. gingivalis but increased expression of mitochondrion, lipid metabolism, carbohydrate and amino acid metabolism, and PPAR pathways in response to C. pneumoniae; WD was associated with increased expression of immune and inflammatory pathways. DAVID analysis of gene clusters identified by two-way ANOVA at acute and chronic time points revealed a set of core genes that exhibited altered expression during the natural progression of atherosclerosis in ApoE-/- mice; these changes were enhanced in P. gingivalis-treated mice but attenuated in C. pneumoniae-treated mice. Notable differences in the expression of genes associated with unstable plaques were also observed among the three pro-atherogenic stimuli. CONCLUSIONS: Despite the common outcome of P. gingivalis, C. pneumoniae, and WD on the induction of vascular inflammation and atherosclerosis, distinct gene signatures and pathways unique to each pro-atherogenic stimulus were identified. Our results suggest that pathogen exposure results in dysregulated cellular responses that may impact plaque progression and regression pathways.
Assuntos
Aorta/metabolismo , Apolipoproteínas E/deficiência , Chlamydophila pneumoniae/fisiologia , Dieta Ocidental/efeitos adversos , Perfilação da Expressão Gênica , Porphyromonas gingivalis/fisiologia , Animais , Aorta/patologia , Cinética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Família Multigênica/genética , Placa Aterosclerótica/etiologia , Placa Aterosclerótica/genética , Placa Aterosclerótica/microbiologia , Placa Aterosclerótica/patologiaRESUMO
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.
Assuntos
Plaquetas/imunologia , Infecções por Cardiovirus/imunologia , Vírus da Encefalomiocardite/imunologia , Glicoproteínas de Membrana/imunologia , Trombose , Receptor 7 Toll-Like/imunologia , Animais , Plaquetas/metabolismo , Infecções por Cardiovirus/sangue , Degranulação Celular/imunologia , Vírus da Encefalomiocardite/metabolismo , Feminino , Humanos , Receptores de Lipopolissacarídeos/imunologia , Receptores de Lipopolissacarídeos/metabolismo , Masculino , Glicoproteínas de Membrana/sangue , Camundongos , Camundongos Knockout , Neutrófilos/imunologia , Neutrófilos/metabolismo , Contagem de Plaquetas , Vesículas Secretórias/imunologia , Vesículas Secretórias/metabolismo , Receptor 7 Toll-Like/sangueRESUMO
OBJECTIVE: Interleukin 1 Receptor 1 (IL1R1) and its ligand, IL1ß, are upregulated in cardiovascular disease, obesity, and infection. Previously, we reported a higher level of IL1R1 transcripts in platelets from obese individuals of the Framingham Heart Study (FHS), but its functional effect in platelets has never been described. Additionally, IL1ß levels are increased in atherosclerotic plaques and in bacterial infections. The aim of this work is to determine whether IL1ß, through IL1R1, can activate platelets and megakaryocytes to promote atherothrombosis. APPROACH AND RESULTS: We found that IL1ß-related genes from platelets, as measured in 1819 FHS participants, were associated with increased body mass index, and a direct relationship was shown in wild-type mice fed a high-fat diet. Mechanistically, IL1ß activated nuclear factor-κB and mitogen-activated protein kinase signaling pathways in megakaryocytes. IL1ß, through IL1R1, increased ploidy of megakaryocytes to 64+ N by 2-fold over control. IL1ß increased agonist-induced platelet aggregation by 1.2-fold with thrombin and 4.2-fold with collagen. IL1ß increased adhesion to both collagen and fibrinogen, and heterotypic aggregation by 1.9-fold over resting. High fat diet-enhanced platelet adhesion was absent in IL1R1(-/-) mice. Wild-type mice infected with Porphyromonas gingivalis had circulating heterotypic aggregates (1.5-fold more than control at 24 hours and 6.2-fold more at 6 weeks) that were absent in infected IL1R1(-/-) and IL1ß(-/-) mice. CONCLUSIONS: In summary, IL1R1- and IL1ß-related transcripts are elevated in the setting of obesity. IL1R1/IL1ß augment both megakaryocyte and platelet functions, thereby promoting a prothrombotic environment during infection and obesity; potentially contributing to the development of atherothrombotic disease.
Assuntos
Inflamação/patologia , Interleucina-1beta/fisiologia , Megacariócitos/citologia , Obesidade/sangue , Ativação Plaquetária/fisiologia , Receptores Tipo I de Interleucina-1/fisiologia , Transcrição Gênica/fisiologia , Animais , Aterosclerose/etiologia , Infecções por Bacteroidaceae/sangue , Infecções por Bacteroidaceae/patologia , Linhagem Celular , Colágeno/farmacologia , Gorduras na Dieta/toxicidade , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Humanos , Imidazóis/farmacologia , Inflamação/etiologia , Inflamação/genética , Interleucina-1beta/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Camundongos , Camundongos Knockout , NF-kappa B/metabolismo , Obesidade/complicações , Obesidade/genética , Fosforilação/efeitos dos fármacos , Ativação Plaquetária/efeitos dos fármacos , Adesividade Plaquetária/efeitos dos fármacos , Adesividade Plaquetária/fisiologia , Porphyromonas gingivalis , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Piridinas/farmacologia , Receptores Tipo I de Interleucina-1/deficiência , Receptores Tipo I de Interleucina-1/genética , Trombina/farmacologia , Transcrição Gênica/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
OBJECTIVE: Cardiovascular disease is a complex disorder influenced by interactions of genetic variants with environmental factors. However, there is no information from large community-based studies examining the relationship of circulating cell-specific RNA to inflammatory proteins. In light of the associations among inflammatory biomarkers, obesity, platelet function, and cardiovascular disease, we sought to examine the relationships of C-reactive protein (CRP) and interleukin-6 (IL-6) to the expression of key inflammatory transcripts in platelets. APPROACH AND RESULTS: We quantified circulating levels of CRP and IL-6 in 1625 participants of the Framingham Heart Study (FHS) Offspring cohort examination 8 (mean age, 66.6 ± 6.6 years; 46% men). We measured the expression of 15 relevant genes by high-throughput quantitative reverse transcriptase polymerase chain reaction from platelet-derived RNA and used multivariable regression to relate serum concentrations of CRP and IL-6 with gene expression. Levels of CRP and IL-6 were associated with 10 of the 15 platelet-derived inflammatory transcripts, ALOX5, CRP, IFIT1, IL6, PTGER2, S100A9, SELENBP1, TLR2, TLR4, and TNFRSF1B (P<0.001). Associations between platelet mRNA expression with CRP and IL-6 persisted after multivariable adjustment for potentially confounding factors. Six genes positively associated with CRP or IL-6 in the FHS sample were also upregulated in megakaryocytes in response to CRP or IL-6 exposure. CONCLUSIONS: Our data highlight the strong connection between the circulating inflammatory biomarkers CRP and IL-6 and platelet gene expression, adjusting for cardiovascular disease risk factors. Our results also suggest that body weight may directly influence these associations.
Assuntos
Plaquetas/imunologia , Proteína C-Reativa/metabolismo , Doenças Cardiovasculares , Interleucina-6/sangue , Obesidade , Idoso , Peso Corporal , Doenças Cardiovasculares/epidemiologia , Doenças Cardiovasculares/imunologia , Doenças Cardiovasculares/metabolismo , Feminino , Expressão Gênica/imunologia , Humanos , Fatores Imunológicos/sangue , Masculino , Massachusetts/epidemiologia , Pessoa de Meia-Idade , Obesidade/epidemiologia , Obesidade/imunologia , Obesidade/metabolismo , RNA/sangue , Fatores de RiscoRESUMO
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.
Assuntos
Plaquetas/fisiologia , Comunicação Celular/fisiologia , Micropartículas Derivadas de Células/fisiologia , Transferência Genética Horizontal/fisiologia , RNA/metabolismo , Animais , Plaquetas/citologia , Plaquetas/metabolismo , Comunicação Celular/genética , Micropartículas Derivadas de Células/metabolismo , Células Cultivadas , Técnicas de Cocultura , Células Endoteliais da Veia Umbilical Humana/citologia , Células Endoteliais da Veia Umbilical Humana/fisiologia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Comunicação Parácrina/fisiologiaRESUMO
TLR2, a functional, inflammatory-related receptor, is known to be expressed on megakaryocytes and platelets and to lead to infection and immune-mediated activation of platelets; however, the role of this receptor in megakaryocytes is not understood. Using Meg-01 cells and mouse megakaryocytes, we found that NFκB, ERK-MAPK, and PI3K/Akt pathways, known downstream pathways of TLRs, are activated by Pam3CSK4, a TLR2-specific ligand. In addition, transcription factors associated with megakaryocyte maturation, GATA-1, NF-E2, and mammalian target of rapamycin (mTOR), are all increased in the presence of Pam3CSK4. The effect of Pam3CSK4 on megakaryocyte maturation was verified by the increase in DNA content and adhesion to extracellular matrix proteins by TLR2-dependent stimulation. In addition, TLR2 stimulation resulted in an increase in reactive oxygen species (ROS) production. Gene expression and protein levels of GP1b, CD41, MCP-1, COX2, NFκB1, and TLR2 were up-regulated in megakaryocytes after TLR2 stimulation through NFκB, PI3K/Akt, and ERK-MAPK pathways. Treatment of wild-type mice with Pam3CSK4 resulted in a return to normal platelet levels and an increase in megakaryocyte maturation, which did not occur in the TLR2(-/-) mice. Therefore, inflammation, through TLR2, can increase maturation and modulate the phenotype of megakaryocytes, contributing to the interrelationship between inflammation and hemostasis.
Assuntos
Megacariócitos/metabolismo , NF-kappa B/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor 2 Toll-Like/fisiologia , Animais , Western Blotting , Adesão Celular , Células Cultivadas , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Megacariócitos/citologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína Quinase 3 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , NF-kappa B/genética , Fosfatidilinositol 3-Quinases/genética , Ativação Plaquetária , Ploidias , Proteínas Proto-Oncogênicas c-akt/genética , RNA Mensageiro/genética , Espécies Reativas de Oxigênio/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Activated protein C (APC), an anticoagulant serine protease, has been shown to have non-hemostatic functions related to inflammation, cell survival, and cell migration. In this study we investigate the mechanism by which APC promotes angiogenesis and breast cancer invasion using ex vivo and in vitro methods. When proteolytically active, APC promotes cell motility/invasion and tube formation of endothelial cells. Ex vivo aortic ring assays verify the role of APC in promoting angiogenesis, which was determined to be dependent on EGFR and MMP activation. Given the capacity of APC to promote angiogenesis and the importance of this process in cancer pathology, we investigated whether the mechanisms by which APC promotes angiogenesis can also promote motility and invasion in the MDA-MB-231 breast cancer cell line. Our results indicate that, extracellularly, APC engages EPCR, PAR-1, and EGFR in order to increase the invasiveness of MDA-MB-231 cells. APC activation of matrix metalloprotease (MMP) -2 and/or -9 is necessary but not sufficient to increase invasion, and APC does not utilize the endogenous plasminogen activation system to increase invasion. Intracellularly, APC activates ERK, Akt, and NFkappaB, but not the JNK pathway to promote MDA-MB-231 cell motility. Similar to the hemostatic protease thrombin, APC has the ability to enhance both endothelial cell motility/angiogenesis and breast cancer cell migration.
Assuntos
Neoplasias da Mama/patologia , Movimento Celular/efeitos dos fármacos , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Espaço Intracelular/metabolismo , Proteína C/farmacologia , Transdução de Sinais/efeitos dos fármacos , Animais , Antígenos CD/metabolismo , Neoplasias da Mama/irrigação sanguínea , Neoplasias da Mama/enzimologia , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Quimiotaxia/efeitos dos fármacos , Células Endoteliais/enzimologia , Receptor de Proteína C Endotelial , Ativação Enzimática/efeitos dos fármacos , Receptores ErbB/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Técnicas In Vitro , Espaço Intracelular/efeitos dos fármacos , Metaloproteinases da Matriz/genética , Metaloproteinases da Matriz/metabolismo , Camundongos , Invasividade Neoplásica , Neovascularização Patológica/enzimologia , Fosfatidilinositol 3-Quinases/metabolismo , Plasminogênio/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor PAR-1/metabolismo , Receptores de Superfície Celular/metabolismoRESUMO
Platelets have been extensively studied as hemostatic regulators, stopping uncontrolled flow of blood from an injured vessel and allowing for repair. However, multiple studies have shown that platelets can interact with bacterial proteins, particularly seen during sepsis and inflammation. Immune cells recognize pathogens through Toll-like Receptors (TLRs). These same receptors allow platelets to recognize bacterial proteins and regulate platelet immunity and function. This review examines the TLRs expressed on platelets and megakaryocytes and how these receptors affect the function of these cells. Through TLRs, platelets go beyond hemostatic regulation and play a pivotal role in inflammation and infection.
Assuntos
Plaquetas/metabolismo , Inflamação/metabolismo , Megacariócitos/metabolismo , Receptores Toll-Like/metabolismo , Animais , CamundongosRESUMO
In 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 platelet-dependent thrombosis. It remains unknown if platelet activation by immune versus thrombotic pathways leads to the differential regulation of signal transduction, protein-protein interactions, and alpha-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 Pam(3)CSK(4) stimulation on alpha-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 Pam(3)CSK(4)- 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.
Assuntos
Plaquetas , Ativação Plaquetária/fisiologia , Transdução de Sinais/fisiologia , Trombose , Difosfato de Adenosina/metabolismo , Difosfato de Adenosina/farmacologia , Plaquetas/citologia , Plaquetas/imunologia , Plaquetas/metabolismo , Comunicação Celular/fisiologia , Grânulos Citoplasmáticos/fisiologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Fator XIIIa/metabolismo , Humanos , Lipopeptídeos/metabolismo , Lipopeptídeos/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação/fisiologia , Ativação Plaquetária/efeitos dos fármacos , Proteômica , Proteínas Proto-Oncogênicas c-akt/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Trombina/metabolismo , Trombina/farmacologia , Trombose/imunologia , Trombose/metabolismo , Trombose/patologia , Receptor 2 Toll-Like/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Toll-like receptors (TLRs) are germline-encoded receptors that recognize various pathogen-associated molecular patterns (PAMPs). They are key components of the innate immunity which are activated in response to pathogens as well as non-pathogenic components of damaged tissues. TLR agonists have been developed to treat allergies, cancers, and chronic infections by upregulating the innate immune system. TLR antagonists may be used to treat a number of inflammatory conditions, such as rheumatoid arthritis and systemic lupus erythematosus. Recent research also has shown that TLRs are involved in the pathogenesis of atherosclerosis, thrombosis, myocardial remodeling, ischemic/reperfusion injury, and valvular disease. This article reviews the current experimental and clinical evidence for the role of TLRs in the cardiovascular system, and examines the mechanisms by which TLR antagonists could potentially be used in targeted therapy.
Assuntos
Anti-Inflamatórios/uso terapêutico , Fármacos Cardiovasculares/uso terapêutico , Doenças Cardiovasculares/tratamento farmacológico , Imunidade Inata/efeitos dos fármacos , Receptores Toll-Like/antagonistas & inibidores , Animais , Doenças Cardiovasculares/imunologia , Humanos , Receptores Toll-Like/imunologiaRESUMO
Heparin and low-molecular weight heparin (LMWH) are complex, heterogeneous polysaccharides used in the treatment of arterial and venous thrombosis. M118 is a novel LMWH with low polydispersity and pronounced anti-Xa and anti-thrombin (IIa) activity as compared to current LMWHs. To determine if M118 is effective in preventing thrombosis in the setting of a vascular plaque, apolipoprotein E knockout mice fed a high fat diet were injected with M118, enoxaparin, unfractionated heparin, or saline control and examined for arterial thrombosis using a rose bengal laser induced carotid artery injury model. M118 significantly increased the time to occlusion as compared to control and unfractionated heparin but not compared to enoxaparin although fewer M118 treated animals had any vascular occlusion present at the time of protocol completion. Platelet-neutrophil aggregates were studied by flow cytometry and were found to be decreased with M118 as compared to enoxaparin. This is the first published report examining M118, a novel LMWH designed to have low polydispersity and enhanced anticoagulant activity. In an animal model of vascular plaque, M118 is a potent inhibitor of arterial thrombosis and, despite lower in vivo anti-Xa and anti-IIa activity levels, M118 was superior to UFH in the prevention of arterial thrombosis.
Assuntos
Anticoagulantes/uso terapêutico , Apolipoproteínas E/deficiência , Modelos Animais de Doenças , Heparina de Baixo Peso Molecular/análogos & derivados , Heparina de Baixo Peso Molecular/uso terapêutico , Tromboembolia/prevenção & controle , Animais , Anticoagulantes/química , Anticoagulantes/farmacocinética , Apolipoproteínas E/genética , Feminino , Heparina de Baixo Peso Molecular/farmacocinética , Masculino , Camundongos , Camundongos Knockout , Tromboembolia/tratamento farmacológico , Tromboembolia/genética , Trombose/tratamento farmacológico , Trombose/genética , Trombose/prevenção & controle , Distribuição Tecidual/efeitos dos fármacos , Distribuição Tecidual/genéticaRESUMO
Plasminogen activator inhibitor-1 (PAI-1) is a physiological inhibitor of urokinase (uPA), a serine protease known to promote cell migration and invasion. Intuitively, increased levels of PAI-1 should be beneficial in downregulating uPA activity, particularly in cancer. By contrast, in vivo, increased levels of PAI-1 are associated with a poor prognosis in breast cancer. This phenomenon is termed the "PAI-1 paradox". Many factors are responsible for the upregulation of PAI-1 in the tumor microenvironment. We hypothesize that there is a breast cancer predisposition to a more aggressive stage when PAI-1 is upregulated as a consequence of Metabolic Syndrome (MetS). MetS exerts a detrimental effect on the breast tumor microenvironment that supports cancer invasion. People with MetS have an increased risk of coronary heart disease, stroke, peripheral vascular disease and hyperinsulinemia. Recently, MetS has also been identified as a risk factor for breast cancer. We hypothesize the existence of the "PAI-1 cycle". Sustained by MetS, adipocytokines alter PAI-1 expression to promote angiogenesis, tumor-cell migration and procoagulant microparticle formation from endothelial cells, which generates thrombin and further propagates PAI-1 synthesis. All of these factors culminate in a chemotherapy-resistant breast tumor microenvironment. The PAI-1 cycle may partly explain the PAI-1 paradox. In this hypothesis paper, we will discuss further how MetS upregulates PAI-1 and how an increased level of PAI-1 can be linked to a poor prognosis.