Small RNA-seq during acute maximal exercise reveal RNAs involved in vascular inflammation and cardiometabolic health: brief report.

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.

Platelet bioreactor: accelerated evolution of design and manufacture.

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.

Platelet-TLR7 mediates host survival and platelet count during viral infection in the absence of platelet-dependent thrombosis.

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.

Interleukin 1 receptor 1 and interleukin 1ß regulate megakaryocyte maturation, platelet activation, and transcript profile during inflammation in mice and humans.

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.

Distinct gene signatures in aortic tissue from ApoE-/- mice exposed to pathogens or Western diet.

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.

Platelets and platelet-like particles mediate intercellular RNA transfer.

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.

Relationship among circulating inflammatory proteins, platelet gene expression, and cardiovascular risk.

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.

Regulatory effects of TLR2 on megakaryocytic cell function.

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.

Activated protein C enhances cell motility of endothelial cells and MDA-MB-231 breast cancer cells by intracellular signal transduction.

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.

Immune versus thrombotic stimulation of platelets differentially regulates signalling pathways, intracellular protein-protein interactions, and alpha-granule release.

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.