BACKGROUND: Platelets store large amounts of serotonin that they release during thrombus formation or acute inflammation. This facilitates hemostasis and modulates the inflammatory response. METHODS:Infarct size, heart function, and inflammatory cell composition were analyzed in mouse models of myocardial reperfusion injury with genetic and pharmacological depletion of platelet serotonin. These studies were complemented by in vitro serotonin stimulation assays of platelets and leukocytes in mice and men, and by measuring plasma serotonin levels and leukocyte activation in patients with acute coronary syndrome. RESULTS:Platelet-derived serotonin induced neutrophil degranulation with release of myeloperoxidase and hydrogen peroxide (H 2 O 2 ) and increased expression of membrane-bound leukocyte adhesion molecule CD11b, leading to enhanced inflammation in the infarct area and reduced myocardial salvage. In patients hospitalized with acute coronary syndrome, plasmatic serotonin levels correlated with CD11b expression on neutrophils and myeloperoxidase plasma levels. Longterm serotonin reuptake inhibition-reported to protect patients with depression from cardiovascular events-resulted in the depletion of platelet serotonin stores in mice. These mice displayed a reduction in neutrophil degranulation and preserved cardiac function. In line, patients with depression using serotonin reuptake inhibition, presented with suppressed levels of CD11b surface expression on neutrophils and lower myeloperoxidase levels in blood. CONCLUSIONS:Taken together, we identify serotonin as a potent therapeutic target in neutrophil-dependent thromboinflammation during myocardial reperfusion injury.

Platelets form complexes with neutrophils during inflammatory processes. These aggregates migrate into affected tissues and also circulate within the organism. Several studies have evaluated platelet-neutrophil complexes as a marker of cardiovascular diseases in human and mouse. Although multiple publications have reported platelet-neutrophil complex counts, we noticed that different methods were used to analyze platelet-neutrophil complex formation, resulting in significant differences, even in baseline values. We established a protocol for platelet-neutrophil complex measurement with flow cytometry in murine and human whole blood samples. In vitro platelet-neutrophil complex formation was stimulated with ADP or PMA. We tested the effect of different sample preparation steps and cytometer settings on platelet-neutrophil complex detection and noticed false-positive counts with increasing acquisition speed. Platelet-neutrophil complex formation depends on platelet P-selectin expression, and antibody blocking of P-selectin consequently prevented ADP-induced platelet-neutrophil complex formation. These findings may help generating more comparable data among different research groups that examine platelet-neutrophil complexes as a marker for cardiovascular disease and novel therapeutic interventions.

ObjectiveActivated platelets release serotonin at sites of inflammation where it acts as inflammatory mediator and enhances recruitment of neutrophils. Chronic treatment with selective serotonin reuptake inhibitors (SSRI) depletes the serotonin storage pool in platelets, leading to reduced leukocyte recruitment in murine experiments. Here, we examined the direct and acute effects of SSRI on leukocyte recruitment in murine peritonitis.MethodsC57Bl/6 and Tph1−/− (Tryptophan hydroxylase1) mice underwent acute treatment with the SSRI fluoxetine or vehicle. Serotonin concentrations were measured by ELISA. Leukocyte rolling and adhesion on endothelium was analyzed by intravital microscopy in mesentery venules with and without lipopolysaccharide challenge. Leukocyte extravasation in sterile peritonitis was measured by flow cytometry of abdominal lavage fluid.ResultsPlasma serotonin levels were elevated 2 hours after fluoxetine treatment (0.70±0.1 µg/ml versus 0.27±0.1, p = 0.03, n = 14), while serum serotonin did not change. Without further stimulation, acute fluoxetine treatment increased the number of rolling leukocytes (63±8 versus 165±17/0.04 mm2min−1) and decreased their velocity (61±6 versus 28±1 µm/s, both p<0.0001, n = 10). In Tph1−/− mice leukocyte rolling was not significantly influenced by acute fluoxetine treatment. Stimulation with lipopolysaccharide decreased rolling velocity and induced leukocyte adhesion, which was enhanced after fluoxetine pretreatment (27±3 versus 36±2/0.04 mm2, p = 0.008, n = 10). Leukocyte extravasation in sterile peritonitis, however, was not affected by acute fluoxetine treatment.ConclusionsAcute fluoxetine treatment increased plasma serotonin concentrations and promoted leukocyte-endothelial interactions in-vivo, suggesting that serotonin is a promoter of acute inflammation. E-selectin was upregulated on endothelial cells in the presence of serotonin, possibly explaining the observed increase in leukocyte-endothelial interactions. However transmigration of neutrophils in sterile peritonitis was not affected by higher serotonin concentrations, indicating that the effect of fluoxetine was restricted to early steps in the leukocyte recruitment. Whether SSRI use in humans alters leukocyte recruitment remains to be investigated.

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miRNAs have shown promise as potential biomarkers for acute myocardial infarction (AMI). However, the current used quantitative real-time PCR (qRT-PCR) allows solely for relative expression of nucleic acids and it is susceptible to day-to-day variability, which has limited the validity of using the miRNAs as biomarkers. In this study we explored the technical qualities and diagnostic potential of a new technique, chip-based digital PCR, in quantifying the miRNAs in patients with AMI and ischaemia-reperfusion injury (I/R). In a dilution series of synthetic C.elegans-miR-39, chip-based digital PCR displayed a lower coefficient of variation (8.9% vs 46.3%) and a lower limit of detection (0.2 copies/μL vs 1.1 copies/μL) compared with qRT-PCR. In the serum collected from 24 patients with ST-elevation myocardial infarction (STEMI) and 20 patients with stable coronary artery disease (CAD) patients after percutaneous coronary intervention (PCI), we used qRT-PCR and multiplexed chip-based digital PCR to quantify the serum levels of miRNA-21 and miRNA-499 as they have been validated in AMI in prior studies. In STEMI, I/R injury was assessed via measurement of ST-segment resolution (ST-R). Chip-based digital PCR revealed a statistical significance in the difference of miR-21 levels between stable CAD and STEMI groups (118.8 copies/μL vs 59 copies/μL; P=0.0300), whereas qRT-PCR was unable to reach significance (136.4 copies/μL vs 122.8 copies/μL; P=0.2273). For miR-499 levels, both chip-based digital PCR and qRT-PCR revealed statistically significant differences between stable CAD and STEMI groups (2 copies/μL vs 8.5 copies/μL, P=0.0011; 0 copies/μL vs 19.4 copies/μL; P<0.0001). There was no association between miR-21/499 levels and ST-R post-PCI. Our results show that the chip-based digital PCR exhibits superior technical qualities and promises to be a superior method for quantifying miRNA levels in the circulation, which may become a more accurate and reproducible method for directly quantifying miRNAs, particularly for use in large multi-centre clinical trials.