Circulating MicroRNA Levels Indicate Platelet and Leukocyte Activation in Endotoxemia Despite Platelet P2Y12 Inhibition

There is evidence for the effects of platelet inhibition on innate immune activation. Circulating microRNAs (miRNAs) have been implicated as markers of platelet and leukocyte activation. In the present study, we assessed the effects of P2Y12 inhibitors on platelet and leukocyte miRNAs during endotoxemia. Healthy volunteers were randomly assigned to receive oral ticagrelor (n = 10), clopidogrel (n = 8) or no drug (n = 8) for one week, followed by an intravenous bolus of 2 ng/kg endotoxin. Serum was collected at baseline, after one week of antiplatelet treatment and 6 and 24 h after endotoxin administration. MiRNAs were screened using LNA-based qPCR, followed by TaqMan-qPCR validation of candidates. Clinical validation was performed in 41 sepsis patients. Platelet-enriched miR-197, miR-223 and miR-223* were decreased in volunteers following antiplatelet therapy. Endotoxin increased platelet miRNAs, whilst the opposite effect was seen for leukocyte-enriched miR-150. Neither of these endotoxin-mediated effects were altered by P2Y12 inhibitors. Sepsis patients with fatal outcomes (n = 12) had reduced miR-150 levels compared with survivors (n = 29). In conclusion, we show that miR-150 is downregulated in experimental endotoxemia and can predict survival in sepsis but is unaffected by P2Y12 inhibition. While P2Y12 inhibition reduces platelet-associated miRNAs in healthy volunteers, it fails to attenuate the response of platelet miRNAs to endotoxemia

Peritoneal fluid reduces angiogenesis-related microRNA expression in cell cultures of endometrial and endometriotic tissues from women with endometriosis.

UNLABELLED: Endometriosis, defined as the presence of endometrium outside the uterus, is one of the most frequent gynecological diseases. It has been suggested that modifications of both endometrial and peritoneal factors could be implicated in this disease. Endometriosis is a multifactorial disease in which angiogenesis and proteolysis are dysregulated. MicroRNAs (miRNAs) are small non-coding RNAs that regulate the protein expression and may be the main regulators of angiogenesis. Our hypothesis is that peritoneal fluid from women with endometriosis could modify the expression of several miRNAs that regulate angiogenesis and proteolysis in the endometriosis development. The objective of this study has been to evaluate the influence of endometriotic peritoneal fluid on the expression of six miRNAs related to angiogenesis, as well as several angiogenic and proteolytic factors in endometrial and endometriotic cell cultures from women with endometriosis compared with women without endometriosis. METHODS: Endometrial and endometriotic cells were cultured and treated with endometriotic and control peritoneal fluid pools. We have studied the expression of six miRNAs (miR-16, -17-5p, -20a, -125a, -221, and -222) by RT-PCR and protein and mRNA levels of vascular endothelial growth factor-A, thrombospondin-1, urokinase plasminogen activator and plasminogen activator inhibitor-1 by ELISA and qRT-PCR respectively. RESULTS: Control and endometriotic peritoneal fluid pools induced a significant reduction of all miRNAs levels in endometrial and endometriotic cell cultures. Moreover, both peritoneal fluids induced a significant increase in VEGF-A, uPA and PAI-1 protein levels in all cell cultures without significant increase in mRNA levels. Endometrial cell cultures from patients treated with endometriotic peritoneal fluid showed lower expression of miRNAs and higher expression of VEGF-A protein levels than cultures from controls. In conclusion , this "in vitro" study indicates that peritoneal fluid from women with endometriosis modulates the expression of miRNAs that could contribute to the angiogenic and proteolytic disequilibrium observed in this disease

Correlation between changes in miR-16 vs. VEGF-A protein levels in eutopic endometrial (A) and endometriotic cell cultures (B) from women with endometriosis after treatment with both peritoneal fluid pools compared to cell cultures without peritoneal fluid treatment.

<p>Change: differences between parameter levels with and without peritoneal fluid treatment.</p

Peritoneal fluid (PF) effects on uPA and PAI-1 expression in stromal cell cultures from control endometrial tissue, and patient endometrial and endometriotic tissues from women with endometriosis.

<p>ØPF, without PF; CPF, control PF; EPF, endometriotic PF. Data are expressed as Mean ± SEM. * <i>p</i><0.05; ** <i>p</i><0.01; *** <i>p</i><0.001; vs ØPF from same tissue culture, ψ <i>p</i><0.05 vs ØPF from control endometrium culture. A: uPA protein; B: uPA mRNA; C: PAI-1 protein; D: PAI-1 mRNA.</p

Peritoneal fluid (PF) effects on miRNA expression in stromal cell cultures from control endometrial tissue, and patient endometrial and endometriotic tissues from women with endometriosis.

<p>ØPF, without PF; CPF, control PF; EPF, endometriotic PF. *<i>p</i><0.05; **<i>p</i><0.01; <i>p</i><0.001 vs ØPF same tissue culture. miRNA expression is presented as fold change relative to control stromal cell culture without peritoneal fluid (control endometrium ØPF = 1). A: miR-16; B: miR-17-5p; C: miR-20a; D: miR-125a; E: miR-221; F: miR-222.</p

Peritoneal fluid (PF) effects on VEGF-A and TSP-1 expression in stromal cell cultures from control endometrial tissue, and patient endometrial and endometriotic tissues from women with endometriosis.

<p>ØPF, without PF; CPF, control PF; EPF, endometriotic PF. Data are expressed as Mean ± SEM. * <i>p</i><0.05; ** <i>p</i><0.01; *** <i>p</i><0.001; vs ØPF from same tissue culture, ψ <i>p</i><0.05 vs ØPF from control endometrium culture. A: VEGF-A protein; B: VEGF-A mRNA; C: TSP-1 protein; D: TSP-1 mRNA.</p