In sepsis, platelets may become activated via toll-like receptors (TLRs), causing microvascular thrombosis. Megakaryocytes (MKs) also express these receptors; thus, severe infection may modulate thrombopoiesis. To explore the relevance of altered miRNAs in platelet activation upon sepsis, we first investigated sepsis-induced miRNA expression in platelets of septic patients. The effect of abnormal Dicer level on miRNA expression was also evaluated. miRNAs were profiled in septic vs. normal platelets using TaqMan Open Array. We validated platelet miR-26b with its target SELP (P-selectin) mRNA levels and correlated them with clinical outcomes. The impact of sepsis on MK transcriptome was analyzed in MEG-01 cells after lipopolysaccharide (LPS) treatment by RNA-seq. Sepsis-reduced miR-26b was further studied using Dicer1 siRNA and calpain inhibition in MEG-01 cells. Out of 390 platelet miRNAs detected, there were 121 significantly decreased, and 61 upregulated in sepsis vs. controls. Septic platelets showed attenuated miR-26b, which were associated with disease severity and mortality. SELP mRNA level was elevated in sepsis, especially in platelets with increased mean platelet volume, causing higher P-selectin expression. Downregulation of Dicer1 generated lower miR-26b with higher SELP mRNA, while calpeptin restored miR-26b in MEG-01 cells. In conclusion, decreased miR-26b in MKs and platelets contributes to an increased level of platelet activation status in sepsis.

Following an intraventricular hemorrhage (IVH), red blood cell lysis and hemoglobin (Hb) oxidation with the release of heme can cause sterile neuroinflammation. In this study, we measured Hb derivates and cellular adhesion molecules ICAM-1 and VCAM-1 with cell-free miRNAs in cerebrospinal fluid (CSF) samples obtained from Grade-III and Grade-IV preterm IVH infants (IVH-III and IVH-IV, respectively) at multiple time points between days 0–60 after the onset of IVH. Furthermore, human choroid plexus epithelial cells (HCPEpiCs) were incubated with IVH and non-IVH CSF (10 v/v %) for 24 h in vitro to investigate the IVH-induced inflammatory response that was investigated via: (i) HMOX1, IL8, VCAM1, and ICAM1 mRNAs as well as miR-155, miR-223, and miR-181b levels by RT-qPCR; (ii) nuclear translocation of the NF-κB p65 subunit by fluorescence microscopy; and (iii) reactive oxygen species (ROS) measurement. We found a time-dependent alteration of heme, IL-8, and adhesion molecules which revealed a prolonged elevation in IVH-IV vs. IVH-III with higher miR-155 and miR-181b expression at days 41–60. Exposure of HCPEpiCs to IVH CSF samples induced HMOX1, IL8, and ICAM1 mRNA levels along with increased ROS production via the NF-κB pathway activation but without cell death, as confirmed by the cell viability assay. Additionally, the enhanced intracellular miR-155 level was accompanied by lower miR-223 and miR-181b expression in HCPEpiCs after CSF treatment. Overall, choroid plexus epithelial cells exhibit an abnormal cell phenotype after interaction with pro-inflammatory CSF of IVH origin which may contribute to the development of later clinical complications in preterm IVH.

Intraventricular hemorrhage (IVH) represents a high risk of neonatal mortality and later neurodevelopmental impairment in prematurity. IVH is accompanied with inflammation, hemolysis, and extracellular hemoglobin (Hb) oxidation. However, microRNA (miRNA) expression in cerebrospinal fluid (CSF) of preterm infants with IVH has been unknown. Therefore, in the present study, candidate pro-inflammatory cell-free miRNAs were analyzed in CSF samples from 47 preterm infants with grade III or IV IVH vs. clinical controls (n = 14). miRNAs were quantified by RT-qPCR, normalized to “spike-in” cel-miR-39. Oxidized Hb and total heme levels were determined by spectrophotometry as well as IL-8, VCAM-1, ICAM-1, and E-selectin concentrations by ELISA. To reveal the origin of the investigated miRNAs, controlled hemolysis experiments were performed in vitro; in addition, human choroid plexus epithelial cell (HCPEpiC) cultures were treated with metHb, ferrylHb, heme, or TNF-α to replicate IVH-triggered cellular conditions. Levels of miR-223, miR-155, miR-181b, and miR-126 as well as Hb metabolites along with IL-8 were elevated in CSF after the onset of IVH vs. controls. Significant correlations were observed among the miRNAs, oxidized Hb forms, and the soluble adhesion molecules. During the post-IVH follow-up, attenuated expression of miRNAs and protein biomarkers in CSF was observed upon elimination of Hb metabolites. These miRNAs remained unaffected by a series of artificially induced hemolysis, which excluded red blood cells as their origin, while stimulation of HCPEpiCs with oxidized Hb fractions and heme resulted in increased extracellular miRNA levels in the cell culture supernatant. Overall, the hemorrhage-induced CSF miRNAs reflected inflammatory conditions as potential biomarkers in preterm IVH.

B cells express various ion channels, but the presence of voltage-gated sodium (Na_V) channels has not been confirmed in the plasma membrane yet. In this study, we have identified several Na_V channels, which are expressed in the human B cell membrane, by electrophysiological and molecular biology methods. The sensitivity of the detected sodium current to tetrodotoxin was between the values published for TTX-sensitive and TTX-insensitive channels, which suggests the co-existence of multiple Na_V1 subtypes in the B cell membrane. This was confirmed by RT-qPCR results, which showed high expression of TTX-sensitive channels along with the lower expression of TTX-insensitive Na_V1 channels. The biophysical characteristics of the currents also supported the expression of multiple Na_V channels. In addition, we investigated the potential functional role of Na_V channels by membrane potential measurements. Removal of Na⁺ from the extracellular solution caused a reversible hyperpolarization, supporting the role of Na_V channels in shaping and maintaining the resting membrane potential. As this study was mainly limited to electrophysiological properties, we cannot exclude the possible non-canonical functions of these channels. This work concludes that the presence of voltage-gated sodium channels in the plasma membrane of human B cells should be recognized and accounted for in the future.

BCR-ABL tyrosine kinase inhibitors (TKIs) are effective drugs in the treatment of patients with chronic myeloid leukemia. However, based on clinical studies, ponatinib was associated with the development of thrombotic complications. Since endothelial cells (ECs) regulate blood coagulation, their abnormal phenotype may play a role in the development of thrombotic events. We here aimed to investigate the effect of ponatinib on the procoagulant activity of cultured endothelial cells in vitro. Human coronary artery endothelial cells (HCAECs) were incubated with 50, 150, and 1000 nM of ponatinib. Subsequently, phosphatidylserine (PS) exposure and endothelial microvesicles (EMVs) were measured by flow cytometry. In addition, EC- and EMV-dependent thrombin generation was analyzed. To investigate pro-apoptotic effects of ponatinib, the level of Bax and Bcl-xL proteins were studied using Western blot and F3, THBD, and VCAM1 mRNAs were quantified by qPCR. Therapeutic concentrations of ponatinib significantly increased PS expression on ECs and the amount of EMVs which significantly shortened the time parameters of thrombin generation. In addition, these changes were associated with an increased ratio of Bax and Bcl-xL proteins in the presence of the decreased THBD mRNA level. Overall, ponatinib enhances the procoagulant activity of ECs via inducing apoptosis, which may contribute to thrombotic events.

Severe SARS-CoV-2 elicits a hyper-inflammatory response that results in intravascular inflammation with endothelial injury, which contributes to increased mortality in COVID-19. To predict the outcome of severe SARS-CoV-2 infection, we analyzed the baseline level of different biomarkers of vascular disorders in COVID-19 subjects upon intensive care unit (ICU) admission and prior to any vaccination. A total of 70 severe COVID-19 patients (37 survivors and 33 non-survivors) were included with 16 age- and sex-matched controls. Vascular dysfunction was monitored via soluble VCAM-1, E-selectin, ACE2 and Lp-PLA2, while abnormal platelet activation was evaluated by soluble P-selectin and CD40L in parallel. These results were correlated with routine laboratory parameters and disease outcomes. Among these parameters, VCAM-1 and ACE2 showed significantly higher serum levels in COVID-19 patients with early death vs. convalescent subjects. VCAM-1 was significantly correlated with the Horowitz index (r = 0.3115) and IL-6 (r = 0.4599), while ACE2 was related to E-selectin (r = 0.4143) and CD40L (r = 0.2948). Lp-PLA2 was altered in none of these COVID-19 subcohorts and showed no relationship with the other parameters. Finally, the pre-treatment level of VCAM-1 (≥1420 ng/mL) and ACE2 activity (≥45.2 μU/mL) predicted a larger risk for mortality (Log-Rank p = 0.0031 and p = 0.0117, respectively). Vascular dysfunction with endothelial cell activation is linked to lethal COVID-19, and highly elevated soluble VCAM-1 and ACE2 at admission to ICU may predict unfavorable outcomes.