Regulation of Endothelial Cell Permeability by Platelet-Derived Extracellular Vesicles

BACKGROUND:Platelet (Plt) derived-extracellular vesicles (Plt-EVs) have hemostatic properties similar to Plts. In addition to hemostasis, Plts also function to stabilize the vasculature and maintain endothelial cell (EC) barrier integrity. We hypothesized that Plt-EVs would inhibit vascular endothelial cell permeability, similar to fresh Plts. To investigate this hypothesis we utilized in vitro and in vivo models of vascular endothelial compromise and bleeding. METHODS:In vitro: Plt-EVs were isolated by ultracentrifugation and characterized for Plt markers and particle size distribution. Effects of Plts and Plt-EVs on endothelial barrier function was assessed by trans - endothelial electrical resistance (TEER) measurements and histological analysis of endothelial junction proteins. Hemostatic potential of Plt-EVs and Plts were assessed by multiple electrode Plt aggregometry. In vivo: The effects of Plts and Plt-EVs on vascular permeability and bleeding were assessed in NOD-SCID mice by an established Miles Assay of vascular permeability and a tail snip bleeding assay. RESULTS:In vitro: Plt-EVs displayed exosomal size distribution and expressed Plt specific surface markers. Plts and Plt-EVs decreased EC permeability and restored EC junctions after thrombin challenge. Multiplate aggregometry revealed that Plt-EVs enhanced Thrombin Receptor Activating Peptide (TRAP) mediated aggregation of whole blood, whereas Plts enhanced TRAP, Arachidonic Acid (ASPI), Collagen, and Adenosine Diphosphate (ADP) mediated aggregation. In vivo: Plt-EVs are equivalent to Plts in attenuating VEGF-A induced vascular permeability and uncontrolled blood loss in a tail snip hemorrhage model. CONCLUSION:Our study is the first to report that Plt-EVs might provide a feasible product for transfusion in trauma patients to attenuate bleeding, inhibit vascular permeability and mitigate the endotheliopathy of trauma (EOT). STUDY TYPE:Original Article LEVEL OF EVIDENCE: This is a pre-clinical study so it does not confirm to the level of evidence table for all clinical studies and case reports

Serum from dengue virus-infected patients with and without plasma leakage differentially affects endothelial cells barrier function <i>in vitro</i>

<div><p>Background</p><p>Although most of cases of dengue infections are asymptomatic or mild symptomatic some individuals present warning signs progressing to severe dengue in which plasma leakage is a hallmark.</p><p>Methodology/Principal findings</p><p>The present study used Electric Cell-substrate Impedance Sensing (ECIS^®) which allows for electrical monitoring of cellular barrier function measuring changes in Transendothelial Electric Resistance (TEER) to investigate the parameters associated with dengue induced leakage. Three groups of individuals were tested: dengue-positives with plasma leakage (leakage), dengue-positives without plasma leakage (no leakage), and dengue-negatives (control). Data show that TEER values of human umbilical vein endothelial cells (HUVECs) was significantly lower after incubation with serum from subjects of the leakage group in comparison to the no leakage or control groups. The serum levels of CXCL1, EGF, eotaxin, IFN-γ, sCD40L, and platelets were significantly decreased in the leakage group, while IL-10, IL-6, and IP-10 levels were significantly increased. We also found a strong correlation between TEER values and augmented levels of IP-10, GM-CSF, IL-1α, and IL-8, as well as decreased levels of CXCL1 and platelets.</p><p>Conclusions/Significance</p><p>The present work shows that the magnitude of the immune response contributes to the adverse plasma leakage outcomes in patients and that serum components are important mediators of changes in endothelial homeostasis during dengue infections. In particular, the increased levels of IP-10 and the decreased levels of CXCL1 and platelets seem to play a significant role in the disruption of vascular endothelium associated with leakage outcomes after DENV infection. These findings may have important implications for both diagnostic and therapeutic approaches to predict and mitigate vascular permeabilization in those experiencing the most severe clinical disease outcomes after dengue infection.</p></div

Effect of serum from DENV-positive patients on TEER of endothelial cells.

<p>Confluent monolayers of HUVECs cultured in ECIS arrays were treated or not with 10% serum from three different groups: Healthy blood donors (control, n = 13), DENV infected patients without leakage (no leakage, n = 13), or DENV infected patients with leakage (leakage, n = 13). (*) Asterisks indicate statistically significant differences (ANOVA/ Tukey’s test) between groups with <i>p</i><0.05. TEER values were obtained at 30 (A) and 120 (B) min after treatment.</p

Serum albumin levels.

<p>Albumin levels of serum from the three different groups: Healthy blood donors (control, n = 13), DENV infected patients without leakage (no leakage, n = 13), or DENV infected patients with leakage (leakage, n = 13) were quantified by the bromocresol purple method. Groups were compared by the ANOVA/Tukey’s test. *Statistically significant difference (<i>p</i><0.05).</p

Clinical and laboratory data of study participants.

<p>Clinical and laboratory data of study participants.</p

Real-time impedance measurement throughout 5 h after treatment.

<p>Confluent monolayers of HUVECs cultured in ECIS arrays were treated or not with 10% serum from three different groups: Healthy blood donors (control, n = 13), DENV infected patients without leakage (no leakage, n = 13), or DENV infected patients with leakage (leakage, n = 13). Graph A shows results from the first experiment and graph B from the second experiment.</p

Comparison of immune components levels in serum from control, no leakage, and leakage groups.

<p>Comparison of immune components levels in serum from control, no leakage, and leakage groups.</p