Platelet Extracellular Vesicles Research Articles

Platelet Vesicles Synergetic with Biosynthetic Cellulose Aerogels for Ultra-Fast Hemostasis and Wound Healing.

Achieving hemostasis in penetrating and irregular wounds is challenging because the hemostasis factor cannot arrive at the bleeding site, and substantial bleeding will wash away the blood clot. Since the inherently gradual nature of blood clot formation takes time, a physical barrier is needed before blood clot formation. Herein, an ultra-light and shape memory hemostatic aerogel consisting of oxidized bacterial cellulose (OBC) and platelet extracellular vesicles (pVEs) is reported. The OBC-pVEs aerogel provides a physical barrier for the bleeding site by self-expansion, absorbing the liquid from blood to concentrate platelets and clotting factors and accelerating the clot formation by activating platelets and transforming fibrinogen into fibrin. In the rat liver and tail injury models, the blood loss decreases by 73% and 59%, and the bleeding times are reduced by 55% and 62%, respectively. OBC-pVEs aerogel has also been shown to accelerate wound healing. In conclusion, this work introduces an effective tool for treating deep, non-compressible, and irregular wounds and offers valuable strategies for trauma bleeding and wound treatment.

PLATELET-DERIVED EXTRACELLULAR VESICLES PROMOTE STEM CELL TENOGENIC COMMITMENT IN A BIOENGINEERED TENDON 3D MODEL

Worldwide, tendon disorders are one of the main causes of disability that decrease the quality of life of individuals and represent a substantial economic burden on society. Currently, the main therapies used for tendon injuries are not able to restore tendon functionality, and due to tendons' hypovascular and hypocellular nature, they present a reduced healing capacity, which also limits the success of the available therapies. In order to discover new therapies, extracellular vesicles (EVs), key players in cell-cell communication, have been widely explored for tissue engineering and regenerative medicine applications. Thus, the aim of this study is to assess the role of EVs derived from platelets in stem cell tenogenic commitment using a bioengineered tendon in vitro model for potential use as tendon therapeutic agents. Biomimetic platelet-derived EVs were produced by freeze-thaw cycles of platelets and isolation at different centrifugation speed. To recreate the architecture of tendons, a 3D system consisting of electrospun anisotropic nanofiber scaffolds coated with collagen encapsulating human adipose stem cells (hASCs) and different types of platelet-derived EVs, were produced. Then, the influence of the tendon-mimetic constructs and the distinct EVs populations in the hASCs tenogenic differentiation were assessed over culture time. We observed that the hASCs on the nanofibrous tendon scaffolds, show high cytoskeleton anisotropic organization that is characteristic of tenocytes. Moreover, acting as biological cues, platelet-derived EVs boosted hASCs tenogenic commitment, supported by the increased gene expression of tendon-related markers (SCX and TNMD). Additionally, EVs enhanced the deposition of tendon like extracellular matrix (ECM), as evidenced by the increased gene expression of ECM-related markers such as COL1, COL3, DCN, TNC, and MMP-3, which are fundamental for ECM synthesis and degradation balance. Moreover, EVs induced lower collagen matrix contraction on hASCs, which has been related with lower myofibroblast differentiation. Overall, the results revealed that EVs are capable of modulating stem cells' behavior boosting their tenogenic commitment, through the increased expression of healthy tendon cell markers, potentiating ECM deposition and decreasing cell contractility. Therefore, platelet EVs are a promising biochemical tool, worthy to be further explored, as paracrine signaling that might potentiate tendon repair and regeneration.

Pilot study of frozen platelet extracellular vesicles as a therapeutic agent in hemorrhagic shock in rats.

Hemorrhage accounts for the most preventable deaths after trauma. Resuscitation is guided by studies that demonstrate improved outcomes in patients receiving whole blood or balanced administration of blood products. Platelets present a logistical challenge due to short shelf life and need for refrigeration. Platelet-derived extracellular vesicles (PEVs) are a possible platelet alternative. Platelet-derived extracellular vesicles are secreted from platelets, have hemostatic effects and mitigate inflammation and vascular injury, similar to platelets. This pilot study aimed to elucidate the therapeutic effects of PEVs in a rat model of uncontrolled hemorrhage. Male rats were anesthetized and femoral vessels cannulated. Vital signs (MAP, HR, and RR) were monitored. Electrolytes, lactate and ABG were obtained at baseline, 1-hour and 3-hours post injury. Laparotomy was performed, 50% of the middle hepatic lobe excised and the abdomen packed with gauze. Rats received 2 mL PEVs or lactated Ringers (LR) over 6 minutes immediately after injury. Peritoneal blood loss was quantified using preweighed gauze at 5 minutes, 15 minutes, 30 minutes, 45 minutes, and 60 minutes. Laparotomy was closed 1-hour postinjury. Animals were monitored for 3 hours postinjury then euthanized. Generalized Linear Mixed Effects models were performed to assess effects of treatment and time on lactate and MAP. Twenty-one rats were included (11 LR, 10 PEV). Overall blood loss was between 6 mL and 10 mL and not significantly different between groups. There was a 36% mortality rate in the LR group and 0% mortality in the PEV group ( p = 0.03). The LR group had significantly higher lactates at 1 hour ( p = 0.025). At 15 minutes, 45 minutes, 60 minutes, and 180 minutes, the MAP of the PEV group was significantly higher than the LR group. Early studies are encouraging regarding the potential use of PEVs in uncontrolled hemorrhagic shock based on improved survival and hemodynamics.

Recipient Platelet-Derived Extracellular Vesicles (PEV) Suppress Antibody-Mediated Transfusion-Related Acute Lung Injury (TRALI)

Transfusion-related acute lung injury (TRALI) is a leading cause of transfusion-related fatalities with a 5 to 14% mortality rate. Symptoms range from mild dyspnea, cough, fever to full-blown respiratory failure and severe hypoxemia within 6 hours from the transfusion. To date, the pathogenesis is not fully understood, but it appears that TRALI requires two hits in order to be initiated; the first-hit is the patient's clinical condition often reflected by a state of inflammation while the second hit is the transfusion itself (often containing anti-donor antibodies). Several reports have described platelet involvement in antibody-mediated TRALI, however, there are significant controversies that have arisen as to whether intact platelets actually play a role or not. On the other hand, platelet extracellular vesicles (PEV) have been found to accumulate in stored platelet units and are positively correlated with significant adverse transfusion reactions. Furthermore, in a murine antibody-independent model of TRALI, it was recently demonstrated that PEV increase in stored murine platelet concentrates and are associated with TRALI-induction via excess ceramide production. We have used an in vivo murine model of TRALI where liposaccharide (LPS)-primed BALB/c mice are injected with a murine monoclonal anti-MHC class I antibody (34-1-2s). This antibody-mediated TRALI model exhibits severe acute lung injury that closely mimics the human condition. We attempted to understand what role recipient PEV may play in antibody-dependent TRALI. Male BALB/c mice were administered LPS ip (0.1 mg/kg) 18 hrs prior to initiating TRALI. At time zero, the mice are injected iv with 34-1-2s (1 mg/kg) and subsequently, TRALI manifestations were monitored every 30 min, and at 90 min post-injection, blood and lungs were harvested for the indicated measurements. Results show that compared with isotype-control treated mice, animals administered 34-1-2s had significantly elevated lung Wet/Dry (W/D) ratios (measure of lung edema, Figure 1a) and pulmonary neutrophil accumulation. The TRALI mice had significant thrombocytopenia and elevated levels of PEV in their plasma as detected by High-sensitivity Flow Cytometry (Hs-FCM). To determine the role of in vivo generated PEV in antibody-mediated TRALI, mice were first administered amitriptyline (AMI), a potent inhibitor of PEV formation. AMI treatment significantly reduced plasma PEV levels, but after administration of 34-1-2s, TRALI responses (lung edema) were significantly enhanced in the AMI-treated mice (Figure 1). To determine if this effect was due to PEV-derived serotonin, mice were next treated with Prozac to deplete platelet/PEV stores of serotonin. Prozac-treated mice had normal levels of PEV after 34-12s administration and TRALI induction was not affected. Our results suggest that in antibody-mediated TRALI, recipient generated PEV play a suppressive role that is independent of serotonin. Further studies are underway to determine the mechanism(s) of PEV-mediated TRALI suppression. Thus, modulating PEV formation in recipients may be an effective therapy for reducing TRALI reactions.

Platelet extracellular vesicles and their mitochondrial content improve the mitochondrial bioenergetics of cellular immune recipients.

Mitochondria play a critical role in the production of cell energy and the regulation of cell death. Therefore, mitochondria orchestrate numerous cell effector functions, including fine-tuning the immune system. While mitochondria are mainly found intracellularly, they can escape the confine of the cell during the process of extracellular vesicle release. Platelets patrol blood vessels to ensure vasculature integrity and to support the immune system. In blood, platelets are the primary source of circulating mitochondria. Activated platelets produce extracellular vesicles, including a subset of mitochondria-containing vesicles. We characterized mitochondrial functions in platelet-derived extracellular vesicles, and examined whether they could impact the bioenergetics of cellular immune recipients using an extracellular flux analyzer to measure real-time bioenergetics. We validated that extracellular vesicles derived from activated platelets contain the necessary mitochondrial machinery to respirate and generate energy. Moreover, neutrophils and monocytes efficiently captured platelet-derived extracellular vesicles, enhancing their mitochondrial fitness. This process required functional mitochondria from donor platelets, as it was abolished by the inactivation of extracellular mitochondria using mitochondrial poison. Together, the data suggest that extracellular mitochondria produced by platelets may support other metabolic functions through transcellular bioenergetics.

Platelet extracellular vesicles are efficient delivery vehicles of doxorubicin, an anti-cancer drug: preparation and in vitro characterization

Platelet extracellular vesicles (PEVs) are an emerging delivery vehi for anticancer drugs due to their ability to target and remain in the tumor microenvironment. However, there is still a lack of understanding regarding yields, safety, drug loading efficiencies, and efficacy of PEVs. In this study, various methods were compared to generate PEVs from clinical-grade platelets, and their properties were examined as vehicles for doxorubicin (DOX). Sonication and extrusion produced the most PEVs, with means of 496 and 493 PEVs per platelet (PLT), respectively, compared to 145 and 33 by freeze/thaw and incubation, respectively. The PEVs were loaded with DOX through incubation and purified by chromatography. The size and concentration of the PEVs and PEV-DOX were analyzed using dynamic light scattering and nanoparticle tracking analysis. The results showed that the population sizes and concentrations of PEVs and PEV-DOX were in the ranges of 120–150 nm and 1.2–6.2 × 1011 particles/mL for all preparations. The loading of DOX determined using fluorospectrometry was found to be 2.1 × 106, 1.7 × 106, and 0.9 × 106 molecules/EV using freeze/thaw, extrusion, and sonication, respectively. The internalization of PEVs was determined to occur through clathrin-mediated endocytosis. PEV-DOX were more efficiently taken up by MDA-MB-231 breast cancer cells compared to MCF7/ADR breast cancer cells and NIH/3T3 cells. DOX-PEVs showed higher anticancer activity against MDA-MB-231 cells than against MCF7/ADR or NIH/3T3 cells and better than acommercial liposomal DOX formulation. In conclusion, this study demonstrates that PEVs generated by PLTs using extrusion, freeze/thaw, or sonication can efficiently load DOX and kill breast cancer cells, providing a promising strategy for further evaluation in preclinical animal models. The study findings suggest that sonication and extrusion are the most efficient methods to generate PEVs and that PEVs loaded with DOX exhibit significant anticancer activity against MDA-MB-231 breast cancer cells.

Development of tandem cation exchange chromatography for high purity extracellular vesicle isolation: The effect of ligand steric availability

Purification of extracellular vesicles for research and therapeutic applications requires updated methodology to address the limitations of traditional ultracentrifugation and other size-based separation techniques. Their downfalls include induced extracellular vesicle aggregation, low yields, poor scalability and one-dimensionality of the separation process, as the size or sedimentation speed of extracellular vesicles is often the only selection criterion. Ion exchange chromatography is a promising alternative or supplementary method candidate, as it offers a different approach for extracellular vesicle separation, which is surface charge. For now, mostly anion exchange chromatography has been evaluated for extracellular vesicle purification, as it successfully relies on the strongly negative surface charge of extracellular vesicles. However, as extracellular vesicles are very complex in their structure, also cation exchange chromatography could be applicable, due to individual cationic domains on the extracellular vesicle surface. Here, we compare anion exchange chromatography to different types of cation exchange chromatography for the purification of platelet extracellular vesicle samples also containing plasma-derived impurities. We found that the choice of resin structure used for cation exchange chromatography is critical for binding platelet extracellular vesicles, as a conventional-type cation exchanger was found to only capture and elute less than 20% of extracellular vesicles. With the tentacle-type resin, it was possible to obtain comparable platelet extracellular vesicle yields (over 90%) with cation exchange chromatography compared to anion exchange chromatography, as well as superior purity, especially when it was combined to conventional cation exchange resin.

Procoagulant effect of extracellular vesicles in patients after transcatheter aortic valve replacement or transcatheter aortic valve replacement with percutaneous coronary intervention.

Patients with severe aortic stenosis (AS) after replacement of the transcatheter aortic valve (TAVR) are more likely to develop thrombotic complications such as cerebral embolism and artificial valve thrombosis. However, the mechanism is not yet well defined. We aimed to explore the plasma extracellular vesicles (EVs) levels and their role in the induction of procoagulant activity (PCA) in patients receiving TAVR alone or TAVR with percutaneous coronary intervention (PCI). EVs were analyzed with flow cytometer. Markers of platelet and endothelial cell activation were quantified using selective enzyme-linked immunosorbent assay (ELISA) kits. Procoagulant activity (PCA) was assessed by clotting time, purified clotting complex assays, and fibrin production assays. Our results confirmed that EVs with positive phosphatedylserin (PS+EV), platelet EVs (PEVs) and positive tissue factor EVs (TF+EVs) were higher in patients following TAVR than before TAVR, particularly in TAVR with PCI. Furthermore, endothelial-derived EVs (EEVs) were also higher in patients after TAVR with PCI than pre-TAVR, however, the EEVs levels in TAVR alone patients were gradually reduce than pre-TAVR. In addition, we further proved that total EVs contributed to dramatically shortened coagulation time, increased intrinsic/extrinsic factor Xa and thrombin generation in patients after TAVR, especially in TAVR with PCI. The PCA was markedly attenuated by approximately 80% with lactucin. Our study reveals a previously unrecognized link between plasma EV levels and hypercoagulability in patients after TAVR, especially TAVR with PCI. Blockade of PS+EVs may improve the hypercoagulable state and prognosis of patients.

P67: An Evaluation of Aspirin and Platelet Activation in the Presence of Shear Stress

Background: Mechanical circulatory support (MCS) causes high levels of shear stress, which could lead to complications such as platelet activation and thrombosis. Aspirin is an anti-platelet drug that functions through COX-1 inhibition, however, this may not be sufficient to reduce activation via mechanical stimuli. As such, this study evaluated the efficiency of Aspirin to reduce the downstream effects of platelet functioning and the impact this may have on shear stress-induced thrombosis. Methods: Human blood was treated with and without Aspirin (4.5 μg/mL) and subjected to VAD-like shear stress for 4 hours. Platelets were analyzed by flow cytometry, using CD42b and CD62P, to assess multifactorial parameters. Positive events were normalized and assessed for platelet activation, granularity (size), as well as the number of platelet-platelet aggregates and platelet-derived extracellular vesicles (EVs). Results: Aspirin moderately reduced platelet activation and platelet granularity compared to the untreated control, suggesting little to no protective effects against mechanical stimulation. However, the presence of Aspirin had a greater effect on reducing the number of platelet aggregates and platelet-derived EVs in the presence of VAD-like shear stress. Conclusion: The use of Aspirin (and COX-1 inhibition) may prove ineffective for reducing platelet activation by mechanical stimuli in high-risk patients. However, Aspirin may impair the downstream formation of pro-thrombotic platelet aggregates and EVs, thereby reducing the risk of thrombosis in MCS patients. As Aspirin is observed to provide some protection, further research into combination therapy with additional anti-platelet drugs is needed to overcome the negative impact of mechanical stimulation.

Effect of Genetic Factors, Age and Sex on Levels of Circulating Extracellular Vesicles and Platelets.

Extracellular vesicles (EVs) mediate cell interactions in biological processes, such as receptor activation or molecule transfer. Estimates of variation by age and sex have been limited by small sample size, and no report has assessed the contribution of genetic factors to levels of EVs. Here, we evaluated blood levels of 25 EV and 3 platelet traits in 974 individuals (933 genotyped) and reported the first genome-wide association study (GWAS) on levels of these traits. EV levels all decreased with age, whereas the trend for their surface markers was more heterogeneous. Platelets and CD31dim platelet EVs significantly increased in females compared to males, although CD31 expression on both platelets and platelet EVs decreased in females. Levels of the other EV subsets were similar between sexes. GWAS revealed three statistically significant genetic signals associated with EV levels in the F10 and GBP1 genes and in the intergenic region between LRIG1 and KBTBD8. These add to a signal in the 3'UTR of RHOF associated with CD31 expression on platelets that was previously found to be associated with other platelet traits. These findings suggest that EV formation is not a simple, constant adjunct of metabolism but is under both age-related and genetic control that can be independent of the regulation of the levels of the cells from which the EVs derive.

OxLDL sensitizes platelets for increased formation of extracellular vesicles capable of finetuning macrophage gene expression

Platelet extracellular vesicles (PEVs) generated upon platelet activation may play a role in inflammatory pathologies such as atherosclerosis. Oxidized low-density lipoprotein (oxLDL), a well-known contributor to atherogenesis, activates platelets and presensitizes them for activation by other agonists. We studied the effect of oxLDL on the secretion, composition, and inflammatory functions of PEVs using contemporary EV analytics. Platelets were activated by co-stimulation with thrombin (T) and collagen (C) ± oxLDL and characterized by high-resolution flow cytometry, nanoparticle tracking analysis, proximity extension assay, western blot, and electron microscopy. The effect of PEVs on macrophage differentiation and functionality was examined by analyzing macrophage surface markers, cytokine secretion, and transcriptome. OxLDL upregulated TC-induced formation of CD61+, P-selectin+ and phosphatidylserine+ PEVs. Blocking the scavenger receptor CD36 significantly suppressed the oxLDL+TC-induced PEV formation, and HDL caused a slight but detectable suppression. The inflammatory protein cargo differed between the PEVs from stimulated and unstimulated platelets. Both oxLDL+TC- and TC-induced PEVs enhanced macrophage HLA-DR and CD86 expression and decreased CD11c expression as well as secretion of several cytokines. Pathways related to cell cycle and regulation of gene expression, and immune system signaling were overrepresented in the differentially expressed genes between TC PEV -treated vs. control macrophages and oxLDL+TC PEV -treated vs. control macrophages, respectively. In conclusion, we speculate that oxLDL and activated platelets contribute to proatherogenic processes by increasing the number of PEVs that provide an adhesive and procoagulant surface, contain inflammatory mediators, and subtly finetune the macrophage gene expression.

Platelets, Protean Cells with All-Around Functions and Multifaceted Pharmacological Applications.

Platelets, traditionally known for their roles in hemostasis and coagulation, are the most prevalent blood component after erythrocytes (150,000-400,000 platelets/μL in healthy humans). However, only 10,000 platelets/μL are needed for vessel wall repair and wound healing. Increased knowledge of the platelet's role in hemostasis has led to many advances in understanding that they are crucial mediators in many other physiological processes, such as innate and adaptive immunity. Due to their multiple functions, platelet dysfunction is involved not only in thrombosis, mediating myocardial infarction, stroke, and venous thromboembolism, but also in several other disorders, such as tumors, autoimmune diseases, and neurodegenerative diseases. On the other hand, thanks to their multiple functions, nowadays platelets are therapeutic targets in different pathologies, in addition to atherothrombotic diseases; they can be used as an innovative drug delivery system, and their derivatives, such as platelet lysates and platelet extracellular vesicles (pEVs), can be useful in regenerative medicine and many other fields. The protean role of platelets, from the name of Proteus, a Greek mythological divinity who could take on different shapes or aspects, is precisely the focus of this review.

Impact of passive heat stress and passive heat acclimation on circulating extracellular vesicles: An exploratory analysis.

What is the central question of this study? How does passive heat stress and subsequent heat acclimation affect the circulating concentration of extracellular vesicles? What is the main finding and its importance? Passive heat stress increased the circulating concentration of total and platelet extracellular vesicles. Seven days of hot water immersion did not modify the change in circulating concentrations of extracellular vesicles during passive heat stress. This retrospective exploratory analysis aimed to improve our understanding of the effect of passive heat stress and subsequent heat acclimation on the circulating concentration of extracellular vesicles (EVs). Healthy young adults (four females and six males, 25±4 years of age, 1.72±0.08m in height and weighing 71.6±9.0kg) were heated with a water-perfused suit before and after seven consecutive days of hot water immersion. Pre-acclimation, participants were heated until oesophageal temperature increased to ∼1.4°C above baseline values. Post-acclimation, participants were heated until oesophageal temperature reached the same absolute value as the pre-acclimation visit (∼38.2°C). Venous blood samples were obtained before and at the end of passive heating to quantify plasma concentrations of EVs from all cell types (CSFE+ ), all cell types except erythrocytes (CSFE+ MHCI+ ), platelets (CSFE+ MHCI+ CD41+ ), endothelial cells (CSFE+ MHCI+ CD62e+ ), red blood cells (CSFE+ CD235a+ ) and leucocytes (CSFE+ MHCI+ CD45+ ) via flow cytometry. Passive heat stress increased the concentration of CFSE+ EVs (46,150,000/ml [3,620,784, 88,679,216], P=0.036), CFSE+ MHCI+ EVs (28,787,500/ml [9,851,127, 47,723,873], P=0.021) and CSFE+ MHCI+ CD41+ EVs (28,343,500/ml [9,637,432, 47,049,568], P=0.008). The concentration of CSFE+ MHCI+ CD62e+ EVs (94,230/ml [-55,099, 243,559], P=0.187), CSFE+ CD235a+ EVs (-1,414/ml [-15,709, 12,882], P=0.403) or CSFE+ MHCI+ CD45+ EVs (-192,915/ml [-690,166, 304,336], P=0.828) did not differ during heat stress. The change in circulating EVs during passive heat stress did not differ after heat acclimation (thermal state × acclimation interactions, all P≥0.180). These results demonstrate that passive heat stress increases the circulating concentration of total and platelet EVs and that passive heat acclimation does not alter this increase.

Platelet-Inspired Nanotherapeutics for Biomedical Applications

Platelets, as abundant corpuscles in the blood, are extensively involved in normal physiological processes and disease occurrence. Recently, remarkable progress in platelet-based nanotherapeutics has been made, especially with the burgeoning fields of biotechnology and nanomedicine. Herein, we aim to provide an overview on platelet-inspired nanotherapeutics for biomedical applications. First, the recent trends of biomimetic platelet-inspired nanotherapeutics are outlined, mainly including platelet membrane-camouflaged nanosystems, platelets loading with nanoparticles and platelet-mimicking nanocarriers. Then, the emerging nanotherapeutics for functional modulation of platelet aggregation, activation, and inhibition are discussed. Subsequently, platelet derivative-based nanotherapeutics are presented, including platelet extracellular vesicles and platelet polyphosphates. Finally, the clinical application prospects of these emerging nanotherapeutics are highlighted, with particular emphasis on their rationale, advantages, and challenges.

Tumor-Educated Platelet Extracellular Vesicles: Proteomic Profiling and Crosstalk with Colorectal Cancer Cells.

Platelet-cancer cell interactions modulate tumor metastasis and thrombosis in cancer. Platelet-derived extracellular vesicles (EVs) can contribute to these outcomes. We characterized the medium-sized EVs (mEVs) released by thrombin-stimulated platelets of colorectal cancer (CRC) patients and healthy subjects (HS) on the capacity to induce epithelial-mesenchymal transition (EMT)-related genes and cyclooxygenase (COX)-2(PTGS2), and thromboxane (TX)B2 production in cocultures with four colorectal cancer cell lines. Platelet-derived mEVs were assessed for their size distribution and proteomics signature. The mEV population released from thrombin-activated platelets of CRC patients had a different size distribution vs. HS. Platelet-derived mEVs from CRC patients, but not from HS, upregulated EMT marker genes, such as TWIST1 and VIM, and downregulated CDH1. PTGS2 was also upregulated. In cocultures of platelet-derived mEVs with cancer cells, TXB2 generation was enhanced. The proteomics profile of mEVs released from activated platelets of CRC patients revealed that 119 proteins were downregulated and 89 upregulated vs. HS. We show that mEVs released from thrombin-activated platelets of CRC patients have distinct features (size distribution and proteomics cargo) vs. HS and promote prometastatic and prothrombotic phenotypes in cancer cells. The analysis of platelet-derived mEVs from CRC patients could provide valuable information for developing an appropriate treatment plan.

ЭНДОТЕЛИАЛЬНАЯ ДИСФУНКЦИЯ ПРИ ХРОНИЧЕСКОМ ОПИСТОРХОЗЕ

Relevance. The basis of the complex, in some cases, of interrelated pathological processes in opisthorchiasis invasion may be endothelial dysfunction, which provokes the risks of cardiovascular events, and is an important aspect in establishing pathogenetic connections between diseases of the liver and the cardiovascular system. When assessing the inflammatory process in the pathogenesis of invasion, there is increasing interest in the contribution of not only cellular elements and circulating inflammatory markers, but also in intercellular communication and regulation through microvesicles. Purpose of the study. To study the circulation of extracellular vesicles, which are associated with blood and endothelial cells, in patients with chronic opisthorchiasis. Materials and methods. The study was conducted on two groups of people (men and women aged 18 to 55 years (34.3±1.3 years)). The experimental group included patients with chronic opisthorchiasis (n=26). There was the control group (n=14) matched by gender and age with experimental group. The qualitative composition and quantity of extracellular vesicles (EVs) was determined by flow cytometry. Results. In the experimental group, there was a significant increase in the number of CD41+ events (platelet EVs) 2 times (from 120 to 245 events/μl (p = 0.030), CD45+ events (panleukocyte EVs) - more than 120 times (from 95 to 11713 events/μl (p=0. 0330), as well as a trend towards an increase in the rate for CD31+ events by 19.8 times (from 495 to 9818 events/μl (p=0.133) in comparison with control group. Screening assessment of the CD9+ population of platelet EVs (CD41+31+CD9+), endothelial EVs (CD41-31+9+) and leukocyte EVs (CD45+CD9+) showed a significant (p=0.0330) increase in events, while their content corresponded to the data obtained by CD31+41+ events in quantitative terms. For endothelial EVs, there was a significant increase in the proportion of CD9+ events from 23.3% in the control to 46.2% in the experimental group, while for leukocyte EVs there was no statistically significant change in the percentage ratio, and it was 9.9% in the control and 12.9% in the experimental group. Conclusions. These results can be considered from the perspective of platelet activation, as well as the development of the inflammatory process and endothelial dysfunction, where microvesicles can act as potential highly sensitive markers for opisthorchiasis invasion.

Circulating Extracellular Vesicles Are Increased in Newly Diagnosed Celiac Disease Patients.

Extracellular vesicles (EVs) are a class of circulating entities that are involved in intercellular crosstalk mechanisms, participating in homeostasis maintenance, and diseases. Celiac disease is a gluten-triggered immune-mediated disorder, characterized by the inflammatory insult of the enteric mucosa following local lymphocytic infiltration, resulting in villous atrophy. The goal of this research was the assessment and characterization of circulating EVs in celiac disease patients, as well as in patients already on an adequate gluten-free regimen (GFD). For this purpose, a novel and validated technique based on polychromatic flow cytometry that allowed the identification and enumeration of different EV sub-phenotypes was applied. The analysis evidenced that the total, annexin V+, leukocyte (CD45+), and platelet (CD41a+) EV counts were significantly higher in both newly diagnosed celiac disease patients and patients under GFD compared with the healthy controls. Endothelial-derived (CD31+) and epithelial-derived (EpCAM+) EV counts were significantly lower in subjects under gluten exclusion than in celiac disease patients, although EpCAM+ EVs maintained higher counts than healthy subjects. The numbers of EpCAM+ EVs were a statistically significant predictor of intraepithelial leukocytes (IEL). These data demonstrate that EVs could represent novel and potentially powerful disease-specific biomarkers in the context of celiac disease.

Circulating brain-derived extracellular vesicles expressing neuroinflammatory markers are associated with HIV-related neurocognitive impairment.

Neurocognitive impairment remains prevalent in people with HIV (PWH) despite long term virological suppression by antiretroviral therapy (ART) regimens. Systemic and neuro-inflammatory processes are suggested to contribute to the complex pathology leading to cognitive impairment in this population, yet the underlying mechanisms remain unresolved. Extracellular vesicles (EVs) play a central role in intracellular communication and have emerged as key modulators of immunological and inflammatory responses. In this report, we examined the impact of EVs in PWH experiencing cognitive deficits to determine their relevance in HIV associated neuropathology. EV phenotypes were measured in plasma samples from 108 PWH with either cognitive impairment (CI, n=92) or normal cognition (NC, n=16) by flow cytometry. Matched cerebrospinal fluid (CSF)-derived EVs were similarly profiled from a subgroup of 84 individuals who underwent a lumbar puncture. Peripheral blood mononuclear cells were assayed by flow cytometry to measure monocyte frequencies in a subset of 32 individuals. Plasma-EVs expressing CD14, CD16, CD192, C195, and GFAP were significantly higher in HIV-infected individuals with cognitive impairment compared to individuals with normal cognition. Increased CSF-EVs expressing GFAP and CD200 were found in the cognitive impairment group compared to the normal cognition group. Frequencies of patrolling monocytes correlated with plasma-EVs expressing CD14, CD66b, MCSF, MAP2, and GFAP. Frequencies of CD195 expression on monocytes correlated positively with plasma-EVs expressing CD41a, CD62P, and CD63. Expression of CD163 on monocytes correlated positively with CSF-EVs expressing GFAP and CD200. Finally, the expression of CD192 on total monocytes correlated with CSF-EVs expressing CD200, CD62P, and CD63. EVs expressing monocyte activation and neuronal markers associated with HIV associated cognitive impairment, suggesting that distinct EV subsets may serve as novel biomarkers of neuronal injury in HIV infection. Further circulating platelet EV levels were linked to monocyte activation indicating a potential novel interaction in the pathogenesis of HIV-related cognitive impairment.

Evaluation of Circulating Platelet Extracellular Vesicles and Hypertension Mediated Organ Damage.

Elevated circulating platelet-derived extracellular vesicles (pEVs) have been associated with arterial hypertension. The role of hypertension-mediated organ damage (HMOD) to induce EV release is still unknown. We studied the micro- and macro-vascular changes (retinal vascular density and pulse wave velocity), endothelial function (flow-mediated vasodilation of brachial artery and finger plethysmography), and assessed the psychosocial status (anxiety and depression) in hypertensive patients to determine their relationship with EV release. Pulse wave velocity showed a significant positive correlation with pEVs (r = 0.33; p = 0.01). Systolic blood pressure (SBP) negatively correlated with retinal vascularity. The superficial retinal vascular plexus density in the whole image showed a significant negative correlation with 24 h SBP (r = −0.38, p < 0.01), day-SBP (r = −0.35, p = 0.01), and night-SBP (r = −0.27, p = 0.04). pEVs did not show significant associations with microvascular damage (retinal vascular density), endothelial function (flow-mediated vasodilation of brachial artery and finger plethysmography), or psychosocial status (anxiety and depression). Our results indicate that the pEV levels were associated with macrovascular damage measured by PWV, whereas no significant association between pEVs and microvascular damage, endothelial function, or emotional status could be detected. The potential utility of pEV in clinical practice in the context of HMOD may be limited to macrovascular changes.

Impact of acute moderate-intensity aerobic exercise on circulating extracellular vesicles in pregnant and non-pregnant women.

Exercise improves cardiovascular and metabolic health in pregnancy and may represent a non-pharmacological approach to improving pregnancy outcomes. Extracellular vesicles (EVs) are emerging biomarkers of endothelial dysfunction and offer the potential for evaluating vascular health non-invasively during pregnancy. The purpose of this study was to investigate changes in circulating EV levels after an acute bout of moderate-intensity aerobic exercise in healthy pregnant and non-pregnant women. We studied plasma samples from pregnant (N=13, 13-28 weeks) and non-pregnant (N=17) women. A pre-exercise blood sample was obtained followed by a 30min bout of moderate-intensity treadmill-based exercise. Immediately following the exercise, a post-exercise blood draw was collected. Large EVs were isolated from plasma by differential centrifugation and characterized by Western blot and electron microscopy. We quantified circulating EVs by nanoscale flow cytometry. Endothelial EVs were identified as VE-Cadherin+, platelet EVs as CD41+,and leukocyte EVs as CD45+events. Acute exercise was associated with a significant reduction in levels of circulating endothelial EVs in the non-pregnant group (p=0.0232) but not in the pregnant group (p=0.2734). A greater proportion of non-pregnant women (13/17, 76.47%) exhibited a reduction in endothelial EVs compared with their pregnant counterparts (4/13, 30.76%, p<0.05). We also observed a positive association between measures of fitness (average speed) and baseline levels of platelet (r=0.5816, p=0.0159) and total EVs (r=0.5325, p=0.0296) in the non-pregnant group but not in pregnant individuals. Collectively, our study highlights that after a matched acute exercise, changes to circulating EV levels differ depending on pregnancy status.