| 1. |
- Bryl-Górecka, Paulina, et al.
(author)
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Bilberry Supplementation after Myocardial Infarction Decreases Microvesicles in Blood and Affects Endothelial Vesiculation
- 2020
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In: Molecular Nutrition & Food Research. - : Wiley-VCH Verlagsgesellschaft. - 1613-4125 .- 1613-4133. ; 64:20
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Journal article (peer-reviewed)abstract
- Scope: Diet rich in bilberries is considered cardioprotective, but the mechanisms of action are poorly understood. Cardiovascular disease is characterized by increased proatherogenic status and high levels of circulating microvesicles (MVs). In an open-label study patients with myocardial infarction receive an 8 week dietary supplementation with bilberry extract (BE). The effect of BE on patient MV levels and its influence on endothelial vesiculation in vitro is investigated.Methods and results: MVs are captured with acoustic trapping and platelet-derived MVs (PMVs), as well as endothelial-derived MVs (EMVs) are quantified with flow cytometry. The in vitro effect of BE on endothelial extracellular vesicle (EV) release is examined using endothelial cells and calcein staining. The mechanisms of BE influence on vesiculation pathways are studied by Western blot and qRT-PCR. Supplementation with BE decreased both PMVs and EMVs. Furthermore, BE reduced endothelial EV release, Akt phosphorylation, and vesiculation-related gene transcription. It also protects the cells from P2X(7)-induced EV release and increase in vesiculation-related gene expression.Conclusion: BE supplementation improves the MV profile in patient blood and reduces endothelial vesiculation through several molecular mechanisms related to the P2X(7)receptor. The findings provide new insight into the cardioprotective effects of bilberries.
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| 2. |
- Bryl-Górecka, Paulina, et al.
(author)
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Effect of exercise on the plasma vesicular proteome : A methodological study comparing acoustic trapping and centrifugation
- 2018
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In: Lab on a Chip. - : Royal Society of Chemistry (RSC). - 1473-0197 .- 1473-0189. ; 18:20, s. 3101-3111
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Journal article (peer-reviewed)abstract
- Extracellular vesicles (EVs) are a heterogeneous group of actively released vesicles originating from a wide range of cell types. Characterization of these EVs and their proteomes in the human plasma provides a novel approach in clinical diagnostics, as they reflect physiological and pathological states. However, EV isolation is technically challenging with the current methods having several disadvantages, requiring large sample volumes, and resulting in loss of sample and EV integrity. Here, we use an alternative, non-contact method based on a microscale acoustic standing wave technology. Improved coupling of the acoustic resonator increased the EV recovery from 30% in earlier reports to 80%, also displaying long term stability between experiment days. We report a pilot study, with 20 subjects who underwent physical exercise. Plasma samples were obtained before and 1 h after the workout. Acoustic trapping was compared to a standard high-speed centrifugation protocol, and the method was validated by flow cytometry (FCM). To monitor the device stability, the pooled frozen plasma from volunteers was used as an internal control. A key finding from the FCM analysis was a decrease in CD62E+ (E-selectin) EVs 1 h after exercise that was consistent for both methods. Furthermore, we report the first data that analyse differential EV protein expression before and after physical exercise. Olink-based proteomic analysis showed 54 significantly changed proteins in the EV fraction in response to physical exercise, whereas the EV-free plasma proteome only displayed four differentially regulated proteins, thus underlining an important role of these vesicles in cellular communication, and their potential as plasma derived biomarkers. We conclude that acoustic trapping offers a fast and efficient method comparable with high-speed centrifugation protocols. Further, it has the advantage of using smaller sample volumes (12.5 μL) and rapid contact-free separation with higher yield, and can thus pave the way for future clinical EV-based diagnostics.
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| 3. |
- Bryl-Gorecka, Paulina
(author)
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Microvesicle signaling in cardiovascular biology under normal and pathobiological conditions
- 2021
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Doctoral thesis (other academic/artistic)abstract
- Cardiovascular disease (CVD) is one of the leading causes of death worldwide. Atherosclerosis, a chronic pathology directly related to the circulatory system, develops due to an interplay between several molecular events. Atherosclerotic plaque build-up can lead to a reduction in arterial blood flow and finally, myocardial infarction (MI). To prevent or slow down of the CVD progression, it is important to primarily address modifiable risk factors connected with lifestyle and nutrition. Microvesicles (MVs) belong to a larger group, called extracellular vesicles that also includes exosomes and apoptotic bodies. These small (<1 μm) vesicles are released from the cell by blebbing of plasma membrane, a complex process involving numerous signaling pathways. Circulating MVs originate mainly from blood cells, as well as vascular endothelial cells. The release is activated by pro-inflammatory and pro-coagulant conditions. MVs transport bioactive molecules both on their surface and inside the lumen that makes them an interesting target for biomarker discovery and potential diagnostic application. The overall aim of this thesis was to explore the role of MVs in cardiovascular-related pathologies. To analyse plasma MVs, we optimized an acoustic trapping machine based method, employing ultrasonic standing wave, that isolates vesicles in a non-contact manner. We demonstrated that the new method is comparable to standard protocols of MV enrichment and could potentially be used for vesicle-based diagnostics . Furthermore, the results of a clinical study revealed that exercise has a protective effect on the vasculature through decreasing a release of MVs from activated endothelium. The proteomic data showed that exercise changes the pattern of the vesicular cargo. Bilberries (Vaccinium myrtillus) are considered to be a beneficial dietary component for patients with CVD and in the BEARSMART randomized clinical study we explored if there was an effect of bilberry powder supplementation on circulating MVs in MI patients. After eight weeks of dietary intervention, there was a significant reduction in platelet and endothelial MV concentration. Moreover, the in vitro part of the study demonstrated that bilberry extract decreased endothelial vesiculation, which was related to the P2X7 purinergic receptor pathway. The results of this project showed for the first time that nutrional changes can directly affect MV release and underlined the protective influence of berries on vascular health. In the PROFLOW clinical study, we observed a negative relationship between coronary flow reserve (CFR), a parameter depicting blood flow in the heart, and levels of endothelial and platet MVs, circulating in patients with CVD. Proteomic profiling demonstrated similar connection for CFR and several vesicular biomarkers. The outcome of this clinical study pointed at a potential application of MVs for diagnosis of vascular dysfunction. Lastly, we showed that atherosclerotic plaque released MVs, following balloon angioplasty. The results revealed that the vesicles originated from several types of cells and exhibited a pro-atherogenic ‘pattern’ of proteins that represented the pathological processes within the cardiovascular system that can lead to the atheroma formation. We also demonstrated an advantage of analysing isolated EVs, compared to crude plasma samples. The outcome of the studies included in this thesis points at the important role of MVs in the cardiovascular system. Study I and II underlined the importance of exercise and nutrition for prevention of CVD through decrease in MVs, whereas the Western world is still characterized with physical inactivity and imbalanced diet. Study III and IV focused on potential application of MVs as biomarkers or targets of therapeutics, however it needs further, thorough research and possibly, personalized approach.
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| 4. |
- Bryl-Górecka, Paulina, et al.
(author)
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Microvesicles in plasma reflect coronary flow reserve in patients with cardiovascular disease.
- 2021
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In: American journal of physiology. Heart and circulatory physiology. - : American Physiological Society. - 1522-1539 .- 0363-6135. ; 320:5
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Journal article (peer-reviewed)abstract
- High levels of microvesicles (MVs), a type of extracellular vesicles, are detected in several pathological conditions. We investigated the connection between coronary flow reserve (CFR), a prognostic clinical parameter that reflects blood flow in the heart, with levels of MVs and their cargo, from plasma of cardiovascular patients. The PROFLOW study consists of 220 patients with prior myocardial infarction and measured CFR with transthoracic echocardiography. The patients were divided into high and low CFR groups. Plasma MVs were captured with acoustic trapping. Platelet and endothelial-derived MVs were measured with flow cytometry and MV lysates were analyzed with proteomic panels against cardiovascular biomarkers. Flow cytometry was further applied to identify cellular origin of biomarkers. Our data shows a negative correlation between MV concentration and CFR values. Platelet and endothelial MV levels were significantly increased in plasma from the low CFR group. CFR negatively correlates with the levels of several proteomic biomarkers and the low CFR group exhibited higher concentrations of these proteins in MVs. Focused analysis of one of the MV proteins, B Cell Activating Factor (BAFF), revealed platelet and not leukocyte origin and release upon proinflammatory stimulus. Higher levels of MVs carrying an elevated concentration of proatherogenic proteins, circulate in plasma in patients with low CFR, a marker of vascular dysfunction, reduced blood flow and poor prognosis. Our findings demonstrate a potential clinical value of MVs as biomarkers and possible therapeutic targets against endothelial deterioration.
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| 5. |
- Bryl-Górecka, Paulina, et al.
(author)
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Microvesicles in plasma reflect coronary flow reserve in patients with cardiovascular disease
- 2021
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In: American Journal of Physiology - Heart and Circulatory Physiology. - 0363-6135. ; 320:5, s. 2147-2160
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Journal article (peer-reviewed)abstract
- High levels of microvesicles (MVs), a type of extracellular vesicles, are detected in several pathological conditions. We investigated the connection between coronary flow reserve (CFR), a prognostic clinical parameter that reflects blood flow in the heart, with levels of MVs and their cargo, from plasma of patients with cardiovascular disease. The PROFLOW study consists of 220 patients with prior myocardial infarction and measured CFR with transthoracic echocardiography. The patients were divided into high and low CFR groups. Plasma MVs were captured with acoustic trapping. Platelet- and endothelial-derived MVs were measured with flow cytometry, and vesicle lysates were analyzed with proteomic panels against cardiovascular biomarkers. Flow cytometry was further applied to identify cellular origin of biomarkers. Our data show a negative correlation between MV concentration and CFR values. Platelet and endothelial MV levels were significantly increased in plasma from the low CFR group. CFR negatively correlates with the levels of several proteomic biomarkers, and the low CFR group exhibited higher concentrations of these proteins in MVs. Focused analysis of one of the MV proteins, B cell activating factor (BAFF), revealed platelet and not leukocyte origin and release upon proinflammatory stimulus. Higher levels of MVs carrying an elevated concentration of proatherogenic proteins circulate in plasma in patients with low CFR, a marker of vascular dysfunction, reduced blood flow, and poor prognosis. Our findings demonstrate a potential clinical value of MVs as biomarkers and possible therapeutic targets against endothelial deterioration.
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| 6. |
- Bryl-Górecka, Paulina, et al.
(author)
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Reply to Trimaille et al.
- 2021
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In: American Journal of Physiology - Heart and Circulatory Physiology. - : American Physiological Society. - 0363-6135 .- 1522-1539. ; 321:4, s. 750-750
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Journal article (peer-reviewed)
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| 7. |
- James, Kreema, et al.
(author)
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Increased expression of miR-224-5p in circulating extracellular vesicles of patients with reduced coronary flow reserve
- 2022
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In: BMC Cardiovascular Disorders. - : Springer Science and Business Media LLC. - 1471-2261. ; 22:1
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Journal article (peer-reviewed)abstract
- Background: Endothelial and microvascular dysfunction are pivotal causes of major adverse cardiac events predicted by coronary flow reserve (CFR). Extracellular Vesicles (EVs) have been studied extensively in the pathophysiology of coronary artery disease. However, little is known on the impact of the non-coding RNA content of EVs with respect to CFR. Methods: We carried out a study among 120 patients divided by high-CFR and low-CFR to profile the miRNA content of circulating EVs. Results: A multiplex array profiling on circulating EVs revealed mir-224-5p (p-value ≤ 0.000001) as the most differentially expressed miRNA in the Low-CFR group and showed a significantly independent relationship to CFR. Literature survey indicated the origin of the miR from liver cells and not of platelet, leukocyte, smooth muscle or endothelial (EC) origin. A q-PCR panel of the conventional cell type-EVs along with hepatic EVs showed that EVs from liver cells showed higher expression of the miR-224-5p. FACS analysis demonstrated the presence of liver-specific (ASGPR-1+/CD14−) EVs in the plasma of our cohort with the presence of Vanin-1 required to enter the EC barrier. Hepatic EVs with and without the miR-224-5p were introduced to ECs in-vitro, but with no difference in effect on ICAM-1 or eNOS expression. However, hepatic EVs elevated endothelial ICAM-1 levels per se independent of the miR-224-5p. Conclusion: This indicated a role of hepatic EVs identified by the miR-224-5p in endothelial dysfunction in patients with Low CFR.
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| 8. |
- Rezeli, Melinda, et al.
(author)
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Comparative Proteomic Analysis of Extracellular Vesicles Isolated by Acoustic Trapping or Differential Centrifugation
- 2016
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In: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 88:17, s. 8577-8586
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Journal article (peer-reviewed)abstract
- Extracellular vesicles (ECVs), including microparticles and exosomes, are submicrometer membrane vesicles released by diverse cell types upon activation or stress. Circulating ECVs are potential reservoirs of disease biomarkers, and the complexity of these vesicles is significantly lower compared to their source, blood plasma, which makes ECV-based biomarker studies more promising. Proteomic profiling of ECVs is important not only to discover new diagnostic or prognostic markers but also to understand their roles in biological function. In the current study, we investigated the protein composition of plasma-derived ECVs isolated by acoustic seed trapping. Additionally, the protein composition of ECVs isolated with acoustic trapping was compared to that isolated with a conventional differential centrifugation protocol. Finally, the proteome of ECVs originating from ST-elevation myocardial infarction patients was compared with that of healthy controls using label-free LC-MS quantification. The acoustic trapping platform allows rapid and automated preparation of ECVs from small sample volumes, which are therefore well-suited for biobank repositories. We found that the protein composition of trapped ECVs is very similar to that isolated by the conventional differential centrifugation method.
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| 9. |
- Sathanoori, Ramasri, et al.
(author)
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P2Y2 receptor modulates shear stress-induced cell alignment and actin stress fibers in human umbilical vein endothelial cells
- 2017
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In: Cellular and Molecular Life Sciences. - : Springer Science and Business Media LLC. - 1420-682X .- 1420-9071. ; 74:4, s. 731-746
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Journal article (peer-reviewed)abstract
- Endothelial cells release ATP in response to fluid shear stress, which activates purinergic (P2) receptor-mediated signaling molecules including endothelial nitric oxide (eNOS), a regulator of vascular tone. While P2 receptor-mediated signaling in the vasculature is well studied, the role of P2Y2 receptors in shear stress-associated endothelial cell alignment, cytoskeletal alterations, and wound repair remains ill defined. To address these aspects, human umbilical vein endothelial cell (HUVEC) monolayers were cultured on gelatin-coated dishes and subjected to a shear stress of 1 Pa. HUVECs exposed to either P2Y2 receptor antagonists or siRNA showed impaired fluid shear stress-induced cell alignment, and actin stress fiber formation as early as 6 h. Similarly, when compared to cells expressing the P2Y2 Arg-Gly-Asp (RGD) wild-type receptors, HUVECs transiently expressing the P2Y2 Arg-Gly-Glu (RGE) mutant receptors showed reduced cell alignment and actin stress fiber formation in response to shear stress as well as to P2Y2 receptor agonists in static cultures. Additionally, we observed reduced shear stress-induced phosphorylation of focal adhesion kinase (Y397), and cofilin-1 (S3) with receptor knockdown as well as in cells expressing the P2Y2 RGE mutant receptors. Consistent with the role of P2Y2 receptors in vasodilation, receptor knockdown and overexpression of P2Y2 RGE mutant receptors reduced shear stress-induced phosphorylation of AKT (S473), and eNOS (S1177). Furthermore, in a scratched wound assay, shear stress-induced cell migration was reduced by both pharmacological inhibition and receptor knockdown. Together, our results suggest a novel role for P2Y2 receptor in shear stress-induced cytoskeletal alterations in HUVECs.
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| 10. |
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