Browsing by Subject "extracellular vesicle"

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  • Rautiainen, Swarna (Helsingin yliopisto, 2020)
    Endothelial dysfunction is a common characteristic of several diseases including diabetes mellitus, coronary heart disease and stroke. Healthy endothelium ensures vascular homeostasis, regulation of blood flow and the exchange of oxygen and nutrients, as well as immune cell filtration to the surrounding tissues. In many cases, endothelial dysfunction results in ischemia in the surrounding tissues impairing cellular regeneration mechanisms, which can lead to tissue necrosis in the worst case. Therapeutic angiogenesis via stem cell transplantation aims to restore tissue blood flow and thus aid in tissue regeneration and restoration of a functioning tissue. Adipose derived stem/stromal cells (ASC) are a stem cell population with a multilineage differentiation ability. They have been shown to differentiate towards adipogenic, osteogenic, chondrogenic, myogenic and neurogenic lineages among others. Their easy obtainability from liposuction material and abundance in the adipose tissue makes them an especially practical and favorable cell option for stem cell research. In angiogenesis research, ASCs are commonly used in a co-culture with an endothelial cell (EC) type such as human umbilical vein endothelial cell. ASCs secrete extracellular vesicles (EV) that are small membrane bound vesicles with a diameter ranging from 40-1000 nm, and which have the ability to alter the behavior of target cells through their cargo. EV cargo consists of microRNAs, messenger-RNAs and proteins, and the EV cargo of ASCs has been shown to have proangiogenic effects. The aim of this work was to review what is currently known about ASC ability to promote angiogenesis through paracrine secretion and differentiation into endothelial cells or pericytes, interactions between ASCs and endothelial cells in the angiogenesis promoting process and the role of ASC extracellular vesicles in promoting angiogenesis. The methods for this work were database research of related articles using scientific databases and search engines, article categorization and reading, and finally manuscript production. It can be concluded from the current literature that a co-culture environment of ASCs and an endothelial cell type supports the formation of tube-like structures in vitro. Additional insulin like growth factor 1 in culture medium enhances the expression of angiogenesis-related growth factors in both cell types via PI3K/AKT signaling pathway. Further, the activation of platelet derived growth factor receptor β supports ASC ability to promote vascular network formation. On the contrary, the presence of ASC secreted activin A results in the inhibition of vascular network formation. ASCs can differentiate into endothelial cells particularly in three-dimensional culture conditions. In addition, fibroblast growth factor 2 and the activation of the AKT-pathway are crucial for endothelial differentiation. In addition, ASCs have the ability to differentiate into pericytes and assume a stabilizing role on the outside of the microvessels. Concerning ASC derived EVs and their cargo, miR-31, miR-125a and miR-126 have proangiogenic effects in vitro and in vivo. Proangiogenic miRNAs in ASC EV cargo are miR-181b-5p and the let7-family, out of which miR-181b-5p upregulates vascular endothelial growth factor and hypoxia-inducible factor 1α and let7-family influences tube formation ability of ECs. In vivo, ASC derived EVs support fat grafting, enhance wound healing both in healthy and diabetic environment, and provide cardioprotection. Therefore, ASC EVs show potential for therapeutic angiogenesis but currently there is a lack of clinical trials in EV research.
  • Lindelöf, Anna-Emilia (Helsingin yliopisto, 2019)
    Tiivistelmä - Referat - Abstract Background. Platelets are known to contain ample amounts of brain derived neurotrophic factor. Previous spectrophotometric studies carried out in Pia Siljander’s lab have shown that BDNF is secreted from activated platelets packed in extracellular vesicles. For this project we wanted to 1) confirm that BDNF really is secreted in extracellular vesicles (EVs)2) find out how the choice of agonist affected the BDNF cargo of the platelet derived EVs, and 3) find out if the BDNF is packed into EVs of certain densities rather than others. Methods. The platelets were isolated from platelet concentrates by size exclusion chromatography. The isolated platelets were then activated by thrombin and collagen co-stimulation (TC) and by Ca2+ionophore, respectively. The platelet activation produced extracellular vesicles (PEVs) which were isolated by differential ultracentrifugation. The isolated PEVs were then analysed by flow cytometry, ELISA and Western blot for EV typical membrane surface proteins and for their BDNF content. As we were interested finding out whether BDNF is enriched in PEVs to certain populations, density gradient centrifugation was performed. These samples were also analysed by Western blot and by ELISA. The size distribution and concentration of PEVs in all samples was analysed by Nanoparticle tracking analysis. Results and conclusions. This study confirmed that platelets secrete PEVs as a response to agonists. PEVs with higher BDNF concentration were produced using TC co-stimulation as compared to PEVs derived from the Ca2+ionophore. The result implies that BDNF is actively packed into PEVs for instance as a thrombogenic response. Based on the density gradient results it seems that BDNF was packed into certain population of PEVs with a density between 1.112 g ml-1 and 1.132 g ml-1 corresponding to a particle diameter of less than 500 nm. The finding that BDNF is actively packed into TC co-stimulation derived PEVs of a certain population is interesting from a theragnostic point of view, since EVs are likely to be key players in the development of new cell-based therapies. Had there been more time, it would have been interesting to optimize both the density gradient protocol and the ELISA analysis. This optimization of methods would make the process more efficient, less prone to sample loss, not to mention that there would be less intra-assay variation.
  • Clayton, Aled; Boilard, Eric; Buzas, Edit I; Cheng, Lesley; Falcón-Perez, Juan Manual; Gardiner, Chris; Gustafson, Dakota; Gualerzi, Alice; Hendrix, An; Hoffman, Andrew; Jones, Jennifer; Lässer, Cecilia; Lawson, Charlotte; Lenassi, Metka; Nazarenko, Irina; O’Driscoll, Lorraine; Pink, Ryan; Siljander, Pia R-M; Soekmadji, Carolina; Wauben, Marca; Welsh, Joshua A; Witwer, Ken; Zheng, Lei; Nieuwland, Rienk (2019)
    There is an increasing interest in exploring clinically relevant information that is present in body fluids, and extracellular vesicles (EVs) are intrinsic components of body fluids (?liquid biopsies?). In this report, we will focus on blood. Blood contains not only EVs but also cells, and non-EV particles including lipoproteins. Due to the high concentration of soluble proteins and lipoproteins, blood, plasma and serum have a high viscosity and density, which hampers the concentration, isolation and detection of EVs. Because most if not all studies on EVs are single-centre studies, their clinical relevance remains limited. Therefore, there is an urgent need to improve standardization and reproducibility of EV research. As a first step, the International Society on Extracellular Vesicles organized a biomarker workshop in Birmingham (UK) in November 2017, and during that workshop several working groups were created to focus on a particular body fluid. This report is the first output of the blood EV work group and is based on responses by work group members to a questionnaire in order to discover the contours of a roadmap. From the answers it is clear that most respondents are in favour of evidence-based research, education, quality control procedures, and physical models to improve our understanding and comparison of concentration, isolation and detection methods. Since blood is such a complex body fluid, we assume that the outcome of the survey may also be valuable for exploring body fluids other than blood.
  • Nurmi, Katariina; Niemi, Katri; Kareinen, Ilona; Silventoinen, Kristiina; Lorey, Martina B; Chen, Yan; Kouri, Vesa‐Petteri; Parantainen, Jukka; Juutilainen, Timo; Öörni, Katariina; Kovanen, Petri T; Nordström, Dan; Matikainen, Sampsa; Eklund, Kari K (2021)
    Objectives The NLRP3 inflammasome plays a key role in arterial wall inflammation. In this study, we elucidated the role of serum lipoproteins in the regulation of NLRP3 inflammasome activation by serum amyloid A (SAA) and other inflammasome activators. Methods The effect of lipoproteins on the NLRP3 inflammasome activation was studied in primary human macrophages and THP-1 macrophages. The effect of oxidised low-density lipoprotein (LDL) was examined in an in vivo mouse model of SAA-induced peritoneal inflammation. Results Native and oxidised high-density lipoproteins (HDL3) and LDLs inhibited the interaction of SAA with TLR4. HDL3 and LDL inhibited the secretion of interleukin (IL)-1 beta and tumor necrosis factor by reducing their transcription. Oxidised forms of these lipoproteins reduced the secretion of mature IL-1 beta also by inhibiting the activation of NLRP3 inflammasome induced by SAA, ATP, nigericin and monosodium urate crystals. Specifically, oxidised LDL was found to inhibit the inflammasome complex formation. No cellular uptake of lipoproteins was required, nor intact lipoprotein particles for the inhibitory effect, as the lipid fraction of oxidised LDL was sufficient. The inhibition of NLRP3 inflammasome activation by oxidised LDL was partially dependent on autophagy. Finally, oxidised LDL inhibited the SAA-induced peritoneal inflammation and IL-1 beta secretion in vivo. Conclusions These findings reveal that both HDL3 and LDL inhibit the proinflammatory activity of SAA and this inhibition is further enhanced by lipoprotein oxidation. Thus, lipoproteins possess major anti-inflammatory functions that hinder the NLRP3 inflammasome-activating signals, particularly those exerted by SAA, which has important implications in the pathogenesis of cardiovascular diseases.