Browsing by Subject "extracellular vesicle"

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.

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.

Platelets originate from megakaryocytes and therefore contain the same receptors. This also applies to the extracellular vesicles (EVs) they release into the bloodstream. Glycoprotein VI (GPVI) is an activating collagen receptor on platelets. It plays an essential role in platelet biology by binding to collagen and activating platelets, leading to generation of EVs. Regulation of hemostasis involves shedding of GPVI from activated platelets, leading to a soluble fragment of GPVI. Soluble GPVI is used as a biomarker for diseases. According to current literature, GPVI is present on megakaryocyte-derived EVs but not on platelet-derived EVs (pEVs), as it is considered that activation of platelets leads to proteolytic cleavage of GPVI. Research on the presence of GPVI on pEVs is so far limited and the results are inconsistent. Based on alternate finding on the presence of GPVI on pEVs using proteomics (Palviainen et al. 2024), the aims of this project were to investigate the presence of GPVI on pEVs and to compare the presence of GPVI on megakaryocyte-derived EVs and pEVs. The presence of GPVI on pEVs was investigated by using multiple set of samples which could express GPVI differently. Platelets from platelet concentrate were isolated, activated, removed after activation and the samples were analysed with flow cytometry. Isolated pEVs were analysed with dot blot and western blot. To obtain megakaryocyte-derived EVs, K562 cell line was differentiated to megakaryocyte-like cells and EVs were isolated from cell conditioned media. The presence of GPVI on pEVs and megakaryocyte-derived EVs was compared with western blot. GPVI was found on pEVs. However, an expected difference in the presence of GPVI between pEVs from activated and unstimulated platelets was not observed. The results also indicated a higher amount of GPVI in megakaryocyte-derived EVs compared to pEVs, but further optimization of the methods used is required for more reliable results. GPVI, previously thought to exist only on megakaryocyte-derived EVs in the circulation or in soluble form cleaved from activated platelets, may actually be present on pEVs. Distinguishing the presence of GPVI between megakaryocyte-derived EVs and pEVs, is relevant when using the receptor as a biomarker. The results of this study are a foundation for further investigation of GPVI on pEVs to elucidate this exciting discrepancy.