Browsing by Subject "Extracellular vesicles"

Although lung transplantation has become a routine procedure and is optimal therapy for patients with end-stage pulmonary diseases, the lifespan of lung allografts is still shorter than that of other organ transplants. As acute allograft rejection is one of the main risk factors for the development of chronic lung allograft dysfunction (CLAD) which threatens the long-term survival rate of the recipients, it is crucial to predict and diagnose acute lung allograft rejection. However, there are no specific methods established so far to predict acute rejection (AR). Even though the histopathological evaluation of transbronchial biopsies (TBBs) is used as the gold standard to ensure the diagnosis of AR, it is essential to discover novel biomarkers for AR to overcome the limitations of the TBB-based invasive diagnostics. Recently extracellular vesicles (EVs) got noticed as potential biomarkers in various fields of medicine based on the findings that they exist in high concentration in body fluids and deliver functional genetic molecules which can modulate gene expression in target cells. In that regard, this preliminary study was designed with two different approaches; a time-point analysis and a case analysis of rejection and non-rejection episodes to validate their potentials as diagnostic and predictive biomarkers for acute lung allograft rejection. To discover biomarkers, EV RNA was isolated from the plasma of four patients that was collected at different time points, and whole EV mRNA transcriptome sequencing was performed on the Illumina platform to obtain at least 15 million reads. The time-point analysis showed that the mRNA contents of EVs changed according to the time points and clinical presentations of the patients. At the same time, gene expression profiles showed that mRNA molecules inside the EVs change from innate immunity to adaptive immunity related signatures with the time after transplantation. Furthermore, the case analysis identified that EVs contain RNA molecules that are closely related to the migration of leukocytes and adaptive immune system during acute rejection episodes. In conclusion, the profiles of EV RNA may reflect the immune responses that are taking place in the recipient’s body. Therefore, it is speculated that EVs may play an essential role in the development of AR by transferring functional mRNA molecules to the allograft, immune cells, and endothelial cells. On that account, EV transcriptome profiling could be used as a diagnostic tool for AR in the future, as well as a therapeutic tool by engineering EVs to target specific genes that may be involved in the development of AR. Keywords: extracellular vesicles, lung transplantation, transplantation immunology, RNA sequencing, acute lung allograft rejection, biomarkers

Atherosclerosis is a cardiovascular disease characterized by the formation and growth of plaque within the arteries. Lipoproteins, especially LDL, initiate atherosclerosis by accumulating in the intima of arteries and becoming modified, e.g. oxidised. Oxidised LDL (OxLDL) is highly pro-atherogenic and promotes atherosclerosis in multiple ways. The role of platelets in the later stages of atherosclerosis is well-documented, but platelets may also be involved in earlier stages of atherosclerosis. Platelets release extracellular vesicles (PEVs) in the form of microvesicles (microparticles) and exosomes that participate in intercellular signalling and in similar pathophysiological processes as platelets. Lipoproteins are known to activate platelets but their effects on PEV formation have not yet been studied. The aim of this thesis was to investigate the effect of OxLDL on PEV formation and compare it to other potential agonists such as LDL, HDL, ATP, thrombin and collagen. Platelets were activated with these agonists separately or in combination with OxLDL. PEVs were studied from the platelet-depleted supernatant and the isolate, which was obtained by differential centrifugation. PEVs were quantified in terms of CD61+ PEVs and particle count by flow cytometry and nanoparticle tracking analysis, respectively. PEVs were characterized by the relative amounts of CD41 (platelet and PEV marker) and Hsp70 (general EV marker) detected by Western blotting. Lastly, the uptake of the differently induced PEVs by HepG2 hepatoma cells was compared by fluorescence microscopy as a characterization of the PEVs’ functionality. Among the lipoproteins, OxLDL was indicated to be a much more potent inducer of PEVs than LDL or HDL, as shown by flow cytometry of CD61+ PEVs, nanoparticle tracking analysis and CD41 and Hsp70 levels in the isolates. However, OxLDL was not as strong a PEV inducer as the co-stimulation with thrombin and collagen (T&C), which induced the highest PEV formation. Size distribution analysis showed that PEVs smaller than 100 nm in size comprised a larger proportion of the total PEVs in OxLDL-induced PEVs compared to LDL- and T&C-induced PEVs. OxLDL combined with weak PEV inducers such as HDL and ATP had an amplifying effect on the generation of CD61+ PEVs, while the highest PEV formation was observed when OxLDL was combined with thrombin and collagen. When OxLDL-induced PEV formation was tested against a range of HDL concentrations, the extent of PEV formation and relative Hsp70 levels both decreased in a HDL concentration-dependent manner up to 50 µg/mL HDL. Both LDL- and OxLDL-induced PEVs were taken up by HepG2 cells, but there was no statistically significant difference between the two. The results indicated the potency of OxLDL in inducing PEV formation, thereby suggesting a novel mechanism by which OxLDL could contribute to the progression of atherosclerosis. Further studies on OxLDL-induced PEVs are needed, but if significant lipoprotein-specific changes in PEV numbers and properties could be observed, PEVs could then be used as a biomarker to diagnose atherosclerosis already at the early stages.