Browsing by Subject "atherosclerosis"

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.