Microvesicle signaling in cardiovascular biology under normal and pathobiological conditions

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

Increased expression of miR-224-5p in circulating extracellular vesicles of patients with reduced coronary flow reserve

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

P2Y2 receptor modulates shear stress-induced cell alignment and actin stress fibers in human umbilical vein endothelial cells

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