Supervisors info:
Σανούδου Δέσποινα, Αναπλ. Καθηγήτρια, Δ' Παθολογική Κλινική, Ιατρική Σχολή, Εθνικό και Καποδιστριακό Πανεπιστήμιο Αθηνών.
Λιανίδου Ευρύκλεια, Καθηγήτρια Αναλυτικής Χημείας και Κλινικής Χημείας, Τμήμα Χημείας, Σχολή Θετικών Επιστημών, Εθνικό και Καποδιστριακό Πανεπιστήμιο Αθηνών.
Χρόνη Αγγελική, Ερευνήτρια Α', Ινστιτούτο Βιοεπιστημών και Εφαρμογών ΕΚΕΦΕ "Δημόκριτος".
Original Title:
Αξιολόγηση μοριακής δράσης ανασυσταμένης HDL, που περιέχει ανθρώπινες απολιποπρωτεΐνες apoA-I ή apoE3 αγρίου τύπου, στα ανθρώπινα ενδοθηλιακά κύτταρα αορτής και ειδικότερα στην έκφραση miRNAs
Summary:
Despite atherosclerosis being the leading cause of cardiovascular disease worldwide, there is no available treatment without important effectivity and/or safety issues. Damage of the endothelial monolayer integrity is an important initiating event in the pathogenesis of atherosclerosis; however increased endothelial cell (EC) proliferation and migration can restore its integrity following vascular injury. Furthermore, the maintenance of cholesterol homeostasis is essential for EC function and its perturbations can contribute to the emergence of several pathologies. Although it has been shown that reconstituted HDL (rHDL) administration is atheroprotective, partly due to the increased reverse cholesterol transport it mediates and a previous study of our group has studied the effects of rHDL particles containing either apoA-I or apoE3 on EC gene expression, their effect on miRNA expression are still unknown. The aim of this study was to identify potential atheroprotective effects of discoid rHDL particles containing human wild type apoA-I or apoE3 and phospholipids and characterize the potential roles of miRNAs in the mechanisms by which these could be exercised. We studied normal human aortic ECs after exposure to the two types of rHDL, using oligonucleotide miRNA expression microarrays (GeneChip™ miRNA 2.0 Array Affymetrix). Multilevel bioinformatical analysis using 1.5-fold and ≤ 0.05 false discovery rate thresholds identified 8 significantly changed miRNAs: miR-3188, miR-3185, miR-1231, miR-3195, miR-2861, miR-1915 and miR-638 were under-expressed after rHDL-apoA-I treatment and so was miR-503 after the rHDL-apoE3 one. There were no significantly changed miRNAs over-expressed. Next we identified the experimentally validated and predicted targets for each one of the 8 miRNAs. Ingenuity Pathway Analysis indicated that both the miRNAs and their targets affect several cellular functions, some of which could affect EC function and atherosclerosis. The in depth functional characterization of the predicted target genes of miR-3185 and miR-2861, revealed that some of them are key cholesterol biosynthesis genes, while the targets of miR-503 include key genes regulating angiogenesis. PubMed analysis showed that miR-503 is an important regulator of angiogenesis; its over-expression inhibits it by reducing EC proliferation and migration, while its inhibition has the opposite effects. Furthermore, genes with significant effects on EC proliferation (EFNB2, FLT1, NR4A1, and ADAMTS4) and migration (EFNB2, FLT1, PTGIR and COL4A1) can be found among its validated and predicted target genes. Combination of the above with data from the preceding gene expression study, allows us to make the assumption that rHDL-apoE3 can theoretically induce EC proliferation through an rHDL-apoE3 – miR-503 expression reduction – target mRNA increase, axis. Although, there are no reports indicating that miR-3185 and miR-2861 have a part in cholesterol metabolism regulation so far, several genes participating in almost every part of it, could be found among their predicted targets. Of these, HMGCR, MVK and CYP51A1 code for enzymes that directly participate in the cholesterol biosynthesis pathway. Again, combination of the above with data from the preceding gene expression study suggest the existence of an axis were rHDL-apoA-I itself or the cholesterol efflux it causes, down-regulates miR-
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3185 and miR -2861, up-regulating HMGCR, MVK and CYP51A1 expression leading to an increase in cholesterol biosynthesis. In conclusion the present study is the first to examine the effects of rHDL-apoA-I and rHDL-apoE3 on EC miRNA expression, as well as the role of the affected miRNAs in mediating the probable atheroprotective effects of rHDL on ECs. Through this study we found that miR-503 has the potential to induce the proliferation and migration of ECs, contributing to the restoration of endothelial integrity, whereas miR-3185 and miR-2861 could potentially induce cholesterol biosynthesis in a controlled manner, contributing to the maintenance of EC cholesterol homeostasis. Through these effects, miR-3185, miR-2861 and miR-503 could mediate potentially atheroprotective rHDL-apoA-I and rHDL-apoE3 functions. This makes rHDL in general and these miRNAs in particular possible therapeutic targets worth exploring more deeply, since the deeper understanding of their effects on the endothelium at the cellular and molecular level promises to facilitate the development of new therapies to prevent from or treat atherosclerosis.
Keywords:
Atherosclerosis, rΗDL-apoA-I, rHDL-apoE3, miRNAs, endothelial cells, proliferation & cholesterol biosynthesis.
