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Doctoral theses

Current Record: 6 of 2013

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Identifier 000425467
Title Study the signaling cascades and the atheroprotective properties of HDL in endothelial cells
Alternative Title Μελέτη των μηχανισμών μοριακής σηματοδότησης και των αθηροπροστατευτικών δράσεων της HDL στα ενδοθηλιακά κύτταρα
Author Θεοφιλάτος, Δημήτρης
Thesis advisor Καρδάσης, Δημήτριος
Reviewer Τσατσάνης, Χρήστος
Σανούδου, Δέσποινα
Abstract Numerous epidemiological studies have shown that low levels of High Density Lipoprotein Cholesterol (HDL-C) in the serum are a strong and independent risk factor for the development of Cardiovascular Diseases (CVDs). Furthermore, a great number of reports have revealed that HDL exerts pleiotropic functions protecting against the development of atherosclerosis, which is the leading cause of CVDs. However, drugs that were designed to increase HDL-C failed to counteract CVD risk in human clinical studies. In addition, recent studies showed that HDL isolated from patients with CVDs is “dysfunctional” and it is unable to exert its known anti-atherosclerotic functions. These findings indicate that only by raising HDL-C levels is not sufficient for atheroprotection, but it must be accompanied by the presence of functional HDL. Also, it should be mentioned that some drugs aiming to raise HDL-C levels caused significant side-effects, highlighting the complexity of the metabolic pathways as well as their poor understanding. The main goals of this PhD thesis were:  Chapter 1: To investigate the molecular mechanisms underlying the atheroprotective roles of HDL in the endothelium. For this purpose, in parts I and II, we focused on identifying genes targeted by HDL in endothelial cells as well as the mechanisms through which HDL regulates their expression, while in part III we studied the functionality of HDL isolated from patients with a chronic inflammatory disease (Ankylosing Spondylitis, AS).  Chapter 2: To identify novel transcription factors and miRNAs in the liver that control the expression of genes involved in the biogenesis of HDL. In part IV we studied the regulation of the transcription factor LXRα by the master hepatic transcription factor HNF-4α, while in part V we studied the role of the miRNA let-7b in the regulation of the expression of apolipoprotein E (apoE) gene. Endothelium injury or ‘dysfunctional’ endothelium has been associated with the initiation and the progression of the atherosclerotic plaque. HDL not only is a positive regulator of the physiological function of endothelium, but also protects endothelium against injury, while dysfunctional HDL is unable to promote these actions. For this purpose, in Chapter 1, we wanted to elucidate the molecular mechanisms underlying HDL functionality in endothelium. The genes that we focused on in the first two parts were derived from microarray data, which had been generated previously by our research group following the treatment of endothelial cells with reconstituted HDL containing the human apolipoprotein A-I (rHDL-AI). We studied the expression of these genes after treatment with natural HDL, which was isolated from transgenic mice expressing the human apoA-I gene (tgHDL). Specifically, in part I we studied the regulation of the Angiopoietin-like 4 (ANGPTL4) gene, which plays an important role in lipid metabolism and atherosclerosis. We observed that both natural HDL (tgHDL) and reconstituted HDL (rHDL-AI) were able to induce the expression of ANGPTL4. To investigate the molecular mechanisms through which tgHDL activates the expression of this gene, we utilized known inhibitors for signaling cascades and we observed that both the AKT and the p38-MAPK pathways are involved in this regulatory effect of tgHDL. Furthermore, by utilizing the separation of nuclear from cytoplasmic protein extracts technique as well as immunofluorescence, we revealed that tgHDL, through the AKT pathway, promotes the phosphorylation of the nuclear transcription factor Forkhead Box O1 (FOXO1) resulting in its translocation to the cytoplasm and, subsequently, its inactivation. By combining different experimental approaches to inactivate FOXO1 in endothelial cells followed by treatment with tgHDL, we identified that FOXO1 suppresses the expression of ANGPTL4, while the FOXO1-mediated inactivation by HDL results in the induction of ANGPTL4. In part II, we focused on the expression of the endothelial lipase (LIPG) gene, which mediates HDL catabolism. Initially, we observed that natural HDL (tgHDL) inhibited the expression of LIPG in contrast to reconstituted HDL (rHDL-AI) which induced its expression. We focused on the effect of tgHDL and we found that not only tgHDL inhibited the expression of LIPG but also prevented the induction of LIPG by growth deprivation (starvation). Silencing the expression of FOXO1 by utilizing a specific siRNA led to reduction of the LIPG mRNA levels indicating that FOXO1 is an important transcriptional activator of this gene in starvation conditions. TgHDL, by promoting FOXO1 transcriptional inactivation, inhibits LIPG expression. In addition to FOXO1 inactivation, tgHDL may inhibit the expression of an additional gene – still uncharacterized- in order to prevent LIPG-mediated induction by starvation. In part III we studied the functionality of HDL isolated from patients with Ankylosing Spondylitis (AS), a chronic inflammatory disease. We reported that HDL isolated from healthy people was able to phosphorylate AKT kinase, while HDL isolated from patients with AS showed reduced AKT phosphorylation indicating the existence of dysfunctional HDL in AS patients. Although HDL is synthesized extracellularly, liver is the main source of proteins participating in HDL biogenesis. Therefore, in Chapter 2 we used the human liver cell line, HepG2, in order to identify the mechanisms underlying the expression of genes involved in HDL metabolism. In part IV we studied the regulation of the transcription factor LXRα in the liver. LXRα (Liver X Receptor α) is a key regulator of lipid and cholesterol metabolism by controlling directly the expression of genes participating in these pathways (for instance ABCA1, ABCG1, SREBP etc.). We observed that the promoter of human LXRα gene (hLXRa) is activated by HNF-4α, a transcription factor expressed in the liver which regulates the expression of several liver-specific genes. By performing ChiP and DNAP analysis, we identified the binding site of HNF4a in the proximal region of the hLXRa promoter and specifically in the region from -50 to -40. Disruption of this site in the hLXRα promoter with site-specific mutagenesis abolished the transactivation by HNF-4α. Moreover, silencing of HNF-4α in HepG2 resulted in reduction of LXRα protein levels. In part V, we studied the ability of the micro-RNA let-7b to regulate the expression of apolipoprotein E (apoE) gene in HepG2 cells. ApoE is a crucial apolipoprotein since it not only mediates the clearance of triglyceride-rich lipoproteins from the circulation, but also participates in the biogenesis of HDL. Overexpression of let-7b in HepG2 caused a reduction in both mRNA and protein levels of apoE. However, let-7b overexpression did not affect the luciferase activity of a plasmid containing the 3’-UTR region of the mRNA of apoE. This observation indicates that let-7b does not regulate directly, though the 3’-UTR region, the expression of apoE. Furthermore, co-expression of a plasmid containing the (-500/+73) promoter of apoE with let-7b led to reduced luciferase activation confirming the indirect effect of let-7b in the regulation of apoE expression. Interestingly, we identified that let-7b also regulates the expression of apoA-I, the main protein of HDL. We found that let-7b overexpression resulted in a reduction of the mRNA levels of apoA-I as well as in the luciferase activity of a plasmid containing the (-1020/-24)-apoA-I promoter. Bioinformatic analysis in the mRNA region of the human apoA-I gene did not reveal any putative let-7b binding sites suggesting that apoA-I, similar to apoE, is regulated by let-7b independently of the 3’-UTR region. In conclusion, the findings from this PhD thesis provide new insights into the mechanisms that control HDL serum levels as well as its functionality. Genes found in the present study to mediate HDL functions in endothelial and liver cells may be of great diagnostic and/or therapeutic value in patients with low HDL or dysfunctional HDL such as patients with CVDs or chronic inflammatory diseases.
Language English
Subject ANGPTL4
Ankylosing spondylitis
Apolipoprotein A-I
Cardiovascular diseases
Gene regulation
Transcription factors
Αγκυλοποιητική σπονδυλίτιδα
Απολιποπρωτεϊνη Α-Ι
Απολιποπρωτεϊνη Ε
Γονιδιακή ρύθμιση
Ενδοθηλιακή λιπάση
Καρδιαγγειακά νοσήματα
Μεταγραφικοί παράγοντες
Μικρά μη κωδικοποιητικά RNA
Ομοιάζουσα με αγγειοποιητίνη Πρωτεϊνη 4
Σηματοδοτικά μονοπάτια
Υψηλής πυκνότητας λιποπρωτεϊνη
Issue date 2019-12-11
Collection   Faculty/Department--School of Medicine--Department of Medicine--Doctoral theses
  Type of Work--Doctoral theses
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