Laboratory of Cardiovascular Genomics
Dimitris Kardassis, PhD
Atherosclerotic Cardiovascular Disease (ASCVD) is the leading cause of death worldwide. Several risk factors including obesity, type II diabetes, non-alcoholic fatty liver disease and dyslipidemia predispose to ASCVD by mechanisms that are not fully understood. Reliable genetic or non-genetic biomarkers are also needed in order to increase the value of current risk prediction algorithms. In our lab we are studying the genetic determinants of atherosclerosis using animal models combined with virus-mediated gene transfer and omics technologies with the goal to understand better the pathogenesis of atherosclerosis and to identify novel biomarkers and drug targets for ASCVD.
1. Mechanisms that control the transcription of key genes of HDL metabolism
We have been studying the mechanisms that control the transcription of the human apolipoprotein genes (the protein components of lipoproteins) since 1990 and we have pioneered the elucidation of the roles of hormone nuclear receptors such as HNF-4 and LXRs in human lipoprotein metabolism. During the past decade we have focused our attention on the regulation of genes involved in High Density Lipoprotein (HDL) metabolism. One of these genes is apoM that plays a critical role in HDL remodeling and is also the carrier of the bioactive lipid sphingosine 1 phosphate (S1P) in the serum. We showed that in hepatocytes apoM is regulated by the cooperative action of hepatocyte nuclear factors 4 (HNF-4) and HNF-1 which bind to adjacent sites in the proximal apoM promoter. We showed that the HNF-4 binding site is a novel hormone response element (HRE) that is shared by many nuclear receptors such as LXRs, PPARs, RXRs and T3Rs. We also showed that during inflammation the plasma levels of apoM are reduced and we elucidated the mechanism that accounts for this reduction which involves a competition between HNF-1 and the pro-inflammatory transcription factor c-Jun for the same binding site.
|Regulation of the human apoM gene by hormone nuclear receptors, HNF-1 and Jun proteins in hepatic cells. From: Mosialou et al. (2010) J Biol Chem. 285(40):30719-30; Mosialou et al (2011) J Biol Chem. 286(19):17259-69|
The gene encoding the membrane transporter ABCA1 is essential for HDL biogenesis. Mutations in the ABCA1 gene case Tangier’s disease which is characterized by the absence of HDL particles ad premature atherosclerosis. We showed that the transcription factor FOXA2 (HNF-3β) inhibits the basal and the LXR-inducible activity of the human ABCA1 promoter by a novel mechanism which involves binding of FOXA2 to the TATA box and inhibiting the recruitment of the basal transcription machinery.
|Proposed mechanism of transcriptional repression of the gene encoding the lipid transporter ABCA1 in hepatic cells by the transcription factor FOXA2 (HNF-3β). From Thymiakou and Kardassis (2014) Biochim Biophys Acta Gene Regul Mech. 1839(6):526-36|
We studied the regulation of the human lipoprotein lipase (LPL) gene which is essential for the hydrolysis of triglycerides (TG) from TG-rich lipoproteins Mutations in LPL gene cause hypertriglyceridemia and acute pancreatitis. LPL is not normally expressed in the liver but its hepatic expression is strongly increased by the accumulation of cholesterol in the liver (high fat diet or synthetic LXR ligands) that causes insulin resistance. We found that basal hepatic LPL activity is controlled by FOXA2 via binding to a novel FOXA2 binding element in the proximal LPL promoter. We also showed that oxysterols induce LPL gene expression via physical and functional interactions between LXRs and FOXA2 without the requirement for direct binding of LXR to the DNA.
|Proposed mechanism of transcriptional regulation of the gene encoding human lipoprotein lipase (LPL) in hepatic cells by oxysterols and insulin via transcription factors LXR and FOXA2 respectively. From: Kanaki and Kardassis (2017) Biochim Biophys Acta Gene Regul Mech. 1860(3):327-336; Kanaki, et al (2017) Biochim Biophys Acta Gene Regul Mech. 1860(8):848-860|
Our work and of others have established the critical roles of the orphan nuclear receptor Hepatocyte Nuclear Factor 4A (HNF4A) in physiological liver functions including human lipoprotein metabolism. In order to investigate further the role of HNF4A in HDL metabolism in vivo, we generated and characterized mice with liver-specific ablation of Hepatocyte Nuclear Factor 4A (H4LivKO and H4IntKO). We found that H4LivKO mice are characterized by increased liver triglyceride content and decreased concentration of serum total cholesterol, HDL cholesterol, triglycerides, phospholipids and cholesteryl esters. H4LivKO serum was enriched in the smaller, denser HDL particles exhibiting decreased activity of paraoxonase-1 but retaining macrophage cholesterol efflux capacity and phospho-AKT activation in endothelial cells. Global gene expression analysis revealed the association of liver HNF4A with known and novel regulators of HDL metabolism as well as NAFLD-susceptibility genes.
|Schematic representation of the major defects in lipoprotein metabolism due to the ablation of the Hnf4α gene in the liver. From: Thymiakou et al (2020) Metabolism. 110:154307|
2. Identification of gene signatures associated with the progression of the metabolic syndrome in mice before and after treatment with RYGB surgery
The metabolic syndrome (MetS) is a cluster of clinical disorders such as dyslipidemia, diabetes and obesity which are associated with increased risk for cardiovascular disease. We have participated in a European consortium called RESOLVE (http://www.resolve-diabetes.org/) funded by fp7 with the objective to understand the pathophysiology of MetS and to use system biology approaches to create a computer model that will predict MetS pathogenesis based on lipid and lipoprotein fluxes and kinetic of gene expression changes in metabolic organs. For this purpose we have been using the established mouse model of MetS apoE3L.CETP. We have completed a diet intervention study in male apoE3L.CETP mice fed either a High (HFD) or a Low (LFD) Fat Diet for 4, 8 and 12 weeks. The liver and white adipose RNA was analyzed on Affymetrix Mouse Gene 2.0 ST arrays followed by bioinformatics. Microarray analysis revealed differentially expressed transcripts at 4, 8 and 12 weeks of HFD compared to LFD which were classified in biological processes including insulin resistance, hepatic steatosis, glucose and lipid metabolism. In the adipose tissue, pathway analysis revealed that the differentially expressed transcripts were clustered in networks associated with inflammatory/immunological responses and also in cell growth/proliferation and metabolic diseases indicative of enhanced adipose tissue inflammation and metabolic dysfunction. We have also collaborated with the group of T. Lutz (Zurich) to study the transcriptomic changes in the livers of obese apoE3L.CETP mice following Roux-en-Y gastric bypass surgery.
|Differential gene expression analysis comparing HFD vs LFD groups of apoE3L.CETP mice at different time points (4, 8 and 12 weeks). (A) Venn diagram (B) Heatmap. From: Nasias et al. (2019) J Cell Physiol. 234(11):20485-20500|
3. Utilization of animal models combined with adenovirus-mediated gene transfer to functionally characterize mutations in genes of HDL metabolism
In our lab, we have used adenovirus-mediated gene transfer and transgenic technology to study the effects of mutations in proteins of HDL metabolism on HDL structure and functions in vivo. With this approach we showed for the first time that mutations in apoA-I can predict increased risk of ischemic heart disease and total mortality without causing a reduction in HDL cholesterol levels. We also showed that deletion of regions 89-99, 218-222 and 225-230 of apoA-I affected the structure and the stability of apoA-I and inhibited the interaction of apoA-I with lipids or with proteins of HDL biogenesis such as ABCA1 and LCAT resulting in hypertriglyceridemia and low plasma HDL levels. Importantly, these clinical abnormalities could be corrected by co-infecting the mice with AVs expressing LCAT or LPL suggesting novel approaches to the treatment of HDL deficiencies. We also generated transgenic mice expressing the natural mutations in apoA-I L141RPisa and L159RFIN and showed that these mutations affected the biogenesis and the functionality of HDL in vivo and predisposed to diet-induced atherosclerosis in the absence of any other genetic defect.
|Diet-induced atherosclerosis in transgenic mice expressing WT or mutant forms of human apoA-I. From: Tiniakou et al. (2015) Atherosclerosis 243(1):77-85|
4. Genetics studies
We identified by next generation sequencing and characterized exonic single nucleotide polymorphisms (SNPs) in 10 genes of HDL metabolism in a Greek cohort with extreme HDL cholesterol levels. For selected amino acid changes we performed computer-aided structural analysis and modeling. A previously uncharacterized rare apolipoprotein A-IV variant, apoA-IV [V336M], present in a subject with low HDL-C (14 mg/dL) and CAD, was expressed in recombinant form and structurally and functionally characterized. ApoA-IV [V336M] displayed thermodynamic stabilization, was able to associate with phospholipids but presented reduced kinetics compared to WT apoA-IV.
|A. 3D ribbon representation of ApoA-IV. The position of Ser147 and Asn147 are shown in red. The position of Val336, not present in the experimental structure, is also shown. Close-up on the interaction of the B. Ser147 and C. Asn147 with the neighboring Arg304 and Glu308 residues. Possible hydrogen bonds are shown with yellow dashed lines. From: Chroni et al. (2020) Arch Biochem Biophys. 696:108655|
5. The atheoprotective mechanisms of HDL and how they are compromised in chronic inflammation
HDL has numerous atheroprotective functions which are facilitated by the interaction of apoA-I with cell surface receptors such as SR-BI or ABCA1 in HDL target cells and the triggering of intracellular signaling cascades leading to changes in the activation status of signaling mediators and the regulation of the expression of target genes which are to a very large extent unknown. One of the goals of our team was to study the role of HDL in chronic inflammatory diseases such as rheumatoid arthritis (RA). In collaboration with P. Verginis (BRFAA, Athens) we used a protocol for antigen-induced RA and showed that apoA-I KO mice that lack conventional HDL developed severe arthritis and had elevated Th1 and Th17 cell responses. We showed that HDL exerts its anti-inflammatory properties through modulation of dendritic cell (DC) maturation and function and that HDL-exposed DCs suppress T cell proliferation in vitro. Our findings support a critical role of SR-BI and ABCA1 transporters in the rHDL-mediated anti-inflammatory function of DCs.
|ApoA-I deficiency in mice results in a more severe phenotype of Antigen Induced Arthritis. From: Tiniakou et al (2015) J Immunol. 194(10): 4676-87|
Using gene expression arrays, we showed that treatment of human coronary artery endothelial cells with rHDL-apoA-I was associated with significant changes in their transcriptome and the bioinformatics analysis identified angiopoietin like 4 (ANGPTL4) as one of the most highly upregulated and biologically relevant molecules. Using specific chemical inhibitors we showed that kinases PI3K and AKT and the transcription factor FOXO1 mediate the transcriptional activation of the ANGPTL4 gene by HDL.
|Proposed mechanism for the regulation of the expression of the human ANGPTL4 gene by HDL in endothelial cells. From: Theofilatos et al. (2018) Metabolism. 87:36-47|