Background and Aims

Increased lipoprotein receptor expression in the heart was associated with lipid accumulation, cardiomyocyte mitochondrial impairment and cardiac dysfunction. This project aimed at investigating whether PCSK9 regulates lipoprotein receptors expression in the heart, thus modulating heart metabolism and cardiac function.

Methods

Pcsk9 KO, Albumin CRE PCSK9LoxP/LoxP conditional KO) and double LDLr-Pcsk9 KO male mice undewent an extensive characterization for heart function followed by heart metabolomics and proteomic profiling.

Results

Pcsk9 KO hearts presented heart failure with preserved ejection fraction, reduced running resistance without muscular defects coupled to major adaptations in cardiac metabolism and mitochondrial functionality. This profile was associated with lipid accumulation in the heart. Moreover PCSK9 deficiency resulted in the accumulation of carnitine-conjugated fatty acid (C8; 0,066±0,047 Vs0,195±0,035 and C12; 0,021±0,018Vs0,11±0,069 pg/ug of prot, p<0,05) which was paralleled by a reduction of glucose 6-P, ribose-5P and erythrose-4P levels in the heart, suggesting a shift from fatty acid oxidation toward anaerobic glycolysis. Also Krebs cycle intermediates were reduced compared to WT samples. A similar phenotype was observed in LDLr Double KO confirming an effect independent of LDLr expression. The cardiac phenotype was reverted in the liver selective KO model thus excluding the involvement of circulating PCSK9 in the development of HFpEF observed in full KO mice. Translational studies showed that human subjects carrying the R46L loss of function polymorphism displayed increased left ventricular mass without alterations in ejection fraction compared to R46R BMI-matched controls.

Conclusions

These data suggest that PCSK9 deficiency impacts cardiac lipid metabolism indepedently of LDLR modulation and promotes the development of HFpEF.

Background and Aims

Nonalcoholic steatohepatitis (NASH) is characterized by hepatic steatosis with inflammation and fibrosis. Membrane endoglin (Eng) is shown to participate in fibrosis, and plasma concentrations of soluble endoglin (sEng) are increased in patients with hypercholesterolemia and type 2 diabetes mellitus. We hypothesize that NASH increases both hepatic Eng expression and sEng in blood, and that high levels of sEng modulate cholesterol and bile acid (BA) metabolism and affect NASH progression.

Methods

Three-month-old transgenic male mice overexpressing human sEng and their wild type littermates are fed for six months with either a high-saturated fat, high-fructose high-cholesterol (FFC) diet or a chow diet. Evaluation of NASH, LC-MS analysis of BA, hepatic expression of Eng, inflammation, fibrosis markers, enzymes and transporters involved in hepatic cholesterol and BA metabolism are assessed using qRT-PCR and Western blot.

Results

The FFC diet significantly increased mouse sEng levels and hepatic expression of Eng. High levels of human sEng resulted in increased hepatic deposition of cholesterol due to reduced conversion into BA, as well as redirected the metabolism of triglycerides (TAG) to its accumulation in the liver, via reduced TAG elimination by beta-oxidation combined with reduced hepatic efflux.

Conclusions

We propose that sEng might be a biomarker of NASH development, and the presence of high levels of sEng might support NASH aggravation by impairing the essential defensive mechanism protecting NASH liver against excessive TAG and cholesterol accumulation, suggesting the importance of high sEng levels in patients prone to develop NASH.

Supported by Ministry of Health of the Czech Republic grant AZV 17-31754A and GAUK No.1166119.

Background and Aims

Macrophages play a key role in the atherosclerosis, tissue macrophages can be divided into two types, the classical activation type (type M1) and alternative activation type (M2 type). Recent studies show that the balance of M1/M2 plays an important role in atherosclerotic plaque stability. miR-125a-3p can regulate the differentiation of monocytes to M1 type macrophages. In our previous study , the expression of miR-125a-3p was significantly increased in patients with acute coronary syndrome. This study is to investigate the effect of miR-125a-3p inhibitor on the atherosclerotic plaque formation, M1/M2 macrophages, MMP-2 and VEGF in atherosclerotic plaque in vivo.

Methods

Cholesterol-fed rabbits were applied to study the effect of miR-125a-3p inhibitor on atherosclerotic plaque, the area of atherosclerotic plaque was measured by image analysis after oil red O staining, MMP-2 and VEGF were measured by immunohistochemistry, and the M1 marker CD11c and M2 marker CD206 were detected by immunofluorescence in plaque tissue.

Results

Compared with high-cholesterol group, plaque area percentage was significantly decresed in miR-125a-3p inhibitor group. Compared with the control group, the level of MMP-9、VEGF and CD11c were significantly increased in high-cholesterol group and decreased in miR-125a-3p inhibitor group, the level of CD206 was significantly decreased in high-cholesterol group and increased in miR-125a-3p inhibitor group.

Conclusions

Inhibition of miR-125a-3p could attenuate the development of atherosclerotic plaque, balance of M1/M2 macrophages, and decrease of MMP-9 and VEGF in plaque tissue. miR-125a-3p may be a new targets for the treatment of unstable plaque.

Background and Aims

Diabetic patients have high incidence of cardiovascular disease, which is associated with increased volume and functional alterations of epicardial adipose tissue (EAT). We aimed to study the inflammatory and cytotoxic effects induced by EAT from diabetic patients on cardiomyocytes, and the counteracting effect of two cardioprotective molecules: HDL and apolipoprotein J (apoJ).

Methods

EAT was obtained after heart surgery from non-diabetic (ND) and diabetic patients, referred as DM-C or DM according to the presence or not of coronary disease. EAT explants were incubated for 24 h, and the secretomes were added to AC16 human cardiomyocytes in the presence or absence of HDL or apoJ. After 24 hours’ incubation, inflammation was assessed by measuring the release of IL6 and MCP1 by ELISA, cytotoxicity was determined by annexin V staining by flow-cytometry, and the expression of selected genes was evaluated by real-time PCR

Results

Secretomes induced the release of MCP1 and IL6 in AC16, being that from DM the major inductor. Secretome from ND had no cytotoxic effect, whereas secretome from diabetic patients induced two-fold the mortality in AC16. The addition of HDL and apoJ inhibited the secretome-induced inflammatory and cytotoxic effects. Secretome from diabetic patients, mainly from DM-C, induced increased expression of genes related to lipid metabolism: DGAT2, PLIN2, PPARα and PPARγ (1.5-2 fold versus ND).

Conclusions

In summary, secretome from EAT of diabetic patients induced increased inflammatory and cytotoxic response in AC16 cardiomyocytes compared with EAT from ND subjects. Both effects were partially inhibited by HDL and apoJ.

Background and Aims

Molecular microbiological and metagenomic studies have suggested that atherosclerotic plaques harbor a microbiome. Chronic bacterial infections are usually due to biofilms resistant to antibiotics and capable to evade immune system. Using RT-qPCR we have detected viridans streptococcal group bacterial DNA in thrombus aspirates of clinical MI patients and in ruptured cerebral artery aneurysms.

Methods

We studied by RT-qPCR, immunohistochemistry, genome wide expression (GWE) and in vitro culture of bacterial activated TLR cell lines the signaling pathways and presence of viridans streptococci as well as immune response in different types of coronary samples in a series of 121 victims of sudden out-of-hospital death and endarterectomy samples from 96 surgical patients.

Results

Viridans streptococcal DNA was found in 42.1% of autopsy coronary plaques and in 42.9% of clinical endarterectomy samples. Of coronary samples, 218/360 (60.6 %) and of available surgical endarterectomies, 30/59 (52.5 %) were immunopositive for viridans streptococci, associating (p<0.0001) with severity of atherosclerosis. Streptococci colonized as a biofilm the necrotic core of atheromas, unrecognized by innate immune system. Phenotypically virulent streptococci, dispersed from the biofilm infiltrated the fibrous caps of atheromas co-localizing with the expression of pattern recognizing receptors and molecules of adaptive immunity. In age-adjusted analysis, streptococcal immunopositivity in autopsy series correlated with death due to CHD (p=0.021) or MI (p=0.042). GWE array of surgical atherosclerotic plaques showed that genes in metabolic pathways for recognizing bacteria were highly upregulated.

Conclusions

These results suggest that persistent chronic bacterial inflammation is involved in the pathogenesis of vulnerable coronary atheromas and symptomatic peripheral artery plaques.

Background and Aims

White (WAT) and brown (BAT) adipose tissue, are respectively responsible for lipid storage and non-shivering thermogenesis, a process mediated by mitochondrial uncoupling protein 1 (UCP1) which uncouples oxidative phosphorylation from ATP production, leading to heat production. This process can be triggered by cold exposure, caloric excess, and the immune system. Recently thermogenesis has been associated with plasma lipoprotein transport system. Specifically, APOE3 is shown to have a bimodal effect on WAT thermogenesis while APOE2 and APOE4 differentially affect BAT and WAT thermogenesis in processes highly modulated by APOA1. Furthermore, APOA1 deficiency is associated with no measurable UCP1 levels in WAT of mice fed high fat diet.

Methods

Based on these observations here we sought to investigate the potential roles of APOA1 and APOE3 in BAT and WAT metabolic activation in mice, following a 2-week stimulation by cold exposure (7^oC).

Results

Our data showed that APOA1 deficiency ablated BAT and WAT mitochondrial metabolic response to cold stimuli. Ectopic expression of APOA1 in Apoa1^-/- mice exposed to 7^oC, resulted in virtually undetectable levels of APOE-containing HDL and only BAT thermogenesis induction. On the contrary, WAT mitochondria responded to cold stimuli only under the concomitant expression of both APOA1 and APOE3 in these mice.

Conclusions

In conclusion, APOA1-containing HDL promotes BAT thermogenesis in the presence of very low levels of APOE3-containing HDL, which acts as an inhibitor in this process. In contrast, induction of WAT thermogenesis is subjected to a more complicated regulation which requires the presence of both APOA1- and APOE3-containing HDL.

Background and Aims

Genome-wide association studies (GWAS) have identified hundreds of loci associated with coronary artery disease (CAD). Many of these loci are enriched in cis-regulatory elements (CREs) but not linked to cardiometabolic risk factors nor to candidate causal genes, complicating their functional interpretation.

Methods

We investigated chromatin accessibility profiles of the human atherosclerotic lesions using single nucleus (sn)ATAC-Seq to map cell type-specific patterns of CREs, to understand transcription factors establishing cell identity and to interpret CAD-relevant, non-coding genetic variation.

Results

We identified specific transcription factors associated with macrophage subtypes and the differentiation trajectory of smooth muscle cells transitioning into fibromyocytes. We demonstrate that endothelial and smooth muscle cell enhancers are particularly enriched for blood pressure and CAD associated genetic variants. We prioritized putative target genes and candidate regulatory elements for ~30% of all the CAD loci using single cell co-accessibility and cis-eQTL information. Finally, we performed genome-wide experimental fine-mapping of the CAD GWAS variants using epigenetic molecular QTL analysis in primary human aortic endothelial cells and STARR-Seq massively parallel reporter assay in smooth muscle cells. This analysis identified potential causal SNP(s) and the associated target gene for over 30 CAD loci. We present several examples where the chromatin accessibility and gene expression could be assigned to one cell type predicting the cell type of action for CAD loci.

Conclusions

These findings highlight the potential of applying snATAC-seq to human tissues in revealing relative contributions of distinct cell types to diseases and in identifying genes likely to be influenced by non-coding GWAS variants.