High-density lipoprotein remodelled in hypercholesterolaemic blood induce epigenetically driven down-regulation of endothelial HIF-1α expression in a preclinical animal model

Abstract

Aims: High-density lipoproteins (HDLs) are circulating micelles that transport proteins, lipids, and miRNAs. HDL-transported miRNAs (HDL-miRNAs) have lately received attention but their effects on vascular cells are not fully understood. Additionally, whether cardiovascular risk factors affect HDL-miRNAs levels and miRNA transfer to recipient cells remains equally poorly known. Here, we have investigated the changes induced by hypercholesterolaemia on HDL-miRNA levels and its effect on recipient endothelial cells (ECs).

Methods and results: Pigs were kept on a high-fat diet (HC; n = 10) or a normocholesterolaemic chow (NC; n = 10) for 10 days reaching cholesterol levels of 321.0 (229.7-378.5) mg/dL and 74.0 (62.5-80.2) mg/dL, respectively. HDL particles were isolated, purified, and quantified. HDL-miRNA profiling (n = 149 miRNAs) of HC- and NC-HDLs was performed by multipanel qPCR. Cell cultures of porcine aortic ECs were used to determine whether HDL-miRNAs were delivered to ECs. Potential target genes modulated by miRNAs were identified by bioinformatics and candidate miRNAs were validated by molecular analysis. In vivo effects in the coronary arteries of normocholesterolaemic swine administered HC- or NC-HDLs were analysed. Among the HDL-miRNAs, four were found in different amounts in HC- and NC-HDL (P < 0.05). miR-126-5p and -3p and miR-30b-5p (2.7×, 1.7×, and 1.3×, respectively) were found in higher levels and miR-103a-3p and miR-let-7g-5p (-1.6×, -1.4×, respectively) in lower levels in HC-HDL. miR-126-5p and -3p were transferred from HC-HDL to EC (2.5×; P < 0.05), but not from NC-HDL, by a SRB1-mediated mechanism. Bioinformatics revealed that HIF1α was the miR-126 target gene with the highest predictive value, which was accordingly found to be markedly reduced in HC-HDL-treated ECs and in miR126 mimic transfected ECs. In vivo validation confirmed that HIF1α was diminished in the coronary endothelial layer of NC pigs administered HC-HDL vs. those administered NC-HDL (P < 0.05).

Conclusion: Hypercholesterolaemia induces changes in the miRNA content of HDL enhancing miR126 and its delivery to ECs with the consequent down-regulation of its target gene HIF1α.

Keywords: Dyslipidaemia; Endothelial cells; HDL; Translational research; miRNAs.

Graphical Abstract

Figure 1

HeatMap including the miRNAs detected in all HDL samples isolated from hypercholesterolaemic (HC; n = 10) and normocholesterolaemic (NC; n = 10) pigs.

Figure 2

Endothelial cells incubated with hypercholesterolaemic-HDL show higher miR-126-5p and 3p levels as compared to normocholesterolaemic-HDL [miR126-5p (A) and miR126-3p (B)], whereas no differences were observed for 103a-3p (C), miR-30b-5p (D), or let-7g-5p (E) levels. n = 5. Data are reported as mean ± SD. *ANOVA test P < 0.05 vs. Control and NC-HDL groups. HC: hypercholesterolaemic; NC: normocholesterolaemic.

Figure 3

Hypercholesterolaemic HDL particles transfer miR-126-5p and -3p (A and B, respectively) to endothelial cells through a SRB1-dependent mechanism. No differences are detected for Pre-mir-126 (C) or for miR-126 encoding gene EGFL7 (D) levels among the different groups. n = 5. Data are reported as mean ± SD. *ANOVA test P < 0.05 vs. any other group. HC: hypercholesterolaemic; NC: normocholesterolaemic.

Figure 4

Control ECs transfected with miR126-mimic show lower HIF1α gene levels as compared to ECs transfected with the negative control both at mRNA expression and protein level (A–D). HIF1α-mRNA expression was lower in negative-control transfected ECs incubated with HC- as compared to NC-HDLs and control (E and F). *ANOVA test P < 0.05. HC: hypercholesterolaemic; NC: normocholesterolaemic.

Figure 5

mRNA expression and protein levels of miR126 target HIF1α in endothelial cells. n = 5. Data are reported as mean ± SD. *ANOVA test P < 0.05 vs. Control and NC-HDL groups. HC: hypercholesterolaemic; NC: normocholesterolaemic.

Figure 6

Confocal microscopy analysis of HIF-1α detection in the endothelial layer of the coronary artery of normocholesterolaemic (NC) pigs administered NC- or hypercholesterolaemic (HC)-HDLs. Representative images of HIF1α (green) detection (A) and bar-graph quantification (B). Data are reported as mean ± SD. n = 6 animals/group. *T-test P < 0.05 vs. NC pigs administered HC-HDL.