Search

Investigations into the regulatory mechanisms controlling cholesterol homeostasis have proven fruitful in identifying low-density lipoprotein (LDL)-lowering therapies to reduce the risk of atherosclerotic cardiovascular disease. A major advance was the discovery of proprotein convertase subtilisin/kexin type 9 (PCSK9), a secreted protein that binds the LDL receptor (LDLR) on the cell surface and internalizes it for degradation, thereby blunting its ability to take up circulating LDL. The discovery that loss-of-function mutations in PCSK9 lead to lower plasma levels of LDL cholesterol and protection from cardiovascular disease led to the therapeutic development of PCSK9 inhibitors at an unprecedented pace. However, there remain many gaps in our understanding of PCSK9 regulation and biology, including its posttranscriptional control by microRNAs. Using a high-throughput region(3′-UTR) of human microRNA library screen, we identified microRNAs targeting the 3′ untranslated region of human PCSK9. The top 35 hits were confirmed by large-format PCSK9 3′-UTR luciferase assays, and 10 microRNAs were then selected for further validation in hepatic cells, including effects on PCSK9 secretion and LDLR cell surface expression. These studies identified seven novel microRNAs that reduce PCSK9 expression, including miR-221-5p, miR-342-5p, miR-363-5p, miR-609, miR-765, and miR-3165. Interestingly, several of these microRNAs were also found to target other genes involved in LDLR regulation and potently upregulate LDLR cell surface expression in hepatic cells. Together, these data enhance our understanding of post-transcriptional regulators of PCSK9 and their potential for therapeutic manipulation of hepatic LDLR expression.

An early event in atherogenesis is the recruitment and infiltration of circulating monocytes and macrophage activation in the subendothelial space. Atherosclerosis subsequently progresses as a unresolved inflammatory disease, particularly in hypercholesterolemic conditions. Although physical exercise training has been a widely accepted strategy to inhibit atherosclerosis, its impact on arterial wall inflammation and macrophage phenotype and function has not yet been directly evaluated. Thus, the aim of this study was to investigate the effects of aerobic exercise training on the inflammatory state of atherosclerotic lesions with a focus on macrophages. Hypercholesterolemic LDL-receptor-deficient male mice were subjected to treadmill training for 8 weeks and fed a high-fat diet. Analyses included plasma lipoprotein and cytokine levels; aortic root staining for lipids (oil red O); macrophages (CD68, MCP1 and IL1β); oxidative (nitrotyrosine and, DHE) and endoplasmic reticulum (GADD) stress markers. Primary bone marrow-derived macrophages (BMDM) were assayed for migration activity, motility phenotype (Rac1 and F-actin) and inflammation-related gene expression. Plasma levels of HDL cholesterol were increased, while levels of proinflammatory cytokines (TNFa, IL1b, and IL6) were markedly reduced in the exercised mice. The exercised mice developed lower levels of lipid content and inflammation in atherosclerotic plaques. Additionally, lesions in the exercised mice had lower levels of oxidative and ER stress markers. BMDM isolated from the exercised mice showed a marked reduction in proinflammatory cytokine gene expression and migratory activity and a disrupted motility phenotype. More importantly, bone marrow from exercised mice transplanted into sedentary mice led to reduced atherosclerosis in the recipient sedentary mice, thus suggesting that epigenetic mechanisms are associated with exercise. Collectively, the presented data indicate that exercise training prevents atherosclerosis by inhibiting bone marrow-derived macrophage recruitment and activation.

MicroRNAs (miRNA) have emerged as important post-transcriptional regulators of metabolic pathways that contribute to cellular and systemic lipoprotein homeostasis. Here, we identify two conserved miRNAs, miR-224, and miR-520d, which target gene networks regulating hepatic expression of the low-density lipoprotein (LDL) receptor (LDLR) and LDL clearance. In silico prediction of miR-224 and miR-520d target gene networks showed that they each repress multiple genes impacting the expression of the LDLR, including the chaperone molecules PCSK9 and IDOL that limit LDLR expression at the cell surface and the rate-limiting enzyme for cholesterol synthesis HMGCR, which is the target of LDL-lowering statin drugs. Using gain- and loss-of-function studies, we tested the role of miR-224 and miR-520d in the regulation of those predicted targets and their impact on LDLR expression. We show that overexpression of miR-224 or miR-520d dose-dependently reduced the activity of PCSK9, IDOL, and HMGCR 3′-untranslated region (3′-UTR)-luciferase reporter constructs and that this repression was abrogated by mutation of the putative miR-224 or miR-520d response elements in the PCSK9, IDOL, and HMGCR 3′-UTRs. Compared to a control miRNA, overexpression of miR-224 or miR-520d in hepatocytes inhibited PCSK9, IDOL, and HMGCR mRNA and protein levels and decreased PCSK9 secretion. Furthermore, miR-224 and miR-520d repression of PCSK9, IDOL, and HMGCR was associated with an increase in LDLR protein levels and cell surface expression, as well as enhanced LDL binding. Notably, the effects of miR-224 and miR-520d were additive to the effects of statins in upregulating LDLR expression. Finally, we show that overexpression of miR-224 in the livers of Ldlr+/− mice using lipid nanoparticle-mediated delivery resulted in a 15% decrease in plasma levels of LDL cholesterol, compared to a control miRNA. Together, these findings identify roles for miR-224 and miR-520d in the posttranscriptional control of LDLR expression and function.

Endothelial dysfunction precedes atherosclerosis and is an independent predictor of cardiovascular events. Cholesterol levels and oxidative stress are key contributors to endothelial damage, whereas high levels of plasma high-density lipoproteins (HDL) could prevent it. Cholesteryl ester transfer protein (CETP) is one of the most potent endogenous negative regulators of HDL-cholesterol. However, whether and to what degree CETP expression impacts endothelial function, and the molecular mechanisms underlying the vascular effects of CETP on endothelial cells, have not been addressed. Acetylcholine-induced endothelium-dependent relaxation of aortic rings was impaired in human CETP-expressing transgenic mice, compared to their non-transgenic littermates. However, endothelial nitric oxide synthase (eNOS) activation was enhanced. The generation of superoxide and hydrogen peroxide was increased in aortas from CETP transgenic mice, while silencing CETP in cultured human aortic endothelial cells effectively decreased oxidative stress promoted by all major sources of ROS: mitochondria and NOX2. The endoplasmic reticulum stress markers, known as GADD153, PERK, and ARF6, and unfolded protein response effectors, were also diminished. Silencing CETP reduced endothelial tumor necrosis factor (TNF) α levels, intercellular cell adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1) expression, diminishing monocyte adhesion. These results support the notion that CETP expression negatively impacts endothelial cell function, revealing a new mechanism that might contribute to atherosclerosis.