Abstract 331: Caeolin-1 Deficiency Increases LDL Cholesterol Uptake but Attenuates Inflammation of Endothelial Cells

Caveolin-1 (Cav-1) is the major structural protein essential to the formation of the caveolae in endothelial cells. Cav-1 is thought to play an important role in the regulation of cellular cholesterol homeostasis, a process that needs to be properly controlled to limit and prevent cholesterol accumulation and eventually atherosclerosis. Genetic ablation of Cav-1 on an apoE knockout background inhibits the progression of atherosclerosis, while re-expression of Cav-1 in the endothelium promotes lesion expansion by affecting several processes including endothelial cell proliferation, migration, and nitric oxide production in vitro and increased expression of vascular cell adhesion molecule-1. Specifically, in vivo study shows that loss of Cav-1 reduces LDL infiltration into the artery wall and inhibits progression of atherosclerosis. However, surprisingly, we found that knock down of Cav-1 in human endothelial cells increases the binding and uptake of diI-LDL in vitro suggesting that Cav-1 decreases transcytosis of LDL. Moreover, Caveolin-1 has also been implicated in the regulation of inflammation in endothelial cells. Here we show that Cav-1 affects the induction of cell adhesion molecules in endothelial cells by inflammatory mediators including TLR2 ligand Pam3cys, IL-1B, and TNF. Specifically, knock-down of Cav-1 in human endothelial cells decreases the induction of ICAM-1, VCAM-1 and SELE in both mRNA and protein levels. Furthermore, we also show that silencing of Cav-1 attenuates the major signaling pathways that are involved in the induction of cell adhesion molecules including MAPK, AKT, JNK-AP1 and NF-κB pathways.</jats:p

Lanosterol Modulates TLR4-Mediated Innate Immune Responses in Macrophages

Macrophages perform critical functions in both innate immunity and cholesterol metabolism. Here, we report that activation of Toll-like receptor 4 (TLR4) in macrophages causes lanosterol, the first sterol intermediate in the cholesterol biosynthetic pathway, to accumulate. This effect is due to type I interferon (IFN)-dependent histone deacetylase 1 (HDAC1) transcriptional repression of lanosterol-14 alpha-demethylase, the gene product of Cyp51A1. Lanosterol accumulation in macrophages, because of either treatment with ketoconazole or induced conditional disruption of Cyp51A1 in mouse macrophages in vitro, decreases IFN beta-mediated signal transducer and activator of transcription (STAT)-1-STAT2 activation and IFN beta-stimulated gene expression. These effects translate into increased survival to endotoxemic shock by reducing cytokine secretion. In addition, lanosterol accumulation increases membrane fluidity and ROS production, thus potentiating phagocytosis and the ability to kill bacteria. This improves resistance of mice to Listeria monocytogenes infection by increasing bacterial clearance in the spleen and liver. Overall, our data indicate that lanosterol is an endogenous selective regulator of macrophage immunity

Macrophage deficiency of miR-21 promotes apoptosis, plaque necrosis, and vascular inflammation during atherogenesis

Atherosclerosis, the major cause of cardiovascular disease, is a chronic inflammatory disease characterized by the accumulation of lipids and inflammatory cells in the artery wall. Aberrant expression of microRNAs has been implicated in the pathophysiological processes underlying the progression of atherosclerosis. Here, we define the contribution of miR-21 in hematopoietic cells during atherogenesis. Interestingly, we found that miR-21 is the most abundant miRNA in macrophages and its absence results in accelerated atherosclerosis, plaque necrosis, and vascular inflammation. miR-21 expression influences foam cell formation, sensitivity to ER-stress-induced apoptosis, and phagocytic clearance capacity. Mechanistically, we discovered that the absence of miR-21 in macrophages increases the expression of the miR-21 target gene, MKK3, promoting the induction of p38-CHOP and JNK signaling. Both pathways enhance macrophage apoptosis and promote the post-translational degradation of ABCG1, a transporter that regulates cholesterol efflux in macrophages. Altogether, these findings reveal a major role for hematopoietic miR-21 in atherogenesis

MicroRNA-148a regulates LDL receptor and ABCA1 expression to control circulating lipoprotein levels

The hepatic low-density lipoprotein receptor (LDLR) pathway is essential for clearing circulating LDL cholesterol (LDL-C). Whereas the transcriptional regulation of LDLR is well characterized, the post-transcriptional mechanisms that govern LDLR expression are just beginning to emerge. Here we develop a high-throughput genome-wide screening assay to systematically identify microRNAs (miRNAs) that regulate LDLR activity in human hepatic cells. From this screen we identified and characterized miR-148a as a negative regulator of LDLR expression and activity and defined a sterol regulatory element binding protein 1 (SREBP1)-mediated pathway through which miR-148a regulates LDL-C uptake. In mice, inhibition of miR-148a increased hepatic LDLR expression and decreased plasma LDL-C. Moreover, we found that miR-148a regulates hepatic expression of ATP-binding cassette, subfamily A, member 1 (ABCA1) and circulating high-density lipoprotein cholesterol (HDL-C) levels in vivo. These studies uncover a role for miR-148a as a key regulator of hepatic LDL-C clearance through direct modulation of LDLR expression and demonstrate the therapeutic potential of inhibiting miR-148a to ameliorate an elevated LDL-C/HDL-C ratio, a prominent risk factor for cardiovascular disease

miR-27b inhibits LDLR and ABCA1 expression but does not influence plasma and hepatic lipid levels in mice

Rationale: Recently, there has been significant interest in the therapeutic administration of miRNA mimics and inhibitors to treat cardiovascular disease. In particular, miR-27b has emerged as a regulatory hub in cholesterol and lipid metabolism and potential therapeutic target for treating atherosclerosis. Despite this, the impact of miR-27b on lipid levels in vivo remains to be determined. As such, here we set out to further characterize the role of miR-27b in regulating cholesterol metabolism in vitro and to determine the effect of miR-27b overexpression and inhibition on circulating and hepatic lipids in mice. Methods and results: Our results identify miR-27b as an important regulator of LDLR activity in human and mouse hepatic cells through direct targeting of LDLR and LDLRAP1. In addition, we report that modulation of miR-27b expression affects ABCA1 protein levels and cellular cholesterol efflux to ApoA1 in human hepatic Huh7 cells. Overexpression of pre-miR-27b in the livers of wild-type mice using AAV8 vectors increased pre-miR-27b levels 50efold and reduced hepatic ABCA1 and LDLR expression by 50% and 20%, respectively, without changing circulating and hepatic cholesterol and triglycerides. To determine the effect of endogenous miR-27b on circulating lipids, wild-type mice were fed a Western diet for one month and injected with 5 mg/kg of LNA control or LNA anti-miR-27b oligonucleotides. Following two weeks of treatment, the expression of ABCA1 and LDLR were increased by 10-20% in the liver, demonstrating effective inhibition of miR-27b function. Intriguingly, no differences in circulating and hepatic lipids were observed between treatment groups. Conclusions: The results presented here provide evidence that short-term modulation of miR-27b expression in wild-type mice regulates hepatic LDLR and ABCA1 expression but does not influence plasma and hepatic lipid levels. (C) 2015 Elsevier Ireland Ltd. All rights reserved

Skeletal Muscle–Specific Deletion of MKP-1 Reveals a p38 MAPK/JNK/Akt Signaling Node That Regulates Obesity-Induced Insulin Resistance

Stress responses promote obesity and insulin resistance, in part, by activating the stress-responsive mitogen-activated protein kinases (MAPKs), p38 MAPK, and c-Jun NH2-terminal kinase (JNK). Stress also induces expression of MAPK phosphatase-1 (MKP-1), which inactivates both JNK and p38 MAPK. However, the equilibrium between JNK/p38 MAPK and MKP-1 signaling in the development of obesity and insulin resistance is unclear. Skeletal muscle is a major tissue involved in energy expenditure and glucose metabolism. In skeletal muscle, MKP-1 is upregulated in high-fat diet–fed mice and in skeletal muscle of obese humans. Mice lacking skeletal muscle expression of MKP-1 (MKP1-MKO) showed increased skeletal muscle p38 MAPK and JNK activities and were resistant to the development of diet-induced obesity. MKP1-MKO mice exhibited increased whole-body energy expenditure that was associated with elevated levels of myofiber-associated mitochondrial oxygen consumption. miR-21, a negative regulator of PTEN expression, was upregulated in skeletal muscle of MKP1-MKO mice, resulting in increased Akt activity consistent with enhanced insulin sensitivity. Our results demonstrate that skeletal muscle MKP-1 represents a critical signaling node through which inactivation of the p38 MAPK/JNK module promotes obesity and insulin resistance.</jats:p