Cholesterol metabolism is tightly regulated at the cellular level. Here we show that miR-33, an intronic microRNA (miRNA) located within the gene encoding sterol-regulatory element–binding factor–2 (SREBF-2), a transcriptional regulator of cholesterol synthesis, modulates the expression of genes involved in cellular cholesterol transport. In mouse and human cells, miR-33 inhibits the expression of the adenosine triphosphate–binding cassette (ABC) transporter, ABCA1, thereby attenuating cholesterol efflux to apolipoprotein A1. In mouse macrophages, miR-33 also targets ABCG1, reducing cholesterol efflux to nascent high-density lipoprotein (HDL). Lentiviral delivery of miR-33 to mice represses ABCA1 expression in the liver, reducing circulating HDL levels. Conversely, silencing of miR-33 in vivo increases hepatic expression of ABCA1 and plasma HDL levels. Thus, miR-33 appears to regulate both HDL biogenesis in the liver and cellular cholesterol efflux.

Long-term epigenetic reprogramming of innate immune cells in response to microbes, also termed "trained immunity," causes prolonged altered cellular functionality to protect from secondary infections. Here, we investigated whether sterile triggers of inflammation induce trained immunity and thereby influence innate immune responses. Western diet (WD) feeding of Ldlr mice induced systemic inflammation, which was undetectable in serum soon after mice were shifted back to a chow diet (CD). In contrast, myeloid cell responses toward innate stimuli remained broadly augmented. WD-induced transcriptomic and epigenomic reprogramming of myeloid progenitor cells led to increased proliferation and enhanced innate immune responses. Quantitative trait locus (QTL) analysis in human monocytes trained with oxidized low-density lipoprotein (oxLDL) and stimulated with lipopolysaccharide (LPS) suggested inflammasome-mediated trained immunity. Consistently, Nlrp3/Ldlr mice lacked WD-induced systemic inflammation, myeloid progenitor proliferation, and reprogramming. Hence, NLRP3 mediates trained immunity following WD and could thereby mediate the potentially deleterious effects of trained immunity in inflammatory diseases.

Peroxisome proliferator-activated receptor-gamma (PPAR-gamma), the transcription factor target of the anti-diabetic thiazolidinedione (TZD) drugs, is reported to mediate macrophage differentiation and inflammatory responses. Using PPAR-gamma-deficient stem cells, we demonstrate that PPAR-gamma is neither essential for myeloid development, nor for such mature macrophage functions as phagocytosis and inflammatory cytokine production. PPAR-gamma is required for basal expression of CD36, but not for expression of the other major scavenger receptor responsible for uptake of modified lipoproteins, SR-A. In wild-type macrophages, TZD treatment divergently regulated CD36 and class A macrophage-scavenger receptor expression and failed to induce significant cellular cholesterol accumulation, indicating that TZDs may not exacerbate macrophage foam-cell formation.

High Density Lipoprotein (HDL) mediates reverse cholesterol transport and it is known to be protective against atherosclerosis. In addition, HDL has potent anti-inflammatory properties that may be critical for protection against other inflammatory diseases. The molecular mechanisms of how HDL can modulate inflammation, particularly in immune cells such as macrophages, remain poorly understood. Here we identify the transcriptional repressor ATF3, as an HDL-inducible target gene in macrophages that down-regulates the expression of Toll-like receptor (TLR)-induced pro-inflammatory cytokines. The protective effects of HDL against TLR-induced inflammation were fully dependent on ATF3 in vitro and in vivo. Our findings may explain the broad anti-inflammatory and metabolic actions of HDL and provide the basis for predicting the success of novel HDL-based therapies.

Atherosclerosis is an inflammatory disease linked to elevated blood cholesterol levels. Despite ongoing advances in the prevention and treatment of atherosclerosis, cardiovascular disease remains the leading cause of death worldwide. Continuous retention of apolipoprotein B-containing lipoproteins in the subendothelial space causes a local overabundance of free cholesterol. Since cholesterol accumulation and deposition of cholesterol crystals (CCs) triggers a complex inflammatory response, we tested the efficacy of the cyclic oligosaccharide 2-hydroxypropyl-β-cyclodextrin (CD), a compound that increases cholesterol solubility, in preventing and reversing atherosclerosis. Here we show that CD treatment of murine atherosclerosis reduced atherosclerotic plaque size and CC load, and promoted plaque regression even with a continued cholesterol-rich diet. Mechanistically, CD increased oxysterol production in both macrophages and human atherosclerotic plaques, and promoted liver X receptor (LXR)-mediated transcriptional reprogramming to improve cholesterol efflux and exert anti-inflammatory effects. In vivo, this CD-mediated LXR agonism was required for the anti-atherosclerotic and anti-inflammatory effects of CD as well as for augmented reverse cholesterol transport. Since CD treatment in humans is safe and CD beneficially affects key mechanisms of atherogenesis, it may therefore be used clinically to prevent or treat human atherosclerosis.

The ABCA1 transporter contains two large domains into which many of the genetic mutations in individuals with Tangier disease fall. To investigate the structural requirements for the cellular cholesterol efflux mediated by ABCA1, we have determined the topology of these two domains and generated transporters harboring five naturally occurring missense mutations in them. These mutants, unlike wild type ABCA1, produced little or no apoA-I-stimulated cholesterol efflux when transfected into 293 cells, establishing their causality in Tangier disease. Because all five mutant proteins were well expressed and detectable on the plasma membrane, their interaction with the ABCA1 ligand, apolipoprotein (apo) A-I, was measured using bifunctional cross-linking agents. Four of five mutants had a marked decline in cross-linking to apoA-I, whereas one (W590S) retained full cross-linking activity. Cross-linking of apoA-I was temperature-dependent, rapid in onset, and detectable with both lipidand water-soluble cross-linking agents. These results suggest that apoA-I-stimulated cholesterol efflux cannot occur without a direct interaction between the apoprotein and critical residues in two extracellular loops of ABCA1. The behavior of the W590S mutant indicates that although binding of apoA-I by ABCA1 may be necessary, it is not sufficient for stimulation of cholesterol efflux.

In an effort to elucidate the physiologic role of ABCA7, we generated mice lacking this transporter (Abca7 ؊/؊ mice). Homozygous null mice were produced from intercrosses of heterozygous null mice at the expected Mendelian frequency and developed normally without any obvious phenotypic abnormalities. Cholesterol and phospholipid efflux stimulated by apolipoprotein A-I from macrophages isolated from wild type and Abca7 ؊/؊ mice did not differ, suggesting that these activities may not be central to the physiological role of the transporter in vivo. Abca7 ؊/؊ females, but not males, had significantly less visceral fat and lower total serum and high density lipoprotein cholesterol levels than wild type, gender-matched littermates. ABCA7 expression was detected in hippocampal and cortical neurons by in situ hybridization and in brain and white adipose tissue by Western blotting. Induction of adipocyte differentiation from 3T3 fibroblasts in culture led to a marked increase in ABCA7 expression. These studies suggest that ABCA7 plays a novel role in lipid and fat metabolism that Abca7 ؊/؊ mice can be used to elucidate.

The expression, function, and regulation of the cholesterol efflux molecule, ABCA1, has been extensively examined in peripheral tissues but only poorly studied in the brain. Brain cholesterol metabolism is of interest because several lines of evidence suggest that elevated cholesterol increases the risk of Alzheimer's disease. We found a largely neuronal expression of ABCA1 in normal rat brain by in situ hybridization. ABCA1 message was dramatically up-regulated in neurons and glia in areas of damage by hippocampal AMPA lesion after 3-7 days. Immunoblot analysis demonstrated ABCA1 protein in cultured neuronal and glial cells, and expression was induced by ligands of the nuclear hormone receptors of the retinoid X receptor and liver X receptor family. ABCA1 was induced by treatment with retinoic acid and several oxysterols, including 22(R)-hydroxycholesterol and 24-hydroxycholesterol. Expression of an ABCA1-green fluorescent protein construct in neuroblastoma cells demonstrated fluorescence in perinuclear compartments and on the plasma membrane. Because the A␤ peptide is important in Alzheimer's disease pathogenesis, we examined whether ABCA1 induction altered A␤ levels. Treatment of neuroblastoma cells with retinoic acid and 22(R)-hydroxycholesterol caused significant increases in secreted A␤40 (29%) and A␤42 (65%). Treatment with a nonsteroidal liver X receptor ligand, TO-901317, similarly increased levels of secreted A␤40 (25%) and A␤42 (126%). The increase in secreted A␤ levels was reduced by RNAi blocking of ABCA1 expression. These data suggest that the cholesterol efflux molecule ABCA1 may also be involved in the secretion of the membrane-associated molecule, A␤.ABCA1 encodes an ATP-binding cassette protein that promotes efflux of cholesterol and phospholipids from intracellular compartments to high density lipoprotein, lipid-deficient apoAI, 1 and other apolipoproteins (1). Disruption of ABCA1 byTangier disease mutations in humans or by engineered knockout in mice is associated with a loss of cellular cholesterol efflux, an ablation of circulating high density lipoprotein, and, interestingly, peripheral neuropathies (2-5). Like many genes involved in cholesterol homeostasis, ABCA1 is regulated by the liver X receptors (LXR) (6 -8), nuclear receptors activated by oxysterols. ABCA1 is also regulated by peroxisome proliferatoractivated receptor ␦, which can be activated by fatty acid metabolites (9). Both of these classes of receptors form heterodimers with retinoid X receptors (RXR), which bind retinoic acid; as heterodimers, they alter gene transcription. These systems for ABCA1 induction help decrease cellular cholesterol after cholesterol loading (10). The importance of cerebral cholesterol metabolism in Alzheimer's disease (AD) risk and pathogenesis is supported by genetic, cell culture, mouse model, and epidemiologic data. ApoE in the central nervous system is implicated in supplying appropriate membrane lipid for development, nerve growth, and responses to injury and repair in the central nervous syste...