Effects of 2-Methoxyestradiol, a Main Metabolite of Estradiol on Hepatic ABCA1 Expression in HepG2 Cells

ATP-binding cassette transporter A1 (ABCA1) is a key regulator of lipid efflux, and the absence of ABCA1 induces hepatic lipid accumulation, which is one of the major causes of fatty liver. 2-Methoxyestradiol (2-ME2) has been demonstrated to protect against fatty liver. In this study, we investigated the effects of 2-ME2 on the hepatic lipid content and ABCA1 expression. We found that 2-ME2 dose-dependently increased ABCA1 expression, and therefore, the lipid content was significantly decreased in HepG2 cells. 2-ME2 enhanced the ABCA1 promoter activity; however, this effect was reduced after the inhibition of the PI3K pathway. The overexpression of Akt or p110 induced ABCA1 promoter activity, while dominant-negative Akt diminished the ability of 2-ME2 on ABCA1 promoter activity. Further, 2-ME2 stimulated the rapid phosphorylation of Akt and FoxO1 and reduced the nuclear accumulation of FoxO1. Chromatin immunoprecipitation confirmed that FoxO1 bonded to the ABCA1 promoter region. The binding was reduced by 2-ME2, which facilitated ABCA1 gene transcription. Furthermore, mutating FoxO1-binding sites in the ABCA1 promoter region or treatment with FoxO1-specific siRNA disrupted the effect of 2-ME2 on ABCA1 expression. All of our results demonstrated that 2-ME2 might upregulate ABCA1 expression via the PI3K/Akt/FoxO1 pathway, which thus reduces the lipid content in hepatocytes.

New Strategies to Promote Macrophage Cholesterol Efflux

The capacity of macrophages to dispose of cholesterol deposited in the atherosclerotic plaque depends on their ability to activate cholesterol efflux pathways. To develop athero-protective therapies aimed at promoting macrophage cholesterol efflux, cholesterol metabolism in THP-1 monocyte-derived macrophages has been extensively studied, but the intrinsic sensitivity of monocytes and the lack of a standardized procedure to differentiate THP-1 monocytes into macrophages have made it difficult to utilize THP-1 macrophages in the same or similar degree of differentiation across studies. The variability has resulted in lack of understanding of how the differentiation affects cholesterol metabolism, and here we review and investigate the effects of THP-1 differentiation on cholesterol efflux. The degree of THP-1 differentiation was inversely associated with ATP binding cassette A1 (ABCA1) transporter-mediated cholesterol efflux. The differentiation-associated decrease in ABCA1-mediated cholesterol efflux occurred despite an increase in ABCA1 expression. In contrast, DSC1 expression decreased during the differentiation. DSC1 is a negative regulator of the ABCA1-mediated efflux pathway and a DSC1-targeting agent, docetaxel showed high potency and efficacy in promoting ABCA1-mediated cholesterol efflux in THP-1 macrophages. These data suggest that pharmacological targeting of DSC1 may be more effective than increasing ABCA1 expression in promoting macrophage cholesterol efflux. In summary, the comparison of THP-1 macrophage subtypes in varying degrees of differentiation provided new insights into cholesterol metabolism in macrophages and allowed us to identify a viable target DSC1 for the promotion of cholesterol efflux in differentiated macrophages. Docetaxel and other pharmacological strategies targeting DSC1 may hold significant potential for reducing atherogenic cholesterol deposition.

5,7,3′,4′-tetramethoxyflavone ameliorates cholesterol dysregulation by mediating SIRT1/FOXO3a/ABCA1 signaling in osteoarthritis chondrocytes

Dyslipidemia has been associated with the development of osteoarthritis. Our previous study found that 5,7,3′,4′-tetramethoxyflavone (TMF) exhibited protective activities against the pathological changes of osteoarthritis. Aim: To investigate the roles of TMF in regulating ABCA1-mediated cholesterol metabolism. Methods: Knockdown and overexpression were employed to study gene functions. Protein–protein interaction was investigated by co-immunoprecipitation, and the subcellular locations of proteins were studied by immunofluorescence. Results: IL-1β decreased ABCA1 expression and induced apoptosis. Therapeutically, TMF ameliorated the effects of IL-1β. FOXO3a knockdown expression abrogated the effects of TMF, and FOXO3a overexpression increased ABCA1 expression by interacting with LXRα. TMF promoted FOXO3a nuclear translocation by activating SIRT1 expression. Conclusions: TMF ameliorates cholesterol dysregulation by increasing the expression of FOXO3a/LXRα/ABCA1 signaling through SIRT1 in C28/I2 cells.

Combined LXR and RXR Agonist Therapy Increases ABCA1 Protein Expression and Enhances ApoAI-Mediated Cholesterol Efflux in Cultured Endothelial Cells

Endothelial ABCA1 expression protects against atherosclerosis and this atheroprotective effect is partially attributed to enhancing apoAI-mediated cholesterol efflux. ABCA1 is a target gene for LXR and RXR; therefore, treating endothelial cells with LXR and/or RXR agonists may increase ABCA1 expression. We tested whether treating cultured immortalized mouse aortic endothelial cells (iMAEC) with the endogenous LXR agonist 22(R)-hydroxycholesterol, synthetic LXR agonist GW3965, endogenous RXR agonist 9-cis-retinoic acid, or synthetic RXR agonist SR11237 increases ABCA1 protein expression. We observed a significant increase in ABCA1 protein expression in iMAEC treated with either GW3965 or SR11237 alone, but no significant increase in ABCA1 protein was observed in iMAEC treated with either 22(R)-hydroxycholesterol or 9-cis-retionic acid alone. However, we observed significant increases in both ABCA1 protein expression and apoAI-mediated cholesterol efflux when iMAEC were treated with a combination of either 22(R)-hydroxycholesterol and 9-cis-retinoic acid or GW3965 and SR11237. Furthermore, treating iMAEC with either 22(R)-hydroxycholesterol and 9-cis-retinoic acid or GW3965 and SR11237 did not trigger an inflammatory response, based on VCAM-1, ICAM-1, CCL2, and IL-6 mRNA expression. Based on our findings, delivering LXR and RXR agonists precisely to endothelial cells may be a promising atheroprotective approach.

Oxidized LDL Downregulates ABCA1 Expression via MEK/ERK/LXR Pathway in INS-1 Cells

Impaired insulin secretion is one of the main causes of type 2 diabetes. Cholesterol accumulation-induced lipotoxicity contributes to impaired insulin secretion in pancreatic beta cells. However, the detailed mechanism in this process remains unclear. In this study, we proved that oxidized low-density lipoprotein (OxLDL) reduced insulin content, decreased PDX-1 expression, and impaired glucose-stimulated insulin secretion (GSIS) in INS-1 cells, which were rescued by addition of high-density lipoprotein (HDL). OxLDL receptors and cholesterol content were increased by OxLDL. Consistently, OxLDL suppressed cholesterol transporter ABCA1 expression and transcription in a dose-dependent and time-dependent manner. Inhibition of MEK by its specific inhibitor, PD98059, altered the effect of OxLDL on ABCA1 transcription and activation of ERK. Next, chromatin immunoprecipitation assay demonstrated that liver X receptor (LXR) could directly bind to ABCA1 promoter and this binding was inhibited by OxLDL. Furthermore, OxLDL decreased the nuclear LXR expression, which was prevented by HDL. LXR-enhanced ABCA1 transcription was suppressed by OxLDL, and the effect was cancelled by mutation of the LXR-binding sites. In summary, our study shows that OxLDL down-regulates ABCA1 expression by MEK/ERK/LXR pathway, leading to cholesterol accumulation in INS-1 cells, which may result in impaired insulin synthesis and GSIS.

Rosmarinic Acid Increases Macrophage Cholesterol Efflux through Regulation of ABCA1 and ABCG1 in Different Mechanisms

Lipid dysregulation in diabetes mellitus escalates endothelial dysfunction, the initial event in the development and progression of diabetic atherosclerosis. In addition, lipid-laden macrophage accumulation in the arterial wall plays a significant role in the pathology of diabetes-associated atherosclerosis. Therefore, inhibition of endothelial dysfunction and enhancement of macrophage cholesterol efflux is the important antiatherogenic mechanism. Rosmarinic acid (RA) possesses beneficial properties, including its anti-inflammatory, antioxidant, antidiabetic and cardioprotective effects. We previously reported that RA effectively inhibits diabetic endothelial dysfunction by inhibiting inflammasome activation in endothelial cells. However, its effect on cholesterol efflux remains unknown. Therefore, in this study, we aimed to assess the effect of RA on cholesterol efflux and its underlying mechanisms in macrophages. RA effectively reduced oxLDL-induced cholesterol contents under high glucose (HG) conditions in macrophages. RA enhanced ATP-binding cassette transporter A1 (ABCA1) and G1 (ABCG1) expression, promoting macrophage cholesterol efflux. Mechanistically, RA differentially regulated ABCA1 expression through JAK2/STAT3, JNK and PKC-p38 and ABCG1 expression through JAK2/STAT3, JNK and PKC-ERK1/2/p38 in macrophages. Moreover, RA primarily stabilized ABCA1 rather than ABCG1 protein levels by impairing protein degradation. These findings suggest RA as a candidate therapeutic to prevent atherosclerotic cardiovascular disease complications related to diabetes by regulating cholesterol efflux in macrophages.

GLP-1-Induced AMPK Activation Inhibits PARP-1 and Promotes LXR-Mediated ABCA1 Expression to Protect Pancreatic β-Cells Against Cholesterol-Induced Toxicity Through Cholesterol Efflux

T2DM (Type 2 diabetes) is a complex, chronic disease characterized as insulin resistance and islet β-cell dysfunction. Bariatric surgeries such as Roux-en-Y gastric bypass (RYGB) surgery and laparoscopic sleeve gastrectomy (LSG) have become part of a critical treatment regimen in the treatment of obesity and T2DM. Moreover, GLP-1 increase following bariatric surgery has been regarded as a significant event in bariatric surgery-induced remission of T2DM. In this study, a high concentration cholesterol-induced lipotoxicity was observed in INS-1 cells, including inhibited cell viability and insulin secretion. Enhanced cell apoptosis and inhibited cholesterol efflux from INS-1 cells; meanwhile, ABCA1 protein level was decreased by cholesterol stimulation. Cholesterol-induced toxicity and ABCA1 downregulation were attenuated by GLP-1 agonist EX-4. GLP-1 induced AMPK phosphorylation during the protection against cholesterol-induced toxicity. Under cholesterol stimulation, GLP-1-induced AMPK activation inhibited PARP-1 activity, therefore attenuating cholesterol-induced toxicity in INS-1 cells. In INS-1 cells, PARP-1 directly interacted with LXR, leading to the poly(ADP-ribosyl)ation of LXRα and downregulation of LXR-mediated ABCA1 expression. In the STZ-induced T2DM model in rats, RYGB surgery or EX-4 treatment improved the glucose metabolism and lipid metabolism in rats through GLP-1 inhibition of PARP-1 activity. In conclusion, GLP-1 inhibits PARP-1 to protect islet β cell function against cholesterol-induced toxicity in vitro and in vivo through enhancing cholesterol efflux. GLP-1-induced AMPK and LXR-mediated ABCA1 expression are involved in GLP-1 protective effects.

Protective role of ABCA1 in ischemic preconditioning is mediated by downregulation of miR-33-5p and miR-135-5p

Ischemic preconditioning (IPC) significantly reduces ischemia–reperfusion injury in the brain by inducing ischemic tolerance. Although emerging evidence suggests that microRNAs (miRNAs) contribute to the pathogenesis of brain ischemia and IPC-induced neuroprotection, the role of miRNAs and their underlying mechanisms are still unclear. IPC was induced in male C57BL/6 mice by brief bilateral common carotid artery occlusion. After 24 h, mice underwent transient middle cerebral artery occlusion followed by 3 h of reperfusion. Expression levels of messenger RNAs (mRNAs) and proteins were examined in the ipsilateral cortex, and mimics and inhibitors of selective miRNAs were transfected into Neuro-2a cells before oxygen–glucose deprivation (OGD). Post-IPC miRNA expression profiling identified neuroprotection-associated changes in miRNA expression in the ipsilateral cortex after ischemic stroke. Among them, miR-33-5p and miR-135b-5p were significantly downregulated by IPC. Inhibition of miR-33-5p and miR-135b-5p expression protected Neuro-2a cells from OGD-induced apoptosis. Inhibition of these two miRNAs significantly increased mRNA and protein levels of ATP-binding cassette subfamily A member 1 (ABCA1), and a binding assay showed that these two miRNAs showed specificity for Abca1 mRNA. Overexpression of ABCA1 decreased the Bax/Bcl2 mRNA ratio and activation of caspase-9 and caspase-3, whereas knockdown of ABCA1 expression increased the Bax/Bcl2 mRNA ratio and the percentage of Neuro-2a cells with a loss of mitochondrial membrane potential after OGD-treatment. In conclusion, ABCA1 expression is regulated by miR-33-5p and miR-135b-5p. Increased ABCA1 expression following IPC exerts a protective influence against cerebral ischemia via suppression of a mitochondria-dependent apoptosis pathway.

Low LAL (Lysosomal Acid Lipase) Expression by Smooth Muscle Cells Relative to Macrophages as a Mechanism for Arterial Foam Cell Formation

Objective: We previously reported smooth muscle cells (SMCs) represent ≥50% of foam cells in human coronary and ≈70% in apoE (apolipoprotein E)-deficient mouse aortic atheromas and exhibit reduced expression of the cholesterol exporter ABCA1 (ATP-binding cassette transporter A1). A major stimulus for ABCA1 expression is flux of cholesterol out of lysosomes, generated by hydrolysis of lipoprotein cholesteryl esters by LAL (lysosomal acid lipase). In this study, we investigated the potential role lysosomal dysfunction might play in foam cell formation by arterial SMCs. Approach and Results: Human monocyte-derived macrophages (macrophages) and arterial SMCs were treated with aggregated LDL (low-density lipoprotein) to increase intracellular cholesterol and investigated for lysosomal and postlysosomal cholesterol metabolism defects. Human and mouse atheromas were analyzed for LAL expression. Unlike macrophages, aggregated LDL uptake failed to upregulate ABCA1 expression, downregulate new cholesterol synthesis, or to significantly increase 27-hydroxycholesterol levels in SMCs. Confocal microscopy revealed retention of neutral lipids within lysosomal compartments in SMCs, while macrophages showed most lipids as cytosolic droplets. LIPA mRNA levels and LAL protein were markedly reduced in SMCs. Treatment of SMCs with medium containing LAL resulted in significantly reduced lysosomal lipid accumulation and increased cholesterol efflux to apoA-I (apolipoprotein AI). Human and mouse atheromas exhibited low LAL/ Lipa expression in intimal SMCs when compared with intimal macrophages. Conclusions: These findings indicate the inherently low level of LAL in SMCs compared with macrophages is associated with reduced capacity to catabolize atherogenic lipoproteins and is a mechanism for SMC foam cell formation in atherosclerosis.