apoAI-mediated Cholesterol Efflux Research Articles

Inhibition of miR-33a-5p in Macrophage-like Cells In Vitro Promotes apoAI-Mediated Cholesterol Efflux.

Atherosclerosis is caused by cholesterol accumulation within arteries. The intima is where atherosclerotic plaque accumulates and where lipid-laden foam cells reside. Intimal foam cells comprise of both monocyte-derived macrophages and macrophage-like cells (MLC) of vascular smooth muscle cell (VSMC) origin. Foam cells can remove cholesterol via apoAI-mediated cholesterol efflux and this process is regulated by the transporter ABCA1. The microRNA miR-33a-5p is thought to be atherogenic via silencing ABCA1 which promotes cholesterol retention and data has shown inhibiting miR-33a-5p in macrophages may be atheroprotective via enhancing apoAI-mediated cholesterol efflux. However, it is not entirely elucidated whether precisely inhibiting miR-33a-5p in MLC also increases ABCA1-dependent cholesterol efflux. Therefore, the purpose of this work is to test the hypothesis that inhibition of miR-33a-5p in cultured MLC enhances apoAI-mediated cholesterol efflux. In our study, we utilized the VSMC line MOVAS cells in our experiments, and cholesterol-loaded MOVAS cells to convert this cell line into MLC. Inhibition of miR-33a-5p was accomplished by transducing cells with a lentivirus that expresses an antagomiR directed at miR-33a-5p. Expression of miR-33a-5p was analyzed by qRT-PCR, ABCA1 protein expression was assessed via immunoblotting, and apoAI-mediated cholesterol efflux was measured using cholesterol efflux assays. In our results, we demonstrated that lentiviral vector-mediated knockdown of miR-33a-5p resulted in decreasing expression of this microRNA in cultured MLC. Moreover, reduction of miR-33a-5p in cultured MLC resulted in de-repression of ABCA1 expression, which caused ABCA1 protein upregulation in cultured MLC. Additionally, this increase in ABCA1 protein expression resulted in enhancing ABCA1-dependent cholesterol efflux through increasing apoAI-mediated cholesterol efflux in cultured MLC. From these findings, we conclude that inhibiting miR-33a-5p in MLC may protect against atherosclerosis by promoting ABCA1-dependent cholesterol efflux.

Investigating the Effect of Aspirin on apoAI-Induced ATP Binding Cassette Transporter 1 Protein Expression and Cholesterol Efflux in Human Astrocytes.

Neurons need a high amount of cholesterol to maintain the stability of their membrane-rich structures. Astrocytes synthesize and distribute cholesterol to neurons, and ABCA1 is a key mediator of cholesterol efflux to generate HDL for cholesterol transport in the brain. Several studies imply the effect of aspirin on ABCA1 expression in peripheral cells such as macrophages. Here, we compared the effect of aspirin with apoA-I on ABCA1 protein expression and cholesterol efflux in human astrocytes. Human astrocytes were cultured, and the effects of aspirin on the expression and protein levels of ABCA1 were investigated through RT-PCR and Western blot analysis. Additionally, the effect of co-treatment with apoA-I and aspirin on ABCA1 protein level and cholesterol efflux was evaluated. Dose and time-course experiments showed that the maximum effect of aspirin on ABCA1 expression occurred at a concentration of 0.5 mM after 12 h of incubation. RT-PCR and western blot data showed that aspirin upregulates ABCA1 expression by up to 4.7-fold and its protein level by 67%. Additionally, co-treatment with aspirin and apoA-I increased cholesterol release from astrocytes, indicating an additive effect of aspirin on apoAI-mediated cholesterol efflux. The results suggest a potential role of aspirin in increasing ABCA1 expression and cholesterol efflux in astrocytes, similar to the effect of apoA-I. This indicates that aspirin could potentially regulate brain cholesterol balance and can be considered in certain neurological diseases, in particular in some neurological disorders related to cholesterol accumulation such as Alzheimer's disease.

Dissecting the Impact of Vascular Smooth Muscle Cell ABCA1 versus ABCG1 Expression on Cholesterol Efflux and Macrophage-like Cell Transdifferentiation: The Role of SR-BI.

Cholesterol-laden macrophages are recognized as a major contributor to atherosclerosis. However, recent evidence indicates that vascular smooth muscle cells (VSMC) that accumulate cholesterol and transdifferentiate into a macrophage-like cell (MLC) phenotype also play a role in atherosclerosis. Therefore, removing cholesterol from MLC may be a potential atheroprotective strategy. The two transporters which remove cholesterol from cells are ABCA1 and ABCG1, as they efflux cholesterol to apoAI and HDL, respectively. In this study, the well-characterized immortalized VSMC line MOVAS cells were edited to generate ABCA1- and ABCG1-knockout (KO) MOVAS cell lines. We cholesterol-loaded ABCA1-KO MOVAS cells, ABCG1-KO MOVAS cells, and wild-type MOVAS cells to convert cells into a MLC phenotype. When we measured apoAI- and HDL-mediated cholesterol efflux in these cells, we observed a drastic decrease in apoAI-mediated cholesterol efflux within ABCA1-KO MOVAS MLC, but HDL-mediated cholesterol efflux was only partially reduced in ABCG1-KO MOVAS cells. Since SR-BI also participates in HDL-mediated cholesterol efflux, we assessed SR-BI protein expression in ABCG1-KO MOVAS MLC and observed SR-BI upregulation, which offered a possible mechanism explaining why HDL-mediated cholesterol efflux remains maintained in ABCG1-KO MOVAS MLC. When we used lentivirus for shRNA-mediated knockdown of SR-BI in ABCG1-KO MOVAS MLC, this decreased HDL-mediated cholesterol efflux when compared to ABCG1-KO MOVAS MLC with unmanipulated SR-BI expression. Taken together, these major findings suggest that SR-BI expression in MLC of a VSMC origin plays a compensatory role in HDL-mediated cholesterol efflux when ABCG1 expression becomes impaired and provides insight on SR-BI demonstrating anti-atherogenic properties within VSMC/MLC.

Constructing Lipoparticles Capable of Endothelial Cell-Derived Exosome-Mediated Delivery of Anti-miR-33a-5p to Cultured Macrophages.

Atherosclerosis is driven by intimal arterial macrophages accumulating cholesterol. Atherosclerosis also predominantly occurs in areas consisting of proinflammatory arterial endothelial cells. At time of writing, there are no available clinical treatments that precisely remove excess cholesterol from lipid-laden intimal arterial macrophages. Delivery of anti-miR-33a-5p to macrophages has been shown to increase apoAI-mediated cholesterol efflux via ABCA1 upregulation but delivering transgenes to intimal arterial macrophages is challenging due to endothelial cell barrier integrity. In this study, we aimed to test whether lipoparticles targeting proinflammatory endothelial cells can participate in endothelial cell-derived exosome exploitation to facilitate exosome-mediated transgene delivery to macrophages. We constructed lipoparticles that precisely target the proinflammatory endothelium and contain a plasmid that expresses XMOTIF-tagged anti-miR-33a-5p (LP-pXMoAntimiR33a5p), as XMOTIF-tagged small RNA demonstrates the capacity to be selectively shuttled into exosomes. The cultured cells used in our study were immortalized mouse aortic endothelial cells (iMAECs) and RAW 264.7 macrophages. From our results, we observed a significant decrease in miR-33a-5p expression in macrophages treated with exosomes released basolaterally by LPS-challenged iMAECs incubated with LP-pXMoAntimiR33a5p when compared to control macrophages. This decrease in miR-33a-5p expression in the treated macrophages caused ABCA1 upregulation as determined by a significant increase in ABCA1 protein expression in the treated macrophages when compared to the macrophage control group. The increase in ABCA1 protein also simulated ABCA1-dependent cholesterol efflux in treated macrophages-as we observed a significant increase in apoAI-mediated cholesterol efflux-when compared to the control group of macrophages. Based on these findings, strategies that involve combining proinflammatory-targeting lipoparticles and exploitation of endothelial cell-derived exosomes appear to be promising approaches for delivering atheroprotective transgenes to lipid-laden arterial intimal macrophages.

The Impact of MiR-33a-5p Inhibition in Pro-Inflammatory Endothelial Cells.

Evidence suggests cholesterol accumulation in pro-inflammatory endothelial cells (EC) contributes to triggering atherogenesis and driving atherosclerosis progression. Therefore, inhibiting miR-33a-5p within inflamed endothelium may prevent and treat atherosclerosis by enhancing apoAI-mediated cholesterol efflux by upregulating ABCA1. However, it is not entirely elucidated whether inhibition of miR-33a-5p in pro-inflammatory EC is capable of increasing ABCA1-dependent cholesterol efflux. In our study, we initially transfected LPS-challenged, immortalized mouse aortic EC (iMAEC) with either pAntimiR33a5p plasmid DNA or the control plasmid, pScr. We detected significant increases in both ABCA1 protein expression and apoAI-mediated cholesterol efflux in iMAEC transfected with pAntimiR33a5p when compared to iMAEC transfected with pScr. We subsequently used polymersomes targeting inflamed endothelium to deliver either pAntimiR33a5p or pScr to cultured iMAEC and showed that the polymersomes were selective in targeting pro-inflammatory iMAEC. Moreover, when we exposed LPS-challenged iMAEC to these polymersomes, we observed a significant decrease in miR-33a-5p expression in iMAEC incubated with polymersomes containing pAntimR33a5p versus control iMAEC. We also detected non-significant increases in both ABCA1 protein and apoAI-mediated cholesterol in iMAEC exposed to polymersomes containing pAntimR33a5p when compared to control iMAEC. Based on our results, inhibiting miR-33a-5p in pro-inflammatory EC exhibits atheroprotective effects, and so precisely delivering anti-miR-33a-5p to these cells is a promising anti-atherogenic strategy.

Abstract 9535: The Effect of Vascular Smooth Muscle Cell miR-33a Expression on apoAI-Mediated Cholesterol Efflux and Macrophage-Like Cell Transdifferentiation

Background: ABCA1 removes cellular cholesterol by participating in apoAI-mediated cholesterol efflux. MiR-33a is an SREBP-2 intronic miRNA that silences ABCA1, which impairs ABCA1-dependent cholesterol efflux. Thus, miR-33a expression may be atherogenic and previous data suggests inhibiting miR-33a in certain cells is atheroprotective. However, current data is scant on whether miR-33a expression in vascular smooth muscle cells (VSMC) is atherogenic. Moreover, cholesterol accumulation in VSMC results in these cells to transdifferentiate into an atherogenic macrophage-like cell (MLC). Therefore, ablating miR-33a expression in VSMC may be atheroprotective via hindering cholesterol accumulation and MLC transdifferentiation in these cells by enhancing apoAI-mediated cholesterol efflux. Methods: An immortalized VSMC line MOVAS cells were used to generate miR-33a knockout (KO) MOVAS cells via using CRISPR/Cas9. We used these cells and parental wild-type (WT) MOVAS cells in our studies to assess whether apoAI-mediated cholesterol efflux is increased in MLC of VSMC origin when miR-33a expression is ablated. We also analyzed whether miR-33a ablation rapidly restores VSMC phenotype in MLC of VSMC origin when cholesterol-loaded cells were exposed to apoAI. Results: We used PCR to confirm that miR-33a was deleted in miR-33a KO MOVAS cells. We also showed that miR-33a-5p and miR-33a-3p expression was absent in miR-33a KO MOVAS cells via RT-PCR. SREBP-2 functionality remained intact in miR-33a KO MOVAS cells, as determined by upregulation of SREBP-2 target genes in serum-starved conditions. When we cholesterol-loaded miR-33a KO MOVAS cells to induce MLC transdifferentiation, we observed increased apoAI-mediated cholesterol efflux when compared to WT MOVAS cells exposed to the same conditions. Based on RT-qPCR analysis, we also observed a rapid restoration in VSMC phenotype when cholesterol-loaded miR-33a KO MOVAS cells were incubated with apoAI when compared to WT MOVAS cells treated with the same conditions. Conclusions: MiR-33a expression in VSMC appears to promote MLC transdifferentiation by impairing apoAI-mediated cholesterol efflux. Future studies are underway to determine if these effects are mediated by miR-33a-5p and/or miR-33a-3p.

Utilizing the LoxP-Stop-LoxP System to Control Transgenic ABC-Transporter Expression In Vitro.

ABCA1 and ABCG1 are two ABC-transporters well-recognized to promote the efflux of cholesterol to apoAI and HDL, respectively. As these two ABC-transporters are critical to cholesterol metabolism, several studies have assessed the impact of ABCA1 and ABCG1 expression on cellular cholesterol homeostasis through ABC-transporter ablation or overexpressing ABCA1/ABCG1. However, for the latter, there are currently no well-established in vitro models to effectively induce long-term ABC-transporter expression in a variety of cultured cells. Therefore, we performed proof-of-principle in vitro studies to determine whether a LoxP-Stop-LoxP (LSL) system would provide Cre-inducible ABC-transporter expression. In our studies, we transfected HEK293 cells and the HEK293-derived cell line 293-Cre cells with ABCA1-LSL and ABCG1-LSL-based plasmids. Our results showed that while the ABCA1/ABCG1 protein expression was absent in the transfected HEK293 cells, the ABCA1 and ABCG1 protein expression was detected in the 293-Cre cells transfected with ABCA1-LSL and ABCG1-LSL, respectively. When we measured cholesterol efflux in transfected 293-Cre cells, we observed an enhanced apoAI-mediated cholesterol efflux in 293-Cre cells overexpressing ABCA1, and an HDL2-mediated cholesterol efflux in 293-Cre cells constitutively expressing ABCG1. We also observed an appreciable increase in HDL3-mediated cholesterol efflux in ABCA1-overexpressing 293-Cre cells, which suggests that ABCA1 is capable of effluxing cholesterol to small HDL particles. Our proof-of-concept experiments demonstrate that the LSL-system can be used to effectively regulate ABC-transporter expression in vitro, which, in turn, allows ABCA1/ABCG1-overexpression to be extensively studied at the cellular level.

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.

Non-lethal sonodynamic therapy facilitates the M1-to-M2 transition in advanced atherosclerotic plaques via activating the ROS–AMPK–mTORC1–autophagy pathway

Emerging evidence indicates that macrophage functional polarization is critically involved in the development of atherosclerosis (AS). Here, we examined the role of 5-aminolaevulinic acid (ALA)-mediated non-lethal sonodynamic therapy (NL-SDT) in macrophage-subset polarization and atherosclerotic lesion stability and explored the potential underlying mechanisms. Using Western diet-fed apolipoprotein E (apoE)−/− and green fluorescent protein (GFP)-positive bone marrow (BM) chimeric mouse models, we demonstrated that NL-SDT promoted phenotypic switching of both BM-derived and resident macrophages from M1 to M2 and significantly inhibited AS progression. Further mechanistic studies indicated that NL-SDT enhanced macrophage differentiation toward the M2 phenotype by activating the reactive oxygen species (ROS)–5′ AMP-activated protein kinase (AMPK)–mammalian target of rapamycin complex 1 (mTORC1)–autophagy signaling pathway in murine BM-derived M1 macrophages (BMDM1s). Moreover, NL-SDT drastically reduced lipid droplets, mainly by promoting apoAI-mediated cholesterol efflux in vitro. Specifically, administration of pharmacological inhibitors to the animal model showed a reciprocal effect on NL-SDT-induced macrophage polarization. These findings indicate that NL-SDT engages a virtuous cycle that enhances M1-to-M2 polarization, cholesterol efflux, and anti-inflammatory reactions in advanced plaque in vivo and in BMDM1s in vitro by activating the ROS–AMPK–mTORC1–autophagy pathway. This discovery might help elucidate the mechanism underlying NL-SDT as a potential treatment to prevent atherothrombotic events.

Exosome-Mediated Transfer of Anti-miR-33a-5p from Transduced Endothelial Cells Enhances Macrophage and Vascular Smooth Muscle Cell Cholesterol Efflux.

Atherosclerosis is a disease of large- and medium-sized arteries that is caused by cholesterol accumulation in arterial intimal cells, including macrophages and smooth muscle cells (SMC). Cholesterol accumulation in these cells can be prevented or reversed in preclinical models-and atherosclerosis reduced-by transgenesis that increases expression of molecules that control cholesterol efflux, including apolipoprotein AI (apoAI) and ATP-binding cassette subfamily A, member 1 (ABCA1). In a previous work, we showed that transduction of arterial endothelial cells (EC)-with a helper-dependent adenovirus (HDAd) expressing apoAI-enhanced EC cholesterol efflux in vitro and decreased atherosclerosis in vivo. Similarly, overexpression of ABCA1 in cultured EC increased cholesterol efflux and decreased inflammatory gene expression. These EC-targeted gene-therapy strategies might be improved by concurrent upregulation of cholesterol-efflux pathways in other intimal cell types. Here, we report modification of this strategy to enable delivery of therapeutic nucleic acids to cells of the sub-endothelium. We constructed an HDAd (HDAdXMoAntimiR33a5p) that expresses an antagomiR directed at miR-33a-5p (a microRNA that suppresses cholesterol efflux by silencing ABCA1). HDAdXMoAntimiR33a5p contains a sequence motif that enhances uptake of anti-miR-33a-5p into exosomes. Cultured EC release exosomes containing small RNA, including miR-33a-5p. After transduction with HDAdXMoAntimiR33a5p, EC-derived exosomes containing anti-miR-33a-5p accumulate in conditioned medium (CM). When this CM is added to macrophages or SMC, anti-miR-33a-5p is detected in these target cells. Exosome-mediated transfer of anti-miR-33a-5p reduces miR-33a-5p by ∼65-80%, increases ABCA1 protein by 1.6-2.2-fold, and increases apoAI-mediated cholesterol efflux by 1.4-1.6-fold (all p ≤ 0.01). These effects were absent in macrophages and SMC incubated in exosome-depleted CM. EC transduced with HDAdXMoAntimiR33a5p release exosomes that can transfer anti-miR-33a-5p to other intimal cell types, upregulating cholesterol efflux from these cells. This strategy provides a platform for genetic modification of intimal and medial cells, using a vector that transduces only EC.

Abstract 884: Expression of Anti-mir-33a-5p in Cultured Endothelial Cells Increases Macrophage and Smooth Muscle Cell Cholesterol Efflux by Exosome-mediated Transfer of Anti-mir-33a-5p

Background: The microRNA miR-33a-5p impairs apoAI-mediated cholesterol efflux by silencing ABCA1 expression. Systemic injection of antagomirs to miR-33a-5p reduces atherosclerosis but can cause hepatic steatosis and hypertriglyceridemia. In contrast, local delivery of miR-33a-5p antagomirs to lipid-laden intimal cells [smooth muscle cells (SMC) and macrophages (Mϕ)] could potentially treat atherosclerosis without causing metabolic abnormalities. Helper-dependent adenoviral vectors (HDAd) provide durable transgene expression in endothelial cells (EC) in vivo; however, HDAd does not cross an intact large vessel EC layer and therefore cannot deliver antagomirs directly to SMC and Mϕ. We hypothesized that transducing EC with an HDAd expressing anti-miR-33a-5p (HDAdAnti) could result in packaging of anti-miR-33a-5p into EC-derived exosomes that, in turn, deliver anti-miR-33a-5p to neighboring SMC and Mϕ. Inhibition of miR-33a-5p in SMC and Mϕ would upregulate ABCA1, and enhance apoAI-mediated cholesterol efflux. Methods: We transduced cultured EC with either HDAdAnti or a control HDAd expressing a “scrambled” shRNA (HDAdScr), harvested conditioned medium (CM) from the EC, and treated SMC and Mϕ (some of which were cholesterol-loaded) with the CM. We measured levels of anti-miR-33a-5p and miR-33a-5p, ABCA1 protein, and apoAI-mediated cholesterol efflux in the treated SMC and Mϕ. Results: Exosomes containing anti-miR-33a-5p were present only in CM from HDAdAnti-transduced EC. Compared to Mϕ incubated with CM from EC transduced with HDAdScr, Mϕ incubated with CM from HDAdAnti-transduced EC had 80% lower levels of miR-33a-5p, a 2.2-fold increase in ABCA1 protein, and a 1.6-fold increase in apoAI-mediated cholesterol efflux (all P<0.001). Similar results were obtained with SMC: 65% lower levels of miR-33a-5p expression, 1.6-fold increase in ABCA1 protein, and 1.4-fold increase in apoAI-mediated cholesterol efflux (all P≤0.01). Conclusions: EC transduced with HDAdAnti release exosomes containing anti-miR-33a-5p. These exosomes transfer anti-miR-33a-5p to SMC and Mϕ, increasing cholesterol efflux. Application of this approach in vivo may accelerate vessel wall cholesterol efflux, preventing and reversing atherosclerosis.

ABCA1 Overexpression in Endothelial Cells In Vitro Enhances ApoAI-Mediated Cholesterol Efflux and Decreases Inflammation.

Atherosclerosis, a disease of blood vessels, is driven by cholesterol accumulation and inflammation. Gene therapy that removes cholesterol from blood vessels and decreases inflammation is a promising approach for prevention and treatment of atherosclerosis. In previous work, we reported that helper-dependent adenoviral (HDAd) overexpression of apolipoprotein A-I (apoAI) in endothelial cells (ECs) increases cholesterol efflux in vitro and reduces atherosclerosis in vivo. However, the effect of HDAdApoAI on atherosclerosis is partial. To improve this therapy, we considered concurrent overexpression of ATP-binding cassette subfamily A, member 1 (ABCA1), a protein that is required for apoAI-mediated cholesterol efflux. Before attempting combined apoAI/ABCA1 gene therapy, we tested whether an HDAd that expresses ABCA1 (HDAdABCA1) increases EC cholesterol efflux, whether increased cholesterol efflux alters normal EC physiology, and whether ABCA1 overexpression in ECs has anti-inflammatory effects. HDAdABCA1 increased EC ABCA1 protein (∼3-fold; p < 0.001) and apoAI-mediated cholesterol efflux (2.3-fold; p = 0.007). Under basal culture conditions, ABCA1 overexpression did not alter EC proliferation, metabolism, migration, apoptosis, nitric oxide production, or inflammatory gene expression. However, in serum-starved, apoAI-treated EC, ABCA1 overexpression had anti-inflammatory effects: decreased inflammatory gene expression (∼50%; p ≤ 0.02 for interleukin [IL]-6, tumor necrosis factor [TNF]-α, and vascular cell adhesion protein-1); reduced lipid-raft Toll-like receptor 4 (80%; p = 0.001); and a trend towards increased nitric oxide production (∼55%; p = 0.1). In ECs stimulated with lipopolysaccharide, ABCA1 overexpression markedly decreased inflammatory gene expression (∼90% for IL-6 and TNF-α; p < 0.001). Therefore, EC ABCA1 overexpression has no toxic effects and counteracts the two key drivers of atherosclerosis: cholesterol accumulation and inflammation. In vivo testing of HDAdABCA1 is warranted.

Abstract 628: Overexpression of ABCA1 in Cultured Endothelial Cells Using Helper-Dependent Adenovirus Enhances ApoAI-Mediated Cholesterol Efflux and has Anti-Inflammatory Effects

Background: ABCA1 removes cholesterol from vascular wall cells via apoAI-mediated efflux, generating HDL that transports cholesterol to the liver for excretion. This process of reverse cholesterol transport is atheroprotective; therefore, strategies that increase vascular wall ABCA1 may prevent or reverse atherosclerosis. Cholesterol efflux mediated by ABCA1 and apoAI can also have anti-inflammatory effects; however, excess depletion of cellular cholesterol can cause cell stress and apoptosis. We tested whether transducing endothelial cells (EC) with a helper-dependent adenoviral vector that expresses ABCA1 (HDAdABCA1) enhances apoAI-mediated cholesterol efflux and reduces inflammatory markers without causing cellular toxicity. Methods: We cloned rabbit ABCA1, constructed HDAdABCA1, transduced bovine aortic EC (BAEC) with either HDAdABCA1 or empty vector (HDAdNull). We measured ABCA1 protein by immunoblotting and apoAI-mediated cholesterol efflux by loading EC with 3 [H] cholesterol, then adding apoAI protein to serum-free medium. We assessed EC phenotype using MTT (metabolic activity), BrdU (proliferation), wound-healing assay (migration), and flow cytometry (apoptosis). We measured ICAM-1, VCAM-1, IL-6, and TNFα mRNA in transduced EC by qRT-PCR both under basal conditions and after serum-starvation, addition of apoAI, and LPS challenge. Results: We observed a ~3-fold increase in ABCA1 protein in EC transduced with HDAdABCA1 and a ~2-fold increase in apoAI-mediated cholesterol efflux (P&lt;0.01 for both). This level of ABCA1 overexpression did not alter EC metabolic activity, proliferation, migration, or apoptosis. Under basal conditions, HDAdABCA1 had no effect on inflammatory markers. However, after serum starvation and addition of apoAI, HDAdABCA1-transduced EC had reduced expression of inflammatory markers both before and after LPS treatment. Conclusions: HDAdABCA1 increases EC ABCA1 expression and enhances apoAI-mediated cholesterol efflux, but does not cause toxicity or increase inflammatory markers. In contrast, ABCA1 overexpression appears to have anti-inflammatory effects. Future studies will test if HDAdABCA1 decreases lipid rafts, and whether overexpression of ABCA1 in EC in vivo is atheroprotective.

The Impact of Serum Amyloid P-Component on Gene Expression in RAW264.7 Mouse Macrophages.

Serum amyloid P-component (SAP) contributes to host defense and prevents fibrosis. Macrophages are the most abundant inflammatory cell type in atherosclerotic plaques. In the present study, using 3H-cholesterol-labeled counting radioactivity assay, we demonstrated that the apoAI-mediated cholesterol efflux in RAW264.7 macrophages was increased by SAP treatment in a time- and dose-dependent manner. We analyzed global gene expression changes upon SAP treatment using RNA sequencing. As a result, a total of 175 differentially expressed genes were identified, of which 134 genes were downregulated and 41 genes were upregulated in SAP treated cells compared to control cells. Quantitative RT-PCR analysis confirmed decreased expression of 5 genes and an increase in expression of 1 gene upon SAP treatment. Gene ontology analysis showed that genes involved in response to stimulus were significantly enriched in differentially expressed genes. Beyond protein-coding genes, we also identified 8 differentially expressed long noncoding RNAs. Our study may provide new insights into mechanisms underlying the functional role of SAP in macrophages.

A Subregion of Reelin Suppresses Lipoprotein-Induced Cholesterol Accumulation in Macrophages.

Activation of apolipoprotein E receptor-2 (apoER2) and very low density lipoprotein receptor (VLDLR) inhibits foam cell formation. Reelin is a ligand of these receptors. Here we generated two reelin subregions containing the receptor binding domain with or without its C-terminal region (R5-6C and R5-6, respectively) and studied the impact of these peptides on macrophage cholesterol metabolism. We found that both R5-6C and R5-6 can be secreted by cells. Purified R5-6 protein can bind apoER2 and VLDLR. Overexpression of apoER2 in macrophages increased the amount of R5-6 bound to the cell surface. Treatment of macrophages with 0.2 μg/ml R5-6 elevated ATP binding cassette A1 (ABCA1) protein level by ~72% and apoAI-mediated cholesterol efflux by ~39%. In addition, the medium harvested from cells overexpressing R5-6 or R5-6C (R5-6- and R5-6C-conditioned media, respectively) also up-regulated ABCA1 protein expression, which was associated with accelerated cholesterol efflux and enhanced phosphorylation of phosphatidylinositol 3 kinase (PI3K) and specificity protein-1 (Sp1) in macrophages. The increased ABCA1 expression and cholesterol efflux by R5-6- and R5-6C-conditioned media were diminished by Sp1 or PI3K inhibitors mithramycin A and LY294002. Further, the cholesterol accumulation induced by apoB-containing, apoE-free lipoproteins was significantly less in macrophages incubated with R5-6- or R5-6C-conditioned medium than in those incubated with control conditioned medium. Knockdown of apoER2 or VLDLR attenuated the inhibitory role of R5-6-conditioned medium against lipoprotein-induced cholesterol accumulation. These results suggest that the reelin subregion R5-6 can serve as a tool for studying the role of apoER2 and VLDLR in atherogenesis.

Abstract 706: HDL and ApoA-I Inhibit Palmitate-induced Lipid Raft Trafficking of TLR4 and Inflammatory Cytokines Expression in Adipocytes

Background: Adipose tissue inflammation induces inflammatory molecules production leading to insulin resistance and increased cardiovascular disease risk. Recently it has become apparent that innate immune system is activated by saturated fatty acids (SFAs) by a process involved in TLR4 signaling. HDL and apoA-I has anti-inflammatory effects, however the mechanisms are not well understood in adipocytes. Aims: We examined to elucidate the effect of HDL and/or ApoA-I mediated cholesterol efflux on induction of inflammatory gene expression by palmitate in adipocytes via TLR4 dependent immune system. Methods: 3T3-L1 adipocytes are exposure to 250uM palmitate for periods of up to 24 hours with or without HDL or Apo A-I. Antisense directed to ABCG-1 or ABCA-1 is added 24 hours prior to each reagent challenge. Cells were harvested and estimated for expression of Saa 3 gene using real-time PCR or detection of TLR4 in raft or non raft by western blotting. Lipid rafts were purified by ultracentrifugation on discontinuous sucrose gradient, and identified by the presence of caveolin-1. Results: TLR4 was translocated into lipid raft from non raft within 15 minutes and dislocated by 60 minutes after palmitate exposure. Saa 3 gene expression was significantly up-regulated by 24 hours palmitate exposure in adipocytes. TLR4 translocation to raft and Saa 3 gene up- regulation induced by palmitate was significantly inhibited by pretreatment of HDL or Apo A-I for 6 hours. Antisense directed to ABCG-1 or SRB1 abrogated these inhibitory effects by HDL, but not ApoA-I whereas antisense to ABCA-1 did by Apo A-I but not HDL. Conclusions: These findings suggest that TLR4 mediates palmitate-induced Saa 3 gene expression in adipocytes. A reduction in membrane cholesterol mediated by ABCG-1, SRB1 or ABCA-1 can account for the ability of HDL or ApoA-I to disrupt stimulation of inflammatory cytokines by palmitate

Curcumin inhibits monocyte chemoattractant protein-1 expression and enhances cholesterol efflux by suppressing the c-Jun N-terminal kinase pathway in macrophage.

To investigate the effect of curcumin on monocyte chemoattractant protein 1 (MCP-1) production and reverse cholesterol transport (RCT) in macrophage induced by oxidation low-density lipoprotein (ox-LDL), and to identify the signal pathways involved. The macrophages were treated with ox-LDL and various concentrations of curcumin simultaneously. The MCP-1 expression was measured by enzyme-linked immunosorbent assay. The apoAI-mediated cholesterol efflux was measured by (3)H-cholesterol-labeled counting radioactivity. The activation of intracellular signaling pathways was studied by Western blotting. Curcumin decreased the production of MCP-1 induced by ox-LDL in macrophages. MCP-1 expression was restrained by the inhibition of c-Jun N-terminal kinase (JNK) pathway (SP600125) and NF-κB pathway (BAY11-7082). Curcumin suppressed the phosphorylation of JNK and activation of NF-κB. Curcumin also enhanced RCT via up-regulating the expression of liver X receptor alpha (LXRα), ATP-binding cassette transporter A1 (ABCA1) and scavenger receptor class B type I (SR-BI). Additionally, the inhibition of JNK (SP600125) increased cholesterol efflux and increased the expression of ABCA1 and SR-BI, but had no effect on LXRα. Curcumin suppresses MCP-1 production induced by ox-LDL via the JNK pathway and NK-κB pathway, while enhances cholesterol efflux in macrophage via suppressing the JNK pathway and activating the LXR-ABCA1/SR-BI pathway, which indicate that the vascular protective effect of curcumin is related to anti-inflammation and anti-atherosclerosis.

Adoptive transfer of gene-modified T-cells engineered to express high-affinity tcr's for cancer-testis antigens NY-ESO-1 or lage-1, in multiple myeloma (MM) patients post autologous hematopoietic stem cell transplant (ASCT)

Advanced atherosclerotic lesions are characterized by lipid accumulation, chronic inflammation, and defective efferocytosis; an ideal therapy should address all aspects of this multi-factorial disease. Ixmyelocel-T, an expanded autologous multicellular therapy showing clinical promise in the treatment of severe diseases associated with atherosclerosis, includes a novel population of M2-like macrophages. We examined the properties of these macrophages to determine their potential mechanisms of action in atherosclerotic states. Flow cytometry revealed that ixmyelocel-T macrophages express surface receptors of alternative activation (CD206 and CD163) and scavenger receptors involved in lipid and apoptotic cell uptake (CD36, LDLR, CD91, MSR1, and MerTK). ELISA analysis revealed ixmyelocel-T macrophages secrete substantial levels of IL-10 (1060 278 vs. 1978 313 pg/ml, p < 0.05) and IL-1ra (12673 1242 vs. 37576 3650 pg/ml, p < 0.001) before and after overnight LPS stimulation. Baseline secretion of the pro-inflammatory cytokines IL-1b, TNFa, and IL-12 is minimal and remains low after LPS stimulation. Fluorescent microscopy and flow cytometry revealed that ixmyelocel-T readily phagocytosed apoptotic cells. Apoptosis analyzed by a caspase 3/7 assay demonstrated that ixmyelocel-T isn’t sensitive to the lipotoxic effects of modified cholesterol (87 10.78 vs. 100 14.58% of control). Real time PCR revealed that the cholesterol transporter genes ABAC1 (1.49 0.32 vs. 0.42 0.11 fold change, p < 0.01) and ABCG1 (0.41 0.13 vs. 0.07 0.03 fold change, p < 0.05) are both significantly upregulated when ixmyelocel-T macrophages are loaded with cholesterol. Ixmyelocel-T also exhibits enhanced ApoAI mediated cholesterol efflux (13.91 1.90 vs. 3.42 0.31%, p < 0.001 compared to THP-1). In addition, ixmyelocel-T effluxed cholesterol in vivo in using a mouse model for reverse cholesterol transport. These biological properties of ixmyelocel-T macrophages suggest that this cellular therapy may exert beneficial effects on atherosclerotic disease by providing macrophages that potentially limit atheroma development through secretion of anti-inflammatory cytokines, removal of apoptotic cells, and promotion of cholesterol efflux.

Apolipoprotein E4 Is Deficient in Inducing Macrophage ABCA1 Expression and Stimulating the Sp1 Signaling Pathway

ATP binding cassette A1 (ABCA1) is a membrane protein that promotes cellular cholesterol efflux. Using RAW 264.7 macrophages, we studied the relative effects of apolipoprotein (apo) E3 and apoE4 on ABCA1 and on the signaling pathway that regulates its expression. Both lipid-associated and lipid-free apoE4 forms induced ∼30% lower levels of ABCA1 protein and mRNA than apoE3 forms. Phosphorylated levels of phosphoinositol 3-kinase (PI3K), protein kinase Cζ (PKCζ) and specificity protein 1 (Sp1) were also lower when treated with apoE4 compared to apoE3. The reduced ability of apoE4 to induce ABCA1 expression, PKCζ and Sp1 phosphorylation were confirmed in human THP-1 monocytes/macrophages. Sequential phosphorylation of PI3K, PKCζ and Sp1 has been suggested as a mechanism for upregulation of ABCA1 expression. Both apoE3 and apoE4 reduced total cholesterol and cholesterol esters in lipid-laden RAW 264.7 cells, and induced apoAI-mediated cholesterol efflux. However, the cholesterol esters and cholesterol efflux in apoE4-treated cells were ∼50% and ∼24% lower, respectively, compared to apoE3-treated cells. Accumulation of cholesterol esters in macrophages is a mechanism for foam cell formation. Thus the reduced ability of apoE4 to activate the PI3K-PKCζ-Sp1 signaling pathway and induce ABCA1 expression likely impairs cholesterol ester removal, and increases foam cell formation.

Up-regulation of ATP Binding Cassette Transporter A1 Expression by Very Low Density Lipoprotein Receptor and Apolipoprotein E Receptor 2

Activation of very low density lipoprotein receptor (VLDLR) and apolipoprotein E receptor 2 (apoER2) results in either pro- or anti-atherogenic effects depending on the ligand. Using reelin and apoE as ligands, we studied the impact of VLDLR- and apoER2-mediated signaling on the expression of ATP binding cassette transporter A1 (ABCA1) and cholesterol efflux using RAW264.7 cells. Treatment of these mouse macrophages with reelin or human apoE3 significantly increased ABCA1 mRNA and protein levels, and apoAI-mediated cholesterol efflux. In addition, both reelin and apoE3 significantly increased phosphorylated disabled-1 (Dab1), phosphatidylinositol 3-kinase (PI3K), protein kinase Cζ (PKCζ), and specificity protein 1 (Sp1). This reelin- or apoER2-mediated up-regulation of ABCA1 expression was suppressed by 1) knockdown of Dab1, VLDLR, and apoER2 with small interfering RNAs (siRNAs), 2) inhibition of PI3K and PKC with kinase inhibitors, 3) overexpression of kinase-dead PKCζ, and 4) inhibition of Sp1 DNA binding with mithramycin A. Activation of the Dab1-PI3K signaling pathway has been implicated in VLDLR- and apoER2-mediated cellular functions, whereas the PI3K-PKCζ-Sp1 signaling cascade has been implicated in the regulation of ABCA1 expression induced by apoE/apoB-carrying lipoproteins. Taken together, these data support a model in which activation of VLDLR and apoER2 by reelin and apoE induces ABCA1 expression and cholesterol efflux via a Dab1-PI3K-PKCζ-Sp1 signaling cascade.