ABCA1 Expression Research Articles

Abstract P3029: The Role Of Fatty Acid Synthase In The Modulation Of Vascular Smooth Muscle Cells Into A Foam Cell Phenotype

Background: The contractile phenotype of differentiated VSMCs is essential for vascular function. When exposed to Cholesterol (CHO), VSMCs dedifferentiate and acquire characteristics of foam cells, lipid-laden macrophage-like cells that comprise the Atherosclerotic plaques. CHO uptake by VSMCs is either removed by ABCA1-mediated efflux or is stored in lipid droplets as cholesterol esters (CEs). CEs are formed by the reaction of CHO with fatty acids catalyzed by SOAT1. Thus, we hypothesize that fatty acid biosynthesis controls the formation of VSMC-derived foam cells. Results: In VSMCs, CHO treatment upregulates the lipogenic enzymes FASN (3.6±1 vs. 9.0±0.9 AU p<0.01) and acetyl-CoA carboxylase (ACC) (2.8±0.5 vs. 10.2±2.3 AU p=0.02) and induces the phosphorylation of ATP Citrate Lyase (ACLY) (1.4±0 vs. 6.2±1 AU). Consistent with the literature, these changes are accompanied by increased expression of the macrophage-related markers CD68 and KLF4. Significantly, downregulation of FASN inhibits the CHO-induced CD68 (9.1±0.8 vs. 4.6±1,9 p=0.002) and KLF4 (p=0.02) expression upregulation. FASN-deficient VSMCs failed to accumulate lipids and transition to a foam cell phenotype after CHO treatment measured by Oil Red O staining. Interestingly, FASN-deficient VSMCs have decreased expression of SOAT1 (p=0.04) and increased expression of the CHO efflux transporter ABCA1 (p=0.04). Conclusion: Our study shows lipogenic enzymes are upregulated during the VSMC to foam cell transition triggered by CHO treatment. FASN expression is required for VSMC-derived foam cell formation. Our data suggest that FASN participates in the esterification of CHO and foam cell formation and may contribute to atherosclerotic plaque accumulation. Thus, FASN may represent a novel target for therapeutic intervention.

Flindissone, a Limonoid Isolated from Trichilia prieuriana, Is an LXR Agonist.

In this study, the ability of six limonoids from Trichilia prieuriana (Meliaceae) to activate the liver X receptor (LXR) was assessed. One of these limonoids, flindissone, was shown to activate LXR by reporter-gene assays. Flindissone is a ring-intact limonoid, structurally similar to sterol-like LXR ligands. In endogenous cellular settings, flindissone showed an activity profile that is characteristic of LXR agonists. It induced cholesterol efflux in THP-1 macrophages by increasing the cholesterol transporter ABCA1 and ABCG1 gene expression. In HepG2 cells, flindissone induced the expression of IDOL, an LXR-target gene that is associated with the downregulation of the LDL receptor. However, unlike synthetic and similarly to sterol-based LXR agonists, flindissone did not induce the expression of the SREBP1c gene, a major transcription factor regulating de novo lipogenesis. Additionally, flindissone also appeared to be able to inhibit post-translational activation of SREBP1c. The results presented here reveal a natural product as a new LXR agonist and point to an additional property of T. prieuriana and other plant extracts containing flindissone.

The interplay between microbial metabolites and macrophages in cardiovascular diseases: A comprehensive review.

The gut microbiome has emerged as a crucial player in developing and progressing cardiovascular diseases (CVDs). Recent studies have highlighted the role of microbial metabolites in modulating immune cell function and their impact on CVD. Macrophages, which have a significant function in the pathogenesis of CVD, are very vulnerable to the effects of microbial metabolites. Microbial metabolites, such as short-chain fatty acids (SCFAs) and trimethylamine-N-oxide (TMAO), have been linked to atherosclerosis and the regulation of immune functions. Butyrate has been demonstrated to reduce monocyte migration and inhibit monocyte attachment to injured endothelial cells, potentially contributing to the attenuation of the inflammatory response and the progression of atherosclerosis. On the other hand, TMAO, another compound generated by gut bacteria, has been linked to atherosclerosis due to its impact on lipid metabolism and the accumulation of cholesterol in macrophages. Indole-3-propionic acid, a tryptophan metabolite produced solely by microbes, has been found to promote the development of atherosclerosis by stimulating macrophage reverse cholesterol transport (RCT) and raising the expression of ABCA1. This review comprehensively discusses how various microbiota-produced metabolites affect macrophage polarization, inflammation, and foam cell formation in CVD. We also highlight the mechanisms underlying these effects and the potential therapeutic applications of targeting microbial metabolites in treating CVD.

Praeruptorin A inhibits the activation of NF-κB pathway and the expressions of inflammatory factors in poly (I:C)-induced RAW264.7 cells.

Praeruptorin A (PA), a natural coumarin compound, has significant anti-inflammatory effects. In this study, we evaluate the anti-inflammatory effect of PA on RAW 264.7 mouse macrophages induced by Polyinosinic acid-polycytidylic acid (poly (I:C)). RAW 264.7 mouse macrophages induced by poly (I:C) were treated with or without PA, the viability of which was determined to screen working solution of PA. RNA-sequencing was applied to analyze the differentially expressed genes (DEGs). Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis were carried out. The expressions of interleukin (IL)-1β, heme oxygenase 1 (HMOX1), prostaglandin-endoperoxide synthase 2 (PTGS2), ATP binding cassette subfamily A member 1 (Abca1) and NF-κB-related proteins were measured by enzyme-linked immunosorbent assay (ELISA), quantitative reverse transcription polymerase chain reaction (qRT-PCR) and western blot. As a result, PA at 1, 2, 3, 4 and 5 μM slightly affected cell viability, while PA at 6 and 7 μM significantly inhibited cell viability. GO and KEGG analysis results revealed that DEGs were mainly enriched in the pathways related to inflammatory signaling. Through further analysis, we obtained five possible targets of PA, and verified that PA inhibited the expressions of IL-1β, HMOX1, PTGS2 and Abca1 as well as the activation of NF-κB pathway in poly (I:C)-induced RAW264.7 cells. To summarize, PA may inhibit expressions of the inflammation-related genes in poly (I:C)-induced RAW264.7 cells, which demonstrates its potential as a drug against virus related diseases.

Paternal cadmium exposure affects estradiol synthesis by impairing intracellular cholesterol homeostasis and mitochondrial function in offspring female mice

Cadmium (Cd) is a toxic heavy metal commonly found in nature and an endocrine disrupting chemical (EDC). Previous studies found that Cd can damage several organs, including the kidneys, bones, cardiovascular system and reproductive system. However, the effect of paternal Cd exposure on the offspring is unclear. In this study, 1 mg/kg of cadmium chloride (CdCl2) was injected intraperitoneally every other day in 8-week-old C57BL/6 J male mice to study the effects on their female offspring. Our results showed an increase in body weight, water intake and food intake in F1 female mice from the Cd-exposed group. The development of secondary follicles and antral follicles in the ovaries of Cd-treated was inhibited. Serum estradiol (E2) was found to be decreased. Further analysis revealed significant downregulation of StAR, P450scc, 17β-HSD, CYP17A1 and CYP19A1, which are related to E2 synthesis. Serum total cholesterol was increased and free cholesterol was reduced. Total cholesterol in ovarian tissue was decreased. qRT-PCR and Western blot analysis revealed a decrease in the mRNA and protein expression of HMGCR, LDLR, and ABCA1, which are associated with cholesterol homeostasis. Oil red O staining indicated that lipid droplets (LDs) were accumulated in ovarian tissues, while the expression of ATGL and HSL proteins associated with lipid droplet degradation was significantly downregulated. In juvenile female mice, ultrastructural alterations of mitochondria in the ovaries were observed by transmission electron microscopy (TEM). In adult female mice, the expression of proteins associated with mitochondrial dynamics (DRP1 and MFN2) was significantly reduced in the ovaries. Overall, our study suggests that paternal Cd exposure inhibits follicular development, and affects serum E2 synthesis by impairing cholesterol homeostasis and affecting mitochondrial function.

Cholesterol Efflux Drives the Generation of Immunosuppressive Macrophages to Promote the Progression of Human Hepatocellular Carcinoma.

Cholesterol is often enriched in tumor microenvironment (TME); however, its impact on disease progression varies in different tissues and cells. Monocytes/macrophages (Mφ) are major components and regulators of the TME and play pivotal roles in tumor progression and therapeutic responses. We aimed to investigate the profile, effects, and regulatory mechanisms of Mφ cholesterol metabolism in the context of human hepatocellular carcinoma (HCC). Here, we found that patients with high serum levels of cholesterol had shorter survival times and lower response rates to anti-PD-1 treatment. However, the cholesterol content in tumor-infiltrating monocytes/Mφ was significantly lower than that in their counterparts in paired nontumor tissues. The expression of the cholesterol efflux transporter, ABCA1, was upregulated in tumor monocytes/Mφ, and ABCA1 upregulation positively associated with decreased cellular cholesterol content and increased serum cholesterol levels. Mechanistically, autocrine cytokines from tumor-treated monocytes increased LXRα and ABCA1 expression, which led to the generation of immature and immunosuppressive Mφ. Although exogenous cholesterol alone had little direct effect on Mφ, it did act synergistically with tumor-derived factors to promote ABCA1 expression in Mφ with more immunosuppressive features. Moreover, high numbers of ABCA1+ Mφ in HCC tumors associated with reduced CD8+ T-cell infiltration and predicted poor clinical outcome for patients. Our results revealed that dysregulated cholesterol homeostasis, due to the collaborative effects of tumors and exogenous cholesterol, drives the generation of immunosuppressive Mφ. The selective modulation of cholesterol metabolism in Mφ may represent a novel strategy for cancer treatment.

Medium-chain fatty acids modify macrophage expression of metabolic and inflammatory genes in a PPAR β/δ-dependent manner

There is great interest on medium chain fatty acids (MCFA) for cardiovascular health. We explored the effects of MCFA on the expression of lipid metabolism and inflammatory genes in macrophages, and the extent to which they were mediated by the nuclear receptor peroxisome proliferator-activated receptor beta/delta (PPAR β/δ). J774A.1 murine macrophages were exposed to octanoate or decanoate as MCFA, a long-chain fatty acid control (palmitate), or the PPAR β/δ agonist GW501516, with or without lipopolysaccharide (LPS) stimulation, and with or without an siRNA-induced knockdown of PPAR β/δ. MCFA increased the expression of Plin2, encoding a lipid-droplet associated protein with anti-inflammatory effects in macrophages, in a partially PPAR β/δ-dependent manner. Both MCFA stimulated expression of the cholesterol efflux pump ABCA1, more pronouncedly under LPS stimulation and in the absence of PPAR β/δ. Octanoate stimulated the expression of Pltp, encoding a phospholipid transfer protein that aids ABCA1 in cellular lipid efflux. Only palmitate increased expression of the proinflammatory genes Il6, Tnf, Nos2 and Mmp9. Non-stimulated macrophages exposed to MCFA showed less internalization of fluorescently labeled lipoproteins. MCFA influenced the transcriptional responses of macrophages favoring cholesterol efflux and a less inflammatory response compared to palmitate. These effects were partially mediated by PPAR β/δ.

Dickkopf-1 promotes vascular smooth muscle cell foam cell formation and atherosclerosis development through CYP4A11/SREBP2/ABCA1.

Vascular smooth muscle cells (VSMCs) are considered to be a crucial source of foam cells in atherosclerosis due to their low expression level of cholesterol exporter ATP-binding cassette transporter A1 (ABCA1) intrinsically. While the definite regulatory mechanisms are complicated and have not yet been fully elucidated, we previously reported that Dickkopf-1 (DKK1) mediates endothelial cell (EC) dysfunction, thereby aggravating atherosclerosis. However, the role of smooth muscle cell (SMC) DKK1 in atherosclerosis and foam cell formation remains unknown. In this study, we established SMC-specific DKK1-knockout (DKK1SMKO ) mice by crossbreeding DKK1flox/flox mice with TAGLN-Cre mice. Then, DKK1SMKO mice were crossed with APOE-/- mice to generate DKK1SMKO /APOE-/- mice, which exhibited milder atherosclerotic burden and fewer SMC foam cells. In vitro loss- and gain-of-function studies of DKK1 in primary human aortic smooth muscle cells (HASMCs) have proven that DKK1 prevented oxidized lipid-induced ABCA1 upregulation and cholesterol efflux and promoted SMC foam cell formation. Mechanistically, RNA-sequencing (RNA-seq) analysis of HASMCs as well as chromatin immunoprecipitation (ChIP) experiments showed that DKK1 mediates the binding of transcription factor CCAAT/enhancer-binding protein delta (C/EBPδ) to the promoter of cytochrome P450 epoxygenase 4A11 (CYP4A11) to regulate its expression. In addition, CYP4A11 as well as its metabolite 20-HETE-promoted activation of transcription factor sterol regulatory element-binding protein 2 (SREBP2) mediated the DKK1 regulation of ABCA1 in SMC. Furthermore, HET0016, the antagonist of CYP4A11, has also shown an alleviating effect on atherosclerosis. In conclusion, our results demonstrate that DKK1 promotes SMC foam cell formation during atherosclerosis via a reduction in CYP4A11-20-HETE/SREBP2-mediated ABCA1 expression.

Unveiling the role of G-quadruplex structure in promoter region: Regulation of ABCA1 expression in macrophages possibly via NONO protein recruitment

ABCA1 has been found to be critical for cholesterol efflux in macrophages. Understanding the mechanism regulating ABCA1 expression is important for the prevention and treatment of atherosclerosis. In the present study, a G-quadruplex (G4) structure was identified in the ABCA1 promoter region. This G4 was shown to be essential for ABCA1 transcription. Stabilizing the G4 by ligands surprisingly upregulated ABCA1 expression in macrophages. Knocking out the G4 remarkably reduced ABCA1 expression, and abolished the increase of ABCA1 expression induced by the G4 ligand. By pull-down assays, the protein NONO was identified as an ABCA1 G4 binder. Overexpression or repression of NONO significantly induced upregulation and downregulation of ABCA1 expression, respectively. ChIP and EMSA experiments showed that the G4 ligand promoted the binding between the ABCA1 G4 and NONO, which led to more recruitment of NONO to the promoter region and enhanced ABCA1 transcription. Finally, the G4 ligand was shown to significantly reduce the accumulation of cholesterol in macrophages. This study showed a new insight into the regulation of gene expression by G4, and provided a new molecular mechanism regulating ABCA1 expression in macrophages. Furthermore, the study showed a possible novel application of the G4 ligand: preventing and treating atherosclerosis.

260-LB: The Effect of LXR Activation on Liver Metabolism and Diet-Induced Steatohepatitis in Mice

While nonalcoholic fatty liver disease (NAFLD) affects approx. 30% of the US population, there is currently no approved pharmaceutical treatment to inhibit the progression of NAFLD to its more severe stage of nonalcoholic steatohepatitis (NASH). The liver X receptors (LXRs) are key regulators of cholesterol and fatty acid homeostasis in liver. However, the effects of LXR activation on liver metabolism in NASH pathogenesis are not well understood. We hypothesize that LXR activation might reduce NAFLD progression by augmenting lipid composition and metabolism in hepatocytes. To test this hypothesis, 8-week-old male wild type (WT) or obese, melanocortin-4 receptor knockout (Mc4r-/-) mice were placed on either chow or Western diet (WD) for 8 weeks. Half of the WD-fed mice were given WD supplemented with GW3965 (100mg/kg), a selective, orally active agonist for the LXRs. Animals fed GW3965-supplemented diet had increased gene expression of Abca1 and Lpcat3, both transcriptional targets of LXR action. LXR activation significantly decreased body mass of both WT and Mc4r-/- mice, which was correlated with reduction of subcutaneous fat. GW3965 supplementation greatly improved glucose homeostasis of Mc4r-/-. LXR activation significantly decreased hepatic triglycerides, cholesterol esters and ceramides content which was accompanied by decreased expression of lipogenic factors (Fasn, Dgat1 and 2, Fatp5, Elovl1 and 6) in Mc4r-/-. Contrary expression of genes involved in fatty acid β-oxidation together with pyruvate cycling fluxes (VPC, VPK+ME, VPEPCK) were elevated in Mc4r-/- fed diet supplemented with GW3965. These metabolic changes were related to decrease in hepatic fibrosis, expression of NF-κB pathway genes as well as inflammatory (Itgam, Cd274), oxidative (Sod2), and endoplasmic reticulum stress markers (Chop, Atf4 and 6). Taken together, these data suggest that LXR activation improves phenotype of genetically obese WD-fed animals and inhibits progression from NAFLD to NASH. Disclosure T. Bednarski: None. M. Rahim: None. N. Whittenbarger: None. A. J. Olichwier: None. J. Young: Consultant; Pfizer Inc. Stock/Shareholder; Metalytics, Inc. Consultant; Metalytics, Inc. Research Support; Merck Sharp & Dohme Corp. Funding National Institutes of Health (R01DK106348); Vanderbilt University; University of Nebraska-Lincoln Nutrition and Health Sciences Fund

MiR-33a Expression Attenuates ABCA1-Dependent Cholesterol Efflux and Promotes Macrophage-Like Cell Transdifferentiation in Cultured Vascular Smooth Muscle Cells.

Recent evidence suggests that the majority of cholesterol-laden cells found in atherosclerotic lesions are vascular smooth muscle cells (VSMC) that have transdifferentiated into macrophage-like cells (MLC). Furthermore, cholesterol-laden MLC of VSMC origin have demonstrated impaired ABCA1-dependent cholesterol efflux, but it is poorly understood why this occurs. A possible mechanism which may at least partially be attributed to cholesterol-laden MLC demonstrating attenuated ABCA1-dependent cholesterol efflux is a miR-33a expression, as a primary function of this microRNA is to silence ABCA1 expression, but this has yet to be rigorously investigated. Therefore, the VSMC line MOVAS cells were used to generate miR-33a knockout (KO) MOVAS cells, and we used KO and wild-type (WT) MOVAS cells to delineate any possible proatherogenic role of miR-33a expression in VSMC. When WT and KO MOVAS cells were cholesterol-loaded to convert into MLC, this resulted in the WT MOVAS cells to exhibit impaired ABCA1-dependent cholesterol efflux. In the cholesterol-loaded WT MOVAS MLC, we also observed a delayed restoration of the VSMC phenotype when these cells were exposed to the ABCA1 cholesterol acceptor, apoAI. These results imply that miR-33a expression in VSMC drives atherosclerosis by triggering MLC transdifferentiation via attenuated ABCA1-dependent cholesterol efflux.

ABCA1 deficiency contributes to podocyte pyroptosis priming via the APE1/IRF1 axis in diabetic kidney disease

Decreased ATP Binding Cassette Transporter A1 (ABCA1) expression and caspase-4-mediated noncanonical inflammasome contribution have been described in podocytes in diabetic kidney disease (DKD). To investigate a link between these pathways, we evaluated pyroptosis-related mediators in human podocytes with stable knockdown of ABCA1 (siABCA1) and found that mRNA levels of IRF1, caspase-4, GSDMD, caspase-1 and IL1β were significantly increased in siABCA1 compared to control podocytes and that protein levels of caspase-4, GSDMD and IL1β were equally increased. IRF1 knockdown in siABCA1 podocytes prevented increases in caspase-4, GSDMD and IL1β. Whereas TLR4 inhibition did not decrease mRNA levels of IRF1 and caspase-4, APE1 protein expression increased in siABCA1 podocytes and an APE1 redox inhibitor abrogated siABCA1-induced expression of IRF1 and caspase-4. RELA knockdown also offset the pyroptosis priming, but ChIP did not demonstrate increased binding of NFκB to IRF1 promoter in siABCA1 podocytes. Finally, the APE1/IRF1/Casp1 axis was investigated in vivo. APE1 IF staining and mRNA levels of IRF1 and caspase 11 were increased in glomeruli of BTBR ob/ob compared to wildtype. In conclusion, ABCA1 deficiency in podocytes caused APE1 accumulation, which reduces transcription factors to increase the expression of IRF1 and IRF1 target inflammasome-related genes, leading to pyroptosispriming.

Macrophage Gpx4 deficiency aggravates foam cell formation by regulating the expression of scavenger receptors, ABCA1, and ABCG1.

Macrophage-derived foam cell formation is critical for the initiation and development of atherosclerosis, which contributes to atherosclerotic cardiovascular disease (ASCVD). Glutathione peroxidase 4 (GPX4), a crucial ferroptosis regulator, protects cells from excessive oxidative stress by neutralizing lipid peroxidation. However, the role of macrophage GPX4 in foam cell formation remains unknown. We reported that oxidized low-density lipoprotein (oxLDL) upregulated GPX4 expression in macrophages. Using the Cre-loxP system, we generated myeloid cell-specific Gpx4 knockout (Gpx4myel-KO ) mice. Bone marrow-derived macrophages (BMDMs) were isolated from WT and Gpx4myel-KO mice and incubated with modified low-density lipoprotein (LDL). We found that Gpx4 deficiency promoted foam cell formation and increased the internalization of modified LDL. Mechanistic studies unveiled that Gpx4 knockout upregulated scavenger receptor type A and LOX-1 expression and downregulated ABCA1 and ABCG1 expression. Collectively, our study lends a novel insight into the role of GPX4 in suppressing macrophage-derived foam cell formation and suggests GPX4 as a promising therapeutic target to interfere with atherosclerosis-related diseases.

DNA methyltransferase 1 deficiency improves macrophage motility and wound healing by ameliorating cholesterol accumulation

Healing of the cutaneous wound requires macrophage recruitment at the sites of injury, where chemotactic migration of macrophages toward the wound is regulated by local inflammation. Recent studies suggest a positive contribution of DNA methyltransferase 1 (Dnmt1) to macrophage pro-informatory responses; however, its role in regulating macrophage motility remains unknown. In this study, myeloid-specific depletion of Dnmt1 in mice promoted cutaneous wound healing and de-suppressed the lipopolysaccharides (LPS)-inhibited macrophage motility. Dnmt1 inhibition in macrophages eliminated the LPS-stimulated changes in cellular mechanical properties in terms of elasticity and viscoelasticity. LPS increased the cellular accumulation of cholesterol in a Dnmt1-depedent manner; cholesterol content determined cellular stiffness and motility. Lipidomic analysis indicated that Dnmt1 inhibition altered the cellular lipid homeostasis, probably through down-regulating the expression of cluster of differentiation 36 CD36 (facilitating lipid influx) and up-regulating the expression of ATP-binding cassette transporter ABCA1 (mediating lipid efflux) and sterol O-acyltransferase 1 SOAT1 (also named ACAT1, catalyzing the esterification of cholesterol). Our study revealed a Dnmt1-dependent epigenetic mechanism in the control of macrophage mechanical properties and the related chemotactic motility, indicating Dnmt1 as both a marker of diseases and a potential target of therapeutic intervention for wound healing.

The role of platelet-derived growth factor BB signaling pathway in the regulation of stem and progenitor Leydig cell proliferation and steroidogenesis in male rats

Platelet-derived growth factor BB (BB) regulates cell proliferation and function. However, the roles of BB on proliferation and function of Leydig stem (LSCs) and progenitor cells (LPCs) and the underlying signaling pathways remain unclear. This study aimed to analyze the roles of PI3K and MAPK pathways in the regulation of proliferation-related and steroidogenesis-related gene expression in rat LSCs/LPCs. In this experiment, BB receptor antagonist, tyrosine kinase inhibitor IV (PKI), the PI3K inhibitor, LY294002, and the MEK inhibitor, U0126, were used to measure the effects of these pathways on the expression of cell cycle-related genes (Ccnd1 and Cdkn1b) and steroidogenesis-related genes (Star, Cyp11a1, Hsd3b1, Cyp17a1, and Srd5a1), as well as Leydig cell maturation gene Pdgfra [1]. These results showed that BB (10 ng/mL)-stimulated EdU-incorporation into LSCs and BB-mediated inhibition on its differentiation was mediated through the activation of its receptor, PDGFRB, as well as MAPK and PI3K pathways. The results of LPC experiment also showed that LY294002 and U0126 decreased BB (10 ng/mL)-upregulated Ccnd1 expression while only U0126 reversed BB (10 ng/mL)-downregulated Cdkn1b expression. U0126 significantly reversed BB (10 ng/mL)-mediated downregulation of Cyp11a1, Hsd3b1, and Cyp17a1 expression. On the other hand, LY294002 reversed the expression of Cyp17a1 and Abca1. In conclusion, BB-mediated induction of proliferation and suppression of steroidogenesis of LSCs/LPCs are dependent on the activation of both MAPK and PI3K pathways, which show distinct regulation of gene expression.

Aberrant DNA Methylation, Expression, and Occurrence of Transcript Variants of the ABC Transporter ABCA7 in Breast Cancer.

The ABC transporter ABCA7 has been found to be aberrantly expressed in a variety of cancer types, including breast cancer. We searched for specific epigenetic and genetic alterations and alternative splicing variants of ABCA7 in breast cancer and investigated whether these alterations are associated with ABCA7 expression. By analyzing tumor tissues from breast cancer patients, we found CpGs at the exon 5-intron 5 boundary aberrantly methylated in a molecular subtype-specific manner. The detection of altered DNA methylation in tumor-adjacent tissues suggests epigenetic field cancerization. In breast cancer cell lines, DNA methylation levels of CpGs in promoter-exon 1, intron 1, and at the exon 5-intron 5 boundary were not correlated with ABCA7 mRNA levels. By qPCR involving intron-specific and intron-flanking primers, we identified intron-containing ABCA7 mRNA transcripts. The occurrence of intron-containing transcripts was neither molecular subtype-specific nor directly correlated with DNA methylation at the respective exon-intron boundaries. Treatment of breast cancer cell lines MCF-7, BT-474, SK-BR3, and MDA-MB-231 with doxorubicin or paclitaxel for 72 h resulted in altered ABCA7 intron levels. Shotgun proteomics revealed that an increase in intron-containing transcripts was associated with significant dysregulation of splicing factors linked to alternative splicing.

Heterogeneous membrane cholesterol in large and small rat mesenteric endothelial cells

Introduction: Endothelial dysfunction is a common event in cardiovascular disease, characterized by damage to the endothelium and impaired local blood flow regulation, including the regulation by the gasotransmitters, nitric oxide (NO), carbon monoxide (CO), and hydrogen sulfide (H 2 S). We and others have shown that H 2 S is a vasodilator; however, not all arterial segments respond similarly to this gasotransmitter. We have shown H 2 S-sensitivity in large arteries (>200 μm) is dependent on the membrane cholesterol content of endothelial cells (EC), while smaller arteries (<200 μm) are inherently highly sensitive to H 2 S. In addition, we have demonstrated ECs from large arteries contain more cholesterol in the plasma membrane than ECs from smaller arteries. Taken together, these results suggest that EC signaling is dependent on the level of membrane cholesterol and that the cholesterol content of the plasma membrane is differentially regulated in arterial segments within the same organ. This study investigated the mechanism mediating differences in endothelial membrane cholesterol between large and small arteries. Our single-cell RNAseq data suggests that the differences in cholesterol content between small and large arteries may result from increased EC expression of ATP-binding cassette family a1 (Abca1) in small arteries. Therefore, we hypothesized that augmented cholesterol efflux by Abca1 mediates the lower membrane cholesterol in ECs of resistance arteries facilitating vasodilatory signaling pathways. Methods: Endothelial tubes were generated by enzymatic digestion followed by titration of mesenteric arteries from male Sprague-Dawley rats (Envigo 175 – 225g, N=5 for all experiments). Cholesterol in endothelial tubes was measured using filipin-lll (50 μg/ml), while Abca1 was measured using immunofluorescence staining and confocal microscopy. We performed in-vitro studies manipulating membrane cholesterol by treating rat mesenteric ECs with Abca1 inhibitor, probucol (10μM), or Abca1 knockdown (KD) through RNA interference. Results: Our data reveal an increase in filipin-lll fluorescence in large artery ECs compared to ECs from small arteries and that Abca1 expression is greater in small artery ECs. Inhibition and KD of Abca1 using probucol or siRNA, respectively, ­ in vitro increased EC membrane cholesterol. Conclusions: Abca1 is an important regulator of EC membrane cholesterol and mediates the lower EC membrane cholesterol in small arteries compared to large arteries. Future investigations will explore whether hypercholesterolemia causes an increase in EC membrane cholesterol in the microcirculation resulting in endothelial dysfunction and impaired organ blood flow control. Funding: 1) NIH 1R01HL160606 (JSN), 2) T32HL007736 (JRA), and 3) UNM Comprehensive Cancer Center Support Grant NCI P30CA118100 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Abstract 678: Use Of Extracellular Substrates By Alveolar Type 2 Pneumocytes For Surfactant Lipid Synthesis.

Introduction: Pulmonary surfactant is a lipoprotein complex essential for lung function. It reduces surface tension during inspiration and avoids alveolar collapse during expiration. It is synthetized by alveolar type 2 pneumocytes (T2C) and it is composed of phospholipids (~90%), mainly phosphatidylcholine (PC), and specific proteins. Decreased surfactant and altered composition are associated with multiple adult lung diseases. However, T2C lipid metabolism and their use of extracellular glucose and lipid for surfactant synthesis has not been studied. Our prior results showed that the low density lipoprotein receptor-related 1 (LRP1) is needed for surfactant synthesis. We hypothesized that nutritional conditions and LRP1 function impact the pathways used by T2C to synthetize surfactant. Methods: Control and LRP1 knockdown T2C-derived A549 cells were cultured in DMEM (4.5 g/L glucose) with 10% FBS and maintained at the air-liquid interface for 24 hours to promote their differentiation and polarization. Then, cells were cultured in DMEM with delipidated FBS and BSA-complexed palmitic acid (PA) at different concentrations (0-0.25 mM) for 2 hours and mRNA of surfactant synthetic enzymes was analyzed. Results: mRNA expression of Fatty acid synthase (FASN) and Choline kinase (CK) decreased 80% and 93% (p<0.001) respectively when at least 0.05 mM PA was present. Phospholipase A2 ( PLA2) followed the opposite pattern, with 21.5-fold increase (p<0.001) when PA was present. FASN and CK expression was downregulated in the absence of LRP1. mRNA expression of genes involved in cholesterol metabolism was assessed too. HMG-CoA reductase decreased only at the highest concentration of PA (41% decrease, p<0.01), while expression of HMG-CoA synthase and lipid exporters ABCA1 and ABCG1 was not affected. ABCG1 expression was compromised in LRP1 knockdown cells. Conclusions: Extracellular availability of glucose and fatty acids affects mRNA expression of genes involved in surfactant lipid synthesis in T2C, suggesting metabolic pathway flexibility during different nutritional conditions. In the absence of extracellular lipid sources, glucose can be used by T2C for fatty acid synthesis. Furthermore, LRP1 expression affects the metabolic pathways used by T2C.

Abstract 675: Time Restricted Feeding Regulates Cholesterol Efflux And Atherosclerosis In Macrophage-specific Bmal1 Deficient Mice

Background: Macrophages play a critical role in atherosclerosis. We have previously shown that Clock mutant and global or tissue-specific Bmal1 deficient mice show enhanced atherosclerosis. Yet, the role of time restricted feeding (TRF) in cholesterol transport and atherosclerosis in macrophage Bmal1 deficient mice remains unknown. Objectives: To establish a novel regulatory link between TRF, circadian clock, and atherosclerosis. Methods: We studied the effect of day or nighttime TRF on macrophage-specific Bmal1 deficient (M- Bmal1 –/– Apoe –/– ) or control mice on cholesterol efflux to clarify the role of Bmal1 in macrophage function and in atherosclerosis. Results: We found that daytime-TRF increased atherosclerosis. Biochemical studies showed that macrophages from daytime TRF M- Bmal1 –/– Apoe –/– mice exhibit increased expression of Lox-1 and Cd36, and take up more oxLDL compared to nighttime TRF mice. Furthermore, daytime TRF mice showed decreased expression of Abca1 and Abcg1, reduced cellular cholesterol efflux and in vivo reverse cholesterol transport compared to nighttime TRF mice. Thus, TRF regulates macrophage cholesterol metabolism by regulating uptake of oxidized lipoproteins and cholesterol efflux pathways. Molecular studies showed that TRF regulates HDAC3 interactions with p300 to regulate Znf202 gene expression. Thus, TRF regulates Bmal1 and Znf202 to regulate uptake of oxidized lipoproteins and cholesterol efflux. Conclusions: TRF is an important regulator of cholesterol metabolism in macrophages and affects atherosclerosis. It regulates macrophage function by modulating the expression of different transcription factors. It is likely that nighttime-TRF may prevent atherosclerosis.

Effects of liposoluble components of highland barley spent grains on physiological indexes, intestinal microorganisms, and the liver transcriptome in mice fed a high-fat diet.

The purpose of this study was to investigate the effects of the active ingredients of barley lees on the physiological indexes, intestinal flora, and liver transcriptome of mice fed a high-fat diet. Twenty-four male C57BL/6J mice were randomly divided into 4 groups and fed the experimental diets for 5 weeks. The results showed that the fat-soluble components of distillers' grains significantly reduced body weight, abdominal fat, perirenal fat, blood glucose, low-density lipoprotein cholesterol, triglycerides, and total cholesterol in the high-fat diet-fed mice (p < .05), significantly decreased alanine aminotransferase and malondialdehyde levels, and significantly increased total superoxide dismutase, catalase, reduced glutathione and glutathione peroxidase levels (p < .05). At the phylum level, lipid-soluble components significantly increased the abundance of Bacteroidetes and decreased the Firmicutes/Bacteroidetes ratio. At the genus level, the relative abundances of Bacteroidetes and Clostridium were increased. Transcriptomic analysis showed that lipid-soluble components of spent grains reduced the mRNA expression of ANGPTL8, CD36, PLTP, and SOAT1 and increased the mRNA expression of CYP7A1 and ABCA1 in the cholesterol metabolism pathway, promoted the transport of cholesterol, and inhibited the absorption of cholesterol, which can decrease cholesterol levels by speeding up the conversion of cholesterol into bile acids.