Lipid-loaded Macrophages Research Articles

FABP5+ lipid-loaded macrophages process tumour-derived unsaturated fatty acid signal to suppress T-cell antitumour immunity

Tumour-associated macrophages (TAMs) contribute to hepatocellular carcinoma (HCC) progression. However, while the pro-tumour and immunosuppressive roles of lipid-loaded macrophages are well established, the mechanisms by which lipid metabolism enhances the tumour-promoting effects of TAMs remain unclear. Single-cell RNA sequencing was performed on mouse and human HCC tumour samples to elucidate the landscape of HCC TAMs. Macrophages were stimulated with various long-chain unsaturated fatty acids (UFAs) to assess immunosuppressive molecule expression invitro. Additionally, invivo and invitro studies were conducted using mice with macrophage-specific deficiencies in fatty acid-binding protein 5 (FABP5) or peroxisome proliferator-activated receptor γ (PPARγ). Single-cell RNA sequencing identified a subpopulation of FABP5+ lipid-loaded TAMs characterized by enhanced immune checkpoint blocker ligands and immunosuppressive molecules in an oncogene-mutant HCC mouse model and human HCC tumours. Mechanistically, long-chain UFAs released by tumour cells activate PPARγ via FABP5, resulting in immunosuppressive properties in TAMs. FABP5 deficiency in macrophages decreases immunosuppressive molecule expression, enhances T cell-dependent antitumour immunity, diminishes HCC growth, and improves immunotherapy efficacy. This study demonstrates that UFAs promote tumourigenesis by enhancing the immunosuppressive tumour microenvironment via FABP5-PPARγ signalling and provides a proof-of-concept for targeting this pathway to improve the efficacy of tumour immunotherapy. Despite the role of tumour-associated macrophages (TAMs) in promoting tumour progression being well established, the mechanisms by which lipid metabolism enhances the tumour-promoting effects of TAMs remain unclear. Our study reveals that FABP5-mediated unsaturated fatty acid metabolism in TAMs is crucial for modulating antitumour T-cell immunity and influencing the efficacy of immunotherapy. This finding provides novel insights into the immunomodulatory roles of FABP5+ lipid-loaded TAMs in hepatocellular carcinoma and suggests that targeting FABP5 could offer a new approach to liver cancer treatment.

Infiltrating lipid-rich macrophage subpopulations identified as a regulator of increasing prostate size in human benign prostatic hyperplasia.

Macrophages exhibit marked phenotypic heterogeneity within and across disease states, with lipid metabolic reprogramming contributing to macrophage activation and heterogeneity. Chronic inflammation has been observed in human benign prostatic hyperplasia (BPH) tissues, however macrophage activation states and their contributions to this hyperplastic disease have not been defined. We postulated that a shift in macrophage phenotypes with increasing prostate size could involve metabolic alterations resulting in prostatic epithelial or stromal hyperplasia. Single-cell RNA-seq of CD45+ transition zone leukocytes from 10 large (>90 grams) and 10 small (<40 grams) human prostates was conducted. Macrophage subpopulations were defined using marker genes. BPH macrophages do not distinctly categorize into M1 and M2 phenotypes. Instead, macrophages with neither polarization signature preferentially accumulate in large versus small prostates. Specifically, macrophage subpopulations with altered lipid metabolism pathways, demarcated by TREM2 and MARCO expression, significantly accumulate with increased prostate volume. TREM2+ and MARCO+ macrophage abundance positively correlates with patient body mass index and urinary symptom scores. TREM2+ macrophages have significantly higher neutral lipid than TREM2- macrophages from BPH tissues. Lipid-rich macrophages were observed to localize within the stroma in BPH tissues. In vitro studies indicate that lipid-loaded macrophages increase prostate epithelial and stromal cell proliferation compared to control macrophages. These data define two new BPH immune subpopulations, TREM2+ and MARCO+ macrophages, and suggest that lipid-rich macrophages may exacerbate lower urinary tract symptoms in patients with large prostates. Further investigation is needed to evaluate the therapeutic benefit of targeting these cells in BPH.

Abstract 3073: Gene Regulation By RNP Granule Formation In Atherosclerosis

Background: Atherosclerosis, the leading cause of cardiovascular disease related death, is a chronic inflammatory condition incited by the accumulation of low-density lipoproteins (LDL) and recruitment of macrophages in the arterial intima. Macrophages become lipid laden foam cells that persist in the arterial vasculature, inciting chronic inflammation and plaque formation. A better understanding of the mechanisms underlying this maladaptive inflammatory response may reveal novel therapeutic strategies for intervention. Hypothesis: We hypothesize that the transformation into a foam cell state, a process that requires transcriptional rewiring, is facilitated by stress granules (SG). SGs are cytoplasmic membrane-less organelles that sequester translation initiation factors, mRNAs, and noncoding RNAs due to stress. We previously reported that SG formation is increased in plaque macrophages of atherosclerotic mice and can be induced in vitro during cellular cholesterol loading. However, the consequences of SG formation and the transcripts sequestered by these organelles is unknown. Methods and Results: To investigate whether SGs influence macrophage phenotype, we used siRNA to knockdown G3bp1 and treated with Dil-oxLDL. We found that G3bp1 knockdown reduced macrophage lipid loading and induced transcriptional changes associated with canonical M2 anti-inflammatory macrophage polarization. Furthermore, RNA-seq of macrophages treated with si G3bp1 and oxLDL showed downregulation of pathways involved in phagosome formation and S100 family signaling. RNA immunoprecipitation of G3BP1 from macrophages treated with an ER stressor revealed preferential binding of G3BP1 to transcripts implicated in cholesterol metabolism. Altogether, these data support a role for SGs in maintaining a pro-inflammatory state and regulating cholesterol pathways in plaque macrophages. Conclusion: Our data support a role for SGs in repressing the translation of RNA transcripts in lipid-laden foam cells that accumulate in atherosclerotic plaque. Identification of the specific proteins and RNAs sequestered in SGs will provide insight into how they regulate gene expression and cellular phenotypic switching that prevents the resolution of inflammation.

84P Profiling of lipid-loaded macrophages in melanoma

84P Profiling of lipid-loaded macrophages in melanoma

Gypenoside XVII inhibits ox-LDL-induced macrophage inflammatory responses and promotes cholesterol efflux through activating the miR-182-5p/HDAC9 signaling pathway

Ethnopharmacological relevanceThe deposition of lipids in macrophages and the subsequent formation of foam cells significantly increase the risk of developing atherosclerosis (As). Targeting ATP-binding cassette transporter A1/G1 (ABCA1/ABCG1)-mediated reverse cholesterol transport is crucial for regulating foam cell formation. Therefore, the search for natural chemical components with the ability to regulate ABCA1/G1 is a potential drug target to combat the development of atherosclerosis. Gypenoside XVII (GP-17), a gypenoside monomer extracted from gynostemma pentaphyllum, presents an efficient anti-atherosclerosis function. However, the suppressed formation mechanism of foam cells by GP-17 remains elusive. Aim of studyTo explore the protective activities of GP-17 in ox-LDL-induced THP-1 macrophage-derived foam cells through modulating the promotion of cholesterol efflux and alleviation of inflammation. Materials and methodsMTT was used to detect cell viability. Bodipy493/503 and oil red O staining were performed to measure cell lipid deposition. Enzymatic assay was used to measure intracellular cholesterol measurement. Cholesterol efflux/uptake were determined by cholesterol efflux assay and Dil-ox-LDL uptake assay. Inflammatory cytokines were measured by ELISA. Bioinformatics prediction and dual luciferase reporter assay were performed to validate miR-182–5p targeting HDAC9. Relative protein levels were evaluated by immunoblotting and relative gene levels were determined by quantitative real-time PCR. ResultsOur results showed that GP-17 upregulated the expression of ABCA1, ABCG1 and miR-182–5p, but reduced HDAC9 expression levels in lipid-loaded macrophages, which promoted cholesterol efflux and inhibited lipid deposition. Additionally, GP-17 promoted the M2 phenotype of the macrophage and suppressed the inflammatory response in THP-1 macrophage-derived foam cells. Overexpression of HDAC9 or suppression of miR-182–5p eliminated the effects of ABCA1/G1 expression, lipid deposition and pro-inflammatory response. ConclusionThese findings suggest that GP-17 exerts a beneficial effect on macrophage lipid deposition and inflammation responses through activating the miR-182–5p/HDAC9 signaling pathway.

A Lipid-Structured Model of Atherosclerotic Plaque Macrophages with Lipid-Dependent Kinetics

Atherosclerotic plaques are fatty growths in artery walls that cause heart attacks and strokes. Plaque formation is driven by macrophages that are recruited to the artery wall. These cells consume and remove blood-derived lipids, such as modified low-density lipoprotein. Ineffective lipid removal, due to macrophage death and other factors, leads to the accumulation of lipid-loaded macrophages and formation of a necrotic lipid core. Experimental observations suggest that macrophage functionality varies with the extent of lipid loading. However, little is known about the influence of macrophage lipid loads on plaque fate. Extending work by Ford et al. (J Theor Biol 479:48–63, 2019) and Chambers et al. (A lipid-structured model of atherosclerosis with macrophage proliferation, 2022), we develop a plaque model where macrophages are structured by their ingested lipid load and behave in a lipid-dependent manner. The model considers several macrophage behaviours, including recruitment to and emigration from the artery wall; proliferation and apotosis; ingestion of plaque lipids; and secondary necrosis of apoptotic cells. We consider apoptosis, emigration and proliferation to be lipid-dependent and we model these effects using experimentally informed functions of the internalised lipid load. Our results demonstrate that lipid-dependent macrophage behaviour can substantially alter plaque fate by changing both the total quantity of lipid in the plaque and the distribution of lipid between the live cells, dead cells and necrotic core. The consequences of macrophage lipid-dependence are often unpredictable because lipid-dependent effects introduce subtle, nonlinear interactions between the modelled cell behaviours. These observations highlight the importance of mathematical modelling in unravelling the complexities of macrophage lipid accumulation during atherosclerotic plaque formation.

Shenlian extract protected ox-LDL-loaded macrophages against ER stress by promoting LAL-LXRα mediated cholesterol flux

Ethnopharmacological relevanceShenlian (SL) extract is consisted of extracts from Salvia miltiorrhiza Bunge and Andrographis paniculata (Burm.f.) Nees, two herbs commonly used in Chinese clinical formula to treat atherosclerosis by removing blood stasis and clearing away heat. Pharmacologically, the anti-atherosclerotic effects of these two herbs are related to unresolved inflammation and the macrophage anergy or apoptosis in lesions led by the lipid flux blockage and ER stress. However, the deeper understanding of SL extract in protecting macrophage in plaques remains unknown. Aim of the studyThis study aimed to investigate the underlying mechanism of SL extract in protecting ER-stressed macrophages from apoptosis in atherosclerosis. MethodsThe ApoE−/− atherosclerotic mice model and ox-LDL loaded macrophages model were established to assess the effect of SL extract on ER stress in vivo and in vitro. Key markers related to ER stress in plaque were determined by immunohistochemical staining. Proteins involved in apoptosis and ER stress in macrophages loaded by ox-LDL were assessed by Western blot. ER morphology was observed by electron microscope. Lipid flux was temporally and quantitatively depicted by Oil red staining. The LAL and LXRα were blocked by lalistat and Gsk 2033 respectively to investigate whether SL extract protected the function of macrophages by the activation of LAL-LXRα axis. ResultsOur study reported that, in ApoE−/− atherosclerotic mice, SL extract effectively relieved ER stress of carotid artery plaque. In lipid-overloaded macrophage models, SL extract significantly alleviated ER stress by promoting cholesterol degradation and efflux, which finally prevented apoptosis of foam cells induced by ox-LDL. Blockage of ER stress by 4-Phenylbutyric acid (4-PBA), an inhibitor of Endoplasmic Reticulum (ER) stress, largely attenuated the protective effects of SL extract on macrophage. By utilizing the selective antagonists against both LAL and LXRα, this study further revealed that the beneficial effects of SL extract in macrophages was dependent on the proper functionalization of LAL-LXRα axis. ConclusionsBy highlighting the therapeutic significance of macrophage protection in resolving atherosclerosis inflammation, our study pharmacologically provided convincing mechanistic evidence of SL extract in the activation LAL-LXRα axis and revealed its promising potential in the promotion of cholesterol turnover and prevention of ER stress induced apoptosis in lipid-loaded macrophages.

Monocyte and macrophage foam cells in diabetes-accelerated atherosclerosis.

Diabetes results in an increased risk of atherosclerotic cardiovascular disease. This minireview will discuss whether monocyte and macrophage lipid loading contribute to this increased risk, as monocytes and macrophages are critically involved in the progression of atherosclerosis. Both uptake and efflux pathways have been described as being altered by diabetes or conditions associated with diabetes, which may contribute to the increased accumulation of lipids seen in macrophages in diabetes. More recently, monocytes have also been described as lipid-laden in response to elevated lipids, including triglyceride-rich lipoproteins, the class of lipids often elevated in the setting of diabetes.

Oxidized cholesteryl ester induces exocytosis of dysfunctional lysosomes in lipidotic macrophages.

A key event in atherogenesis is the formation of lipid-loaded macrophages, lipidotic cells, which exhibit irreversible accumulation of undigested modified low-density lipoproteins (LDL) in lysosomes. This event culminates in the loss of cell homeostasis, inflammation, and cell death. Nevertheless, the exact chemical etiology of atherogenesis and the molecular and cellular mechanisms responsible for the impairment of lysosome function in plaque macrophages are still unknown. Here, we demonstrate that macrophages exposed to cholesteryl hemiazelate (ChA), one of the most prevalent products of LDL-derived cholesteryl ester oxidation, exhibit enlarged peripheral dysfunctional lysosomes full of undigested ChA and neutral lipids. Both lysosome area and accumulation of neutral lipids are partially irreversible. Interestingly, the dysfunctional peripheral lysosomes are more prone to fuse with the plasma membrane, secreting their undigested luminal content into the extracellular milieu with potential consequences for the pathology. We further demonstrate that this phenotype is mechanistically linked to the nuclear translocation of the MiT/TFE family of transcription factors. The induction of lysosome biogenesis by ChA appears to partially protect macrophages from lipid-induced cytotoxicity. In sum, our data show that ChA is involved in the etiology of lysosome dysfunction and promotes the exocytosis of these organelles. This latter event is a new mechanism that may be important in the pathogenesis of atherosclerosis.

Abstract 462: Transcriptomic Analysis Reveals That Intimal Monocytes Drive Inflammation In Early Atherogenesis

Hypercholesterolemia (HC) is a key risk factor for atherosclerosis, a chronic inflammatory disease that causes myocardial infarction and stroke. Although all regions of arteries are exposed to HC, atherosclerosis develops in discrete regions, such as the lesser curvature (LC) of the mouse aortic arch. In Ldlr -/- mice HC induces intimal myeloid cells to develop into the foam cells of nascent lesions. The purpose of this study is to characterize the transcriptomic changes occurring in the aortic intimal myeloid cells during the earliest stages of atherogenesis, and correlate them to cellular responses. Intimal cells were isolated from the LC of the ascending aortic arch of Ldlr -/- mice at 0, 5, 14, and 56 days of CRD feeding using en face enzymatic digestion and cell microisolation. Bulk RNA-seq and RT-qPCR revealed that genes associated with lipid-loaded macrophages (e.g., Abcg1 , Lgals3 ) were progressively elevated over time. Atherogenesis-associated inflammatory transcripts and pathways, such as leukocyte transendothelial migration, cell adhesion, and cytokine/chemokine signalling were significantly elevated after 14d of CRD, but not at 5d. Single cell transcriptomic analysis of the mouse aortic arch intima of Ldlr -/- mice revealed multiple sub-populations of myeloid cells. With our intima specific approach, we have uncovered a diverse landscape of intimal myeloid cells; including monocytes, macrophages, foam cells, DCs, and neutrophils. Atherogenesis-associated inflammatory transcripts were scarcely detected in macrophage and foam cell clusters, but were prominent in non-macrophage cell types. Ccr2 + intimal monocytes exhibited a hypercholesterolemia induced increase in inflammatory pathways within 5 days of CRD feeding. Furthermore, analysis of monocyte and macrophage clusters suggests that this inflammatory signature dissipates as recently recruited monocytes transition into a macrophage/foam cell fate. We are using Ccr2 + fate mapping models, to interrogate the hypercholesterolemia-induced transition of inflammatory monocytes within a growing lesion. We suggest that the atherogenesis-associated inflammation may be initiated by recently recruited monocytes, and may not originate directly from lipid loaded macrophages.

Capacity of HDL to Efflux Cellular Cholesterol from Lipid-Loaded Macrophages Is Reduced in Patients with Familial Hypercholesterolemia.

This study aimed to evaluate the high-density lipoprotein (HDL) capacity to efflux cellular cholesterol from lipid-loaded macrophages to find a reliable and low-cost biomarker with the purpose of better evaluating the risk of premature cardiovascular (CV) events in FH patients. This case-controlled study comprised 16 homozygous (HOFH) and 18 heterozygous (HEFH) FH patients, as well as 20 healthy subjects recruited as controls. Two main subfractions of HDL (HDL2 (d = 1.063-1.125 g/mL) and HDL3 (d = 1.125-1.210 g/mL)) were isolated from the patients' serum samples using sequential ultracentrifugation. After compositional characterization, the capacity of HDL to efflux cholesterol (CEC%) from lipid-laden macrophages was measured. The HDL2 and HDL3 subfractions showed some differences in lipid and protein composition between the studied groups. In addition, both HDL subfractions (p < 0.001) revealed significantly reduced CEC% in HOFH patients (HDL2: 2.5 ± 0.1 and HDL3: 3.2 ± 0.2) in comparison with the HEFH (HDL2: 3.2 ± 0.1% and HDL3: 4.1 ± 0.2%) and healthy (HDL2: 3.3 ± 0.2% and HDL3: 4.5 ± 0.3%) subjects. Additionally, multinomial logistic regression results indicated that the CEC% of both HDL2 (OR: 0.091; 95% CI: 0.018-0.452, p < 0.01) and HDL3 (OR: 0.118; 95% CI: 0.035-0.399, p < 0.01) subfractions are strongly and inversely associated with the homozygous form of FH. A decreased capacity of HDL particles to efflux cholesterol from macrophages might identify homozygous FH patients who are at elevated risk for premature CVDs. Prospective studies with a large sample size are warranted to evaluate this hypothesis.

Current Understanding on the Role of Lipids in Macrophages and Associated Diseases

Lipid metabolism is the major intracellular mechanism driving a variety of cellular functions such as energy storage, hormone regulation and cell division. Lipids, being a primary component of the cell membrane, play a pivotal role in the survival of macrophages. Lipids are crucial for a variety of macrophage functions including phagocytosis, energy balance and ageing. However, functions of lipids in macrophages vary based on the site the macrophages are residing at. Lipid-loaded macrophages have recently been emerging as a hallmark for several diseases. This review discusses the significance of lipids in adipose tissue macrophages, tumor-associated macrophages, microglia and peritoneal macrophages. Accumulation of macrophages with impaired lipid metabolism is often characteristically observed in several metabolic disorders. Stress signals differentially regulate lipid metabolism. While conditions such as hypoxia result in accumulation of lipids in macrophages, stress signals such as nutrient deprivation initiate lipolysis and clearance of lipids. Understanding the biology of lipid accumulation in macrophages requires the development of potentially active modulators of lipid metabolism.

Macrophage-Derived 25-Hydroxycholesterol Promotes Vascular Inflammation, Atherogenesis, and Lesion Remodeling.

Cross-talk between sterol metabolism and inflammatory pathways has been demonstrated to significantly affect the development of atherosclerosis. Cholesterol biosynthetic intermediates and derivatives are increasingly recognized as key immune regulators of macrophages in response to innate immune activation and lipid overloading. 25-Hydroxycholesterol (25-HC) is produced as an oxidation product of cholesterol by the enzyme cholesterol 25-hydroxylase (CH25H) and belongs to a family of bioactive cholesterol derivatives produced by cells in response to fluctuating cholesterol levels and immune activation. Despite the major role of 25-HC as a mediator of innate and adaptive immune responses, its contribution during the progression of atherosclerosis remains unclear. The levels of 25-HC were analyzed by liquid chromatography-mass spectrometry, and the expression of CH25H in different macrophage populations of human or mouse atherosclerotic plaques, respectively. The effect of CH25H on atherosclerosis progression was analyzed by bone marrow adoptive transfer of cells from wild-type or Ch25h-/- mice to lethally irradiated Ldlr-/- mice, followed by a Western diet feeding for 12 weeks. Lipidomic, transcriptomic analysis and effects on macrophage function and signaling were analyzed in vitro from lipid-loaded macrophage isolated from Ldlr-/- or Ch25h-/-;Ldlr-/- mice. The contribution of secreted 25-HC to fibrous cap formation was analyzed using a smooth muscle cell lineage-tracing mouse model, Myh11ERT2CREmT/mG;Ldlr-/-, adoptively transferred with wild-type or Ch25h-/- mice bone marrow followed by 12 weeks of Western diet feeding. We found that 25-HC accumulated in human coronary atherosclerotic lesions and that macrophage-derived 25-HC accelerated atherosclerosis progression, promoting plaque instability through autocrine and paracrine actions. 25-HC amplified the inflammatory response of lipid-loaded macrophages and inhibited the migration of smooth muscle cells within the plaque. 25-HC intensified inflammatory responses of lipid-laden macrophages by modifying the pool of accessible cholesterol in the plasma membrane, which altered Toll-like receptor 4 signaling, promoted nuclear factor-κB-mediated proinflammatory gene expression, and increased apoptosis susceptibility. These effects were independent of 25-HC-mediated modulation of liver X receptor or SREBP (sterol regulatory element-binding protein) transcriptional activity. Production of 25-HC by activated macrophages amplifies their inflammatory phenotype, thus promoting atherogenesis.

Abstract 9816: Cholesterol Efflux Capacity Mediates The Relationship Between Neutrophils And Coronary Burden In Psoriasis

Introduction: Psoriasis is a chronic inflammatory disease associated with increased levels of activated neutrophils, lower cholesterol efflux capacity (CEC), and subclinical atherosclerosis. High-density lipoprotein cholesterol efflux is the primary method of cholesterol removal from peripheral tissues and is linked to innate immunity and cardiovascular disease. Preclinical models suggest a strong relationship between CEC, neutrophil frequency and activation, and atherosclerosis, yet this relationship is understudied in clinical models. Hypothesis: (1) Psoriasis patients with high neutrophils have lower CEC and high non-calcified coronary burden (NCB); (2) CEC mediates the relationship between neutrophils and NCB. Methods: Coronary computed tomography angiography was used to quantitate NCB in one-hundred seventy-nine untreated psoriasis patients. Circulating neutrophils were measured via flow cytometry, and mean fluorescence intensity of CD62L and CD11b was determined. CEC was quantified using a cell-based ex-vivo assay measuring the ability of apolipoprotein B depleted plasma to mobilize cholesterol from lipid-loaded macrophages. Results: Patients with high neutrophils had decreased CEC (0.93 (0.15) vs 1.00 (0.17); P =0.03) and higher NCB (1.24 (0.55) vs 1.10 (0.50); P =0.003). The relationship between neutrophils and NCB was mediated by CEC beyond adjustment for Framingham risk score, smoking status, and statin treatment (20%, P =&lt;0.001) ( Figure 1 ). CEC associated with neutrophil activation markers, CD11b and CD62L (β: -0.23 P =0.04, β:0.21, P =0.04). Finally, one year of biologic therapy improved CEC (0.08 (0.17) vs (-0.009 (0.19); P =0.008) and neutrophil counts (-0.45 (-1.36- -0.45) vs -0.14 (-0.60-0.42; P =0.004). Conclusion: Patients with more neutrophils had lower CEC and higher NCB. Further, CEC mediates the relationship between neutrophils and NCB, underscoring the relationship between neutrophils and CEC in atherosclerosis.

Dicer prevents activation of the type I interferon pathway in lipid-loaded macrophages

Abstract Background The expression of the endonuclease Dicer in macrophages decreases atherosclerosis and necrotic core formation by producing microRNAs such as miR-10a. This effect of Dicer is associated with enhanced mitochondrial respiration in lipid-loaded macrophages. However, the mechanism by which Dicer-dependent production of miRNAs in lipid-loaded macrophages regulates mitochondrial function is unclear. Purpose We aimed to determine the effect of Dicer on lipid-loaded macrophages in the context of atherosclerosis. Methods Mice with a myeloid cell-specific knockout of Dicer (Lys-Cre/Dicerflox/flox/Apoe−/− mice [M-Dicer−/−]) and control mice (Lys-Cre/DicerWT/WT/Apoe−/− mice [M-Dicer+/+]) were fed a high-fat diet (HFD) for 24 weeks. The oxygen consumption rate (OCR) in aortic arch plaques was studied ex vivo by Seahorse Flux XF 24 Analyzer. Bone marrow-derived macrophages (BMDMs) were stimulated with oxLDL (100 μg/mL) for 72 h. Lipid-loaded macrophages were used for proteomic analysis by mass spectrometry. RIP-prime- seq was performed in tAgo2 immunoprecipitates (IP) from Dicer+/+ and Dicer−/− BMDMs. The miRNA expression profile was determined in lipid-loaded BMDMs by NanoString technology. Stat1 phosphorylation was determined in lipid-loaded macrophages by Jess automated western blot system (ProteinSimple). Result The OCR in aortic arch tissue with plaques was higher than in those without plaques in M-Dicer+/+ mice after 24 weeks of HFD feeding. Dicer knockout in macrophages decreased the OCR in aortic arch tissues with plaques but not in aortic arch tissues without plaques (n=3–4, p&amp;lt;0.05). The proteomic analysis of oxLDL-treated BMDMs indicated that Dicer knockout activated the type I interferon signaling pathway by up-regulating the expression of STAT1/2 and interferon-stimulated genes, such as ISG15. Proteins related to mitochondrial DNA were upregulated (e.g., Dnmt3a) or downregulated (e.g., Tfam) by Dicer knockout (n=5, adj.p&amp;lt;0.05). RIP-prime-seq from BMDMs showed that 1376 transcripts were significantly enriched in the tAgo IP from Dicer+/+ compared with that from Dicer−/− BMDMs (n=3–5, adj.p&amp;lt;0.05) including Dnmt3a and Stat2. STAT1 phosphorylation was increased in Dicer−/− compared with Dicer+/+ BMDMs. Among the 299 miRNAs downregulated by Dicer knockout in lipid-loaded macrophages (n=6, adj.p&amp;lt;0.05), miR-29 and miR-30 have highly conserved binding sites (predicted by TargetScan) for Dnmt3a. Conclusion Our results indicate that Dicer expression in lipid-loaded macrophages limits Stat1/2-driven type I interferon response due to mitochondrial damage. This effect may be mediated by the suppression of Dnmt3a by miRNAs such as miR-29 and miR-30. This suggests that targeting Dnmt3a-mediated mitochondrial damage in lipid-loaded macrophages by miRNAs may be therapeutic strategy to limit atherosclerosis. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): DFG

PLK1 promotes cholesterol efflux and alleviates atherosclerosis by up-regulating ABCA1 and ABCG1 expression via the AMPK/PPARγ/LXRα pathway

Polo-like kinase 1 (PLK1) is a serine/threonine kinase involving lipid metabolism and cardiovascular disease. However, its role in atherogenesis has yet to be determined. The aim of this study was to observe the impact of PLK1 on macrophage lipid accumulation and atherosclerosis development and to explore the underlying mechanisms. We found a significant reduction of PLK1 expression in lipid-loaded macrophages and atherosclerosis model mice. Lentivirus-mediated overexpression of PLK1 promoted cholesterol efflux and inhibited lipid accumulation in THP-1 macrophage-derived foam cells. Mechanistic analysis revealed that PLK1 stimulated the phosphorylation of AMP-activated protein kinase (AMPK), leading to activation of the peroxisome proliferator-activated receptor γ (PPARγ)/liver X receptor α (LXRα) pathway and up-regulation of ATP binding cassette transporter A1 (ABCA1) and ABCG1 expression. Injection of lentiviral vector expressing PLK1 increased reverse cholesterol transport, improved plasma lipid profiles and decreased atherosclerotic lesion area in apoE-deficient mice fed a Western diet. PLK1 overexpression also facilitated AMPK and HSL phosphorylation and enhanced the expression of PPARγ, LXRα, ABCA1, ABCG1 and LPL in the aorta. In summary, these data suggest that PLK1 inhibits macrophage lipid accumulation and mitigates atherosclerosis by promoting ABCA1- and ABCG1-dependent cholesterol efflux via the AMPK/PPARγ/LXRα pathway.

Early atherogenesis: new insights from new approaches.

Purpose of reviewTo highlight recent conceptual and technological advances that have positioned the field to interrogate the cellular and molecular mechanisms contributing to the initiation of atherosclerosis, including intimal lipid accumulation, inflammation, and lesion growth.Recent findingsAdvances in the understanding of endothelial LDL transcytosis and rapid lipid uptake by intimal macrophages provide mechanistic insights into intimal LDL accumulation and the initiation of atherogenesis. Recent studies have used unbiased single-cell approaches, such as single-cell RNA sequencing and CyTOF, to characterize the cellular components of the normal intima and atherosclerotic lesions. In-vitro studies and high-resolution transcriptomic analysis of aortic intimal lipid-loaded versus lipid-poor myeloid populations in vivo suggest that lipid-loaded macrophages may not be the primary drivers of inflammation in atherosclerotic lesions.SummaryA new perspective on the complex cellular landscape of the aorta, specifically the atherosclerosis-prone regions, confirm that intimal accumulation of lipid, monocyte recruitment, and macrophage accumulation are key events in atherogenesis triggered by hypercholesterolemia. Targeting these early events may prove to be a promising strategy for the attenuation of lesion development; however, the specific details of how hypercholesterolemia acts to initiate early inflammatory events remain to be fully elucidated.

Abstract 412: Transcriptomic Analysis Reveals That Lipid Loading Of Distinct Intimal Myeloid Cell Subpopulations Precedes Inflammation In Early Atherogenesis

Hypercholesterolemia (HC) is a key risk factor of atherosclerosis, a chronic inflammatory disease that causes myocardial infarction and stroke. Although all regions of arteries are exposed to HC, atherosclerosis develops in discrete regions, such as the lesser curvature (LC) of the mouse aortic arch. Dietary HC in Ldlr -/- mice induces intimal myeloid cells to develop into foam cells, constituting nascent lesions. The purpose of this study is to characterize the transcriptomic changes occurring in the aortic intima during the earliest stages of atherogenesis, and correlate them to cellular responses. Using en face enzymatic digestion and intimal cell microisolation, transcriptomic analysis was performed on the LC. Intimal cells were isolated from the LC of the ascending aortic arch of Ldlr -/- mice at 0, 5, and 14 days of CRD feeding. Bulk RNA-seq and RT-qPCR found that genes associated with lipid-loaded macrophages (e.g., Abcg1, Lgals3 ) were progressively elevated at 5 and 14 days of CRD. Atherogenesis-associated inflammatory transcripts and pathways, such as leukocyte transendothelial migration, cell adhesion, and cytokine/chemokine signalling were significantly elevated after 14d of CRD, but not detected at 5d. Single cell transcriptomic analysis of the mouse aortic arch intima of Ldlr -/- mice revealed multiple sub-populations of myeloid, endothelial, and other cell types. A shift in the C1qb + , Mmp12 + myeloid subpopulations was observed as the duration of cholesterol feeding increased - with expansion of a macrophage population rich in lipid-responsive transcripts. This finding is consistent with an increase in macrophage number and the presence of Nile Red + CD68 + foam cells observed by immunoconfocal microscopy at 5 days of CRD. However, a robust inflammatory signature was not detected in this macrophage cluster. Interestingly, an inflammatory signature similar to that reported in early atherogenesis was identified in a distinct Ccr2 + myeloid population with no signature of lipid loading. This suggests that the inflammation associated with atherogenesis may be a result of intercellular crosstalk between distinct myeloid subpopulations within the intima and may not originate directly from lipid loaded macrophages.

Abstract 500: Silencing Apolipoprotein C3 Prevents Diabetic Kidney Disease And Atherosclerosis In A Mouse Model Of Type 2 Diabetes

Diabetes increases the risk of both cardiovascular disease and kidney disease. Notably, most of the excess cardiovascular risk in people with diabetes is in those with kidney disease. Apolipoprotein C3 (APOC3) is a small apolipoprotein that is elevated by insulin-insufficiency and regulates plasma triglyceride levels. To test if APOC3, and the dyslipidemia it represents, play a role in diabetic kidney disease (DKD) and associated atherosclerosis, we treated BTBR wildtype (WT) and leptin-deficient (OB; diabetic) mice with an antisense oligonucleotide (ASO) to APOC3 or a control ASO (cASO), all in the setting of human-like dyslipidemia (accomplished by administration of an ASO targeting the LDLR). APOC3 ASO treatment reduced triglycerides, triglyceride-rich lipoproteins. It prevented diabetes-accelerated atherosclerosis in the aortic sinus, brachiocephalic artery, and aorta (sinus lesion: 67926 ± 8486 μm 2 lesion in cASO-treated OB mice compared to 38589 ± 9507 μm 2 in APOC3 ASO-treated OB mice, p&lt;0.05, n=12-15). The reduction in lesion size with APOC3 treatment was associated with fewer macrophages and reduced perilipin 2 staining (a marker for lipid droplet formation). In the kidney, diabetes resulted in a dramatic increase in glomerular hypertrophy, neutral lipid accumulation, APOC3, and APOE-accumulation, which APOC3 ASO-treatment attenuated. Furthermore, diabetes resulted in monocyte recruitment, macrophage accumulation, and macrophage lipid loading in the glomeruli, all of which was dramatically suppressed in the setting of APOC3 silencing (36% of glomerular macrophages were lipid loaded in OB cASO mice whereas only 9% were lipid loaded in APOC3-ASO treated mice, p&lt;0.0001). The recruitment was driven by increased endothelial cell ICAM1 expression, but monocyte lipid loading may also contribute. Intriguingly, APOC3-ASO treatment reduced diabetes-associated urinary albumin creatinine ratio but did not affect it in non-diabetic mice (WT mice: 374 ± 71 μg albumin/mg creatinine, OB cASO mice: 4648 ± 1021 μg/mg and OB mice with APOC3 ASO: 2341 ± 439 μg/mg, p&lt;0.05, n=12-14). Together, this suggests that targeting APOC3 and diabetic dyslipidemia might be beneficial for both diabetes-accelerated atherosclerosis and kidney disease.

Autophagy Is Differentially Regulated in Leukocyte and Nonleukocyte Foam Cells During Atherosclerosis.

Atherosclerosis is characterized by an accumulation of foam cells within the arterial wall, resulting from excess cholesterol uptake and buildup of cytosolic lipid droplets (LDs). Autophagy promotes LD clearance by freeing stored cholesterol for efflux, a process that has been shown to be atheroprotective. While the role of autophagy in LD catabolism has been studied in macrophage-derived foam cells, this has remained unexplored in vascular smooth muscle cell (VSMC)-derived foam cells that constitute a large fraction of foam cells within atherosclerotic lesions. We performed a comparative analysis of autophagy flux in lipid-rich aortic intimal populations to determine whether VSMC-derived foam cells metabolize LDs similarly to their macrophage counterparts. Atherosclerosis was induced in GFP-LC3 (microtubule-associated proteins 1A/1B light chain 3) transgenic mice by PCSK9 (proprotein convertase subtilisin/kexin type 9)-adeno-associated viral injection and Western diet feeding. Using flow cytometry of aortic digests, we observed a significant increase in dysfunctional autophagy of VSMC-derived foam cells during atherogenesis relative to macrophage-derived foam cells. Using cell culture models of lipid-loaded VSMCs and macrophages, we show that autophagy-mediated cholesterol efflux from VSMC foam cells was poor relative to macrophage foam cells, and largely occurs when HDL (high-density lipoprotein) was used as a cholesterol acceptor, as opposed to apoA-1 (apolipoproteinA-1). This was associated with the predominant expression of ABCG1 in VSMC foam cells. Using metformin, an autophagy activator, cholesterol efflux to HDL was significantly increased in VSMC, but not in macrophage, foam cells. These data demonstrate that VSMC and macrophage foam cells perform cholesterol efflux by distinct mechanisms, and that autophagy flux is highly impaired in VSMC foam cells, but can be induced by pharmacological means. Further investigation is warranted into targeting autophagy specifically in VSMC foam cells, the predominant foam cell subtype of advanced atherosclerotic plaques, to promote reverse cholesterol transport and resolution of the atherosclerotic plaque.