Abstract 408: Enzyme Modified LDL Induces Migration and Calcification of Human Coronary Artery SMCs

2017 ◽  
Vol 37 (suppl_1) ◽  
Author(s):  
Bijou Chellan ◽  
Marion Hofmann Bowman
Keyword(s):  
Coronary Artery ◽  
Foam Cell ◽  
Oxidized Ldl ◽  
Control Cell ◽  
Foam Cells ◽  
Foam Cell Formation ◽  
Whole Genome ◽  
Human Coronary Artery ◽  
Alizarin Red ◽  
Modified Ldl

Background: Enzyme modified LDL (ELDL) is present in human atherosclerotic lesions and is a major foam cell-forming modified LDL for murine vascular smooth muscle cells (SMC) as reported by us previously. Here we study ELDL and its effects on human coronary artery SMC (HCASMC) in vitro. Methods and Results: Incubation of HCASMC with 10 μg/ml ELDL (trypsin, cholesterol esterase modified) resulted in significant foam cell formation (analyzed by Oil Red O, lipid measurement) compared to HCASMC incubated with oxidized LDL (200 μg/ml; -75%, p<0.01) or native LDL (200 μg/ml; -50%, p<0.01). Whole genome gene expression (Illumina Bead Chip HT12v4, analyzed by DAVID v6.8 and IPA) of HCASMC treated with ELDL, oxLDL, LDL, and control (cell culture medium only) showed several top canonical pathways specifically induced by ELDL, together with activated upstream regulators including p38MAPK, NFkB. ERK. Upregulation of ANGPTL4 and BMP-2 -mRNA (22 and 2 fold respectively over native LDL) was verified by qRT-PCR and immunoblotting. ELDL-induced foam cells showed dose dependent (1-20 μg/ml ELDL) increase in migration in collagen coated trans well dishes, which was attenuated by Lacidepine, a known inhibitor of ELDL uptake in murine SMCs. Furthermore, rANGPTL4 also upregulated HCASMC migration dose dependently (1-5 μg/ml for 24 h) and was comparable with the migration induced by ELDL. However, Lacidipine had no effect on rANGPLT4 mediated migration, suggesting that ANGPLT-4 independently of ELDL uptake promotes migration of HCASMC. In calcification assays using MEM with 0.2% FCS and 1.5 mM phosphate, ELDL at 2.5 μg/ml induced more calcification native LDL (>25%, p<0.01, analyzed by alizarin red staining and organic extraction, and this was proceeded by increase in BMP-2 mRNA. Conclusions: ELDL is highly potent in inducing foam cells in cultured HCASMC. Whole genome expression and bioinformatics analysis indicate up-regulation of pathways linked to osteochondrogenic transformation. BMP-2 and ANGPTL4 are significantly upregulated in ELDL-induced HCASMC foam cells. These results point to the potential of ELDL to induce migratory and osteoblastic effects in HCASMC with potential implications in SMC migration and calcification in human atherosclerosis.

Abstract 363: Lysosomal Lipid Accumulation and Dysregulation of Downstream Cholesterol Homeostasis in Human Arterial Smooth Muscle Cells

2017 ◽  
Vol 37 (suppl_1) ◽  
Author(s):  
Joshua A Dubland ◽  
Collin S Pryma ◽  
Sima Allahverdian ◽  
Ying Wang ◽  
Teddy Chan ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Lipid Accumulation ◽  
Foam Cell ◽  
Cholesterol Acyltransferase ◽  
Foam Cells ◽  
Cholesterol Homeostasis ◽  
Foam Cell Formation ◽  
Human Coronary Artery ◽  
Coronary Artery Atherosclerosis ◽  
Abca1 Expression

Background and Hypothesis: Previously we reported that ≥ 50% of the foam cells in human coronary artery atherosclerosis are smooth muscle cell (SMC) derived, and that the rate-limiting cholesterol exporter, ATP-binding cassette transporter protein A1 (ABCA1), has reduced expression in SMCs compared to leukocytes in the intima. Upregulation of ABCA1 expression is dependent on normal flux of lipoprotein-derived cholesterol out of lysosomes and the subsequent generation of 27-hydroxycholesterol (27-OHC). Excess lipoprotein-derived cholesterol is also converted to cholesteryl esters (CE) through the actions of acyl-coA cholesterol acyltransferase (ACAT). Processes by which macrophages store lipoprotein-derived cholesterol in cytosolic and lysosomal compartments are well described, whereas less is known about SMCs. Hypothesis: We hypothesize that preferential SMC foam cell formation and the observed derangement in ABCA1 expression are associated with increased lysosomal sequestration of atherogenic lipids. Methods and Results: Human aortic SMCs and human monocyte-derived macrophages (HMMs) were loaded with aggregated LDL (agLDL) for 24 hrs, followed by a 24 hr equilibration without lipids to allow processing of lipoproteins. Data are presented as mean±SEM. In response to agLDL, CE content (nmol/mg protein) measured by LC/MS/MS increased from 2.2±1.3 to 277.0±18.2 in HMMs, and 3.5±5.3 to 46.8±14.5 in SMCs (n=9). Confocal microscopy indicated elevated lysosomal lipid accumulation in SMCs compared to HMMs. Levels of 27-OHC (ng/mg protein) as measured by LC/MS/MS increased from 80.9±9.5 to 281.4±22.7 (3.5-fold) in HMMs, and 0.7±0.2 to 1.5±0.3 (1.4-fold) in SMCs (n=9). ACAT activity, measured by the incorporation of 14 C-labelled oleate (pmol/mg protein), increased from 21.0±1.7 to 678.1±110.9 (32.2-fold) in HMMs, and 55.6±3.3 to 133.7±15.2 (2.4-fold) in SMCs (n=16). By Western blot, fold change in ABCA1 with exposure to agLDL was 2.3±0.3 in HMMs and 1.3±0.2 in SMCs (n=7-10). Conclusions: Accumulation of atherogenic lipids in the lysosomes of SMCs provides a potential mechanism for the reduced ABCA1 expression and preferential formation of foam cells by arterial SMCs.

Abstract 1455: Athero-protective Role of IL10 Involves Regulation of Lipid Metabolism in Macrophages

Circulation ◽  
2007 ◽  
Vol 116 (suppl_16) ◽  
Author(s):  
Xinbing Han ◽  
Shiro Kitamoto ◽  
Qingyu Lian ◽  
William A Boisvert
Keyword(s):  
Lipid Metabolism ◽  
Cholesterol Efflux ◽  
Foam Cell ◽  
Foam Cells ◽  
Foam Cell Formation ◽  
Lipid Uptake ◽  
Modified Ldl ◽  
Ldl Uptake ◽  
Inflammatory Molecules

Introduction Previous studies utilizing interleukin (IL)10-overexpressing mice and IL10-deficient mice have demonstrated an anti-atherogenic role of IL10. Internalization of modified low density lipoprotein (LDL) that leads to foam cell formation has long been considered one of the requisite initiating events in atherogenesis. We sought to determine if IL10 exerts its anti-atherogenic effect by modulating lipid metabolism in the macrophage. Methods & results In lipid uptake studies, IL10 substantially stimulated Dil-acetylated (Ac)LDL uptake by 187% in murine macrophage-like RAW264.7 cells. IL10 induced the expression of SR-AII and CD36 by 15.1 fold and 6.5 fold, respectively, in macrophage-derived foam cells. Moreover, CD36 protein levels were increased by IL10, suggesting that these scavenger receptors account, at least in part, for the increase in modified LDL uptake by the macrophages. Accordingly, IL10 treatment for 24hr significantly increased cholesteryl ester content by 1.5 folds compared with untreated controls (p<0.05). Interestingly, IL10 also markedly promoted ATP-binding cassette protein A1 (ABCA1)-mediated free cholesterol efflux to lipid-free apoAI acting as a cholesterol acceptor. This was peroxisome proliferator-activated receptor (PPAR)γ-dependent because specific PPARγ antagonist GW9226 completely blocked the IL10-triggered cholesterol efflux to lipid-free apoAI. In addition, expression of pro-inflammatory molecules such as TNFα, MCP-1 and iCAM-1 was dramatically inhibited by IL10 in the lipid-laden foam cells. Using immunofluorescence assay of caspase 3 fragment and TUNEL assay, we demonstrated that IL10 significantly suppressed apoptosis of foam cells (27.3 ± 2.1% for AcLDL-treated cells vs. 8.3 ± 1.0 %for AcLDL plus IL10-treated cells, n=8). Conclusion Our results indicate that IL10 can mediate both the uptake of cholesterol from modified LDL and the efflux of stored cholesterol. Therefore, IL10 may facilitate the removal of harmful atherogenic lipoprotein molecules from the vessel wall. These characteristics along with its ability to suppress the expression of inflammatory molecules and apoptosis of foam cells make IL10 a highly anti-atherogenic agent.

scholarly journals Foam Cells as Therapeutic Targets in Atherosclerosis with a Focus on the Regulatory Roles of Non-Coding RNAs

2021 ◽  
Vol 22 (5) ◽  
pp. 2529
Author(s):  
Amin Javadifar ◽  
Sahar Rastgoo ◽  
Maciej Banach ◽  
Tannaz Jamialahmadi ◽  
Thomas P. Johnston ◽  
...  
Keyword(s):  
Lipid Metabolism ◽  
Foam Cell ◽  
Cell Formation ◽  
Foam Cells ◽  
Foam Cell Formation ◽  
Special Focus ◽  
Lipid Uptake ◽  
Therapeutic Goals ◽  
Expression Of Genes

Atherosclerosis is a major cause of human cardiovascular disease, which is the leading cause of mortality around the world. Various physiological and pathological processes are involved, including chronic inflammation, dysregulation of lipid metabolism, development of an environment characterized by oxidative stress and improper immune responses. Accordingly, the expansion of novel targets for the treatment of atherosclerosis is necessary. In this study, we focus on the role of foam cells in the development of atherosclerosis. The specific therapeutic goals associated with each stage in the formation of foam cells and the development of atherosclerosis will be considered. Processing and metabolism of cholesterol in the macrophage is one of the main steps in foam cell formation. Cholesterol processing involves lipid uptake, cholesterol esterification and cholesterol efflux, which ultimately leads to cholesterol equilibrium in the macrophage. Recently, many preclinical studies have appeared concerning the role of non-encoding RNAs in the formation of atherosclerotic lesions. Non-encoding RNAs, especially microRNAs, are considered regulators of lipid metabolism by affecting the expression of genes involved in the uptake (e.g., CD36 and LOX1) esterification (ACAT1) and efflux (ABCA1, ABCG1) of cholesterol. They are also able to regulate inflammatory pathways, produce cytokines and mediate foam cell apoptosis. We have reviewed important preclinical evidence of their therapeutic targeting in atherosclerosis, with a special focus on foam cell formation.

Abstract 57: The Epigenetic Enzyme Kdm6b Controls the Pro-Fibrotic Transcriptome Signature of Foam Cells

2017 ◽  
Vol 37 (suppl_1) ◽  
Author(s):  
Annette E Neele ◽  
Koen H Prange ◽  
Marten A Hoeksema ◽  
Saskia van der Velden ◽  
Tina Lucas ◽  
...  
Keyword(s):  
Foam Cell ◽  
Smooth Muscle Actin ◽  
Cell Formation ◽  
Atherosclerotic Lesion ◽  
Foam Cells ◽  
Foam Cell Formation ◽  
Cell Phenotype ◽  
Sequencing Analysis ◽  
Dependent Manner ◽  
Alpha Smooth Muscle Actin

Aim: Foam cells are a key hallmark of atherosclerotic lesion formation. Within the atherosclerotic lesion macrophages scavenge modified lipoproteins and thereby acquire their foam cell characteristics. Besides their foam cell phenotype, macrophages can have specific inflammation regulatory functions in atherosclerotic lesions. Epigenetic pathways are crucial for monocyte to macrophage differentiation and activation. The H3K27 demethylase Kdm6b (also known as Jmjd3) is regulated in response to various triggers and regulates several modes of macrophage activation. Given the crucial role of macrophage foam cells in atherosclerosis, we here studied Kdm6b in peritoneal foam cells in order to identify regulated pathways. Material and Methods: A myeloid deficient Kdm6b mice (LysMCre-Kdm6b fl/fl ) was generated and bone marrow of Kdm6b wt or Kdm6b del mice was transplanted to irradiated Ldlr -/- mice which were fed a high fat diet for 9 weeks to induce foam cell formation. Peritoneal foam cells from Kdm6b del or Kdm6b wt mice were isolated and used for RNA-sequencing analysis. Results: Among the list of downregulated genes many genes involving fibrosis were affected in Kdm6b deficient foam cells including Collagen genes ( Col1a1 , Col1a2 ), Alpha smooth muscle actin ( Acta2 ) and Fibronectin-1 ( Fn1 ). Pathway analysis on downregulated genes ( P -value < 0.05) indicated that pathways involved in epithelial to mesenchymaltransition (EMT) ( q- value=10 -13 ) and extracellular matrix organization ( q- value=10 -4 ) were significantly downregulated. Pro-fibrotic pathways were thus strongly suppressed in Kdm6b deleted foam cells. Analysis of published datasets of foam cells showed that foam cell formation induces these pro-fibrotic characteristics. Overlay of both data sets indicated that fibrotic genes which are induced upon foam cell formation, are reduced in the absence of Kdm6b. These data suggest that foam cell formation induces a pro-fibrotic gene signature in a Kdm6b-dependent manner. Conclusion: We identified Kdm6b as a novel regulator of the pro-fibrotic signature of peritoneal foam cells.

scholarly journals Anti-Atherosclerotic Activity of (3R)-5-Hydroxymellein from an Endophytic Fungus Neofusicoccum parvum JS-0968 Derived from Vitex rotundifolia through the Inhibition of Lipoproteins Oxidation and Foam Cell Formation

Biomolecules ◽  
2020 ◽  
Vol 10 (5) ◽  
pp. 715
Author(s):  
Jae-Yong Kim ◽  
Soonok Kim ◽  
Sang Hee Shim
Keyword(s):  
Conformational Changes ◽  
Endophytic Fungus ◽  
Foam Cell ◽  
Oxidized Ldl ◽  
Cell Formation ◽  
Density Lipoprotein ◽  
Ldl Oxidation ◽  
Foam Cell Formation ◽  
Macrophage Foam Cell ◽  
Neofusicoccum Parvum

An endophytic fungus, Neofusicoccum parvum JS-0968, was isolated from a plant, Vitex rotundifolia. The chemical investigation of its cultures led to the isolation of a secondary metabolite, (3R)-5-hydroxymellein. It has been reported to have antifungal, antibacterial, and antioxidant activity, but there have been no previous reports on the effects of (3R)-5-hydroxymellein on atherosclerosis. The oxidation of lipoproteins and foam cell formation have been known to be significant in the development of atherosclerosis. Therefore, we investigated the inhibitory effects of (3R)-5-hydroxymellein on atherosclerosis through low-density lipoprotein (LDL) and high-density lipoprotein (HDL) oxidation and macrophage foam cell formation. LDL and HDL oxidation were determined by measuring the production of conjugated dienes and malondialdehyde, the amount of hyperchromicity and carbonyl content, conformational changes, and anti-LDL oxidation. In addition, the inhibition of foam cell formation was measured by Oil red O staining. As a result, (3R)-5-hydroxymellein suppressed the oxidation of LDL and HDL through the inhibition of lipid peroxidation, the decrease of negative charges, the reduction of hyperchromicity and carbonyl contents, and the prevention of apolipoprotein A-I (ApoA-I) aggregation and apoB-100 fragmentation. Furthermore, (3R)-5-hydroxymellein significantly reduced foam cell formation induced by oxidized LDL (oxLDL). Taken together, our data show that (3R)-5-hydroxymellein could be a potential preventive agent for atherosclerosis via obvious anti-LDL and HDL oxidation and the inhibition of foam cell formation.

scholarly journals Role of Phagocytosis in the Pro-Inflammatory Response in LDL-Induced Foam Cell Formation; a Transcriptome Analysis

2020 ◽  
Vol 21 (3) ◽  
pp. 817 ◽  
Author(s):  
Alexander N. Orekhov ◽  
Nikita G. Nikiforov ◽  
Vasily N. Sukhorukov ◽  
Marina V. Kubekina ◽  
Igor A. Sobenin ◽  
...  
Keyword(s):  
Immune Response ◽  
Inflammatory Response ◽  
Foam Cell ◽  
Cell Formation ◽  
Foam Cell Formation ◽  
Master Regulators ◽  
Knock Down ◽  
Modified Ldl ◽  
Naturally Occurring ◽  
Stimulation Of

Excessive accumulation of lipid inclusions in the arterial wall cells (foam cell formation) caused by modified low-density lipoprotein (LDL) is the earliest and most noticeable manifestation of atherosclerosis. The mechanisms of foam cell formation are not fully understood and can involve altered lipid uptake, impaired lipid metabolism, or both. Recently, we have identified the top 10 master regulators that were involved in the accumulation of cholesterol in cultured macrophages induced by the incubation with modified LDL. It was found that most of the identified master regulators were related to the regulation of the inflammatory immune response, but not to lipid metabolism. A possible explanation for this unexpected result is a stimulation of the phagocytic activity of macrophages by modified LDL particle associates that have a relatively large size. In the current study, we investigated gene regulation in macrophages using transcriptome analysis to test the hypothesis that the primary event occurring upon the interaction of modified LDL and macrophages is the stimulation of phagocytosis, which subsequently triggers the pro-inflammatory immune response. We identified genes that were up- or downregulated following the exposure of cultured cells to modified LDL or latex beads (inert phagocytosis stimulators). Most of the identified master regulators were involved in the innate immune response, and some of them were encoding major pro-inflammatory proteins. The obtained results indicated that pro-inflammatory response to phagocytosis stimulation precedes the accumulation of intracellular lipids and possibly contributes to the formation of foam cells. In this way, the currently recognized hypothesis that the accumulation of lipids triggers the pro-inflammatory response was not confirmed. Comparative analysis of master regulators revealed similarities in the genetic regulation of the interaction of macrophages with naturally occurring LDL and desialylated LDL. Oxidized and desialylated LDL affected a different spectrum of genes than naturally occurring LDL. These observations suggest that desialylation is the most important modification of LDL occurring in vivo. Thus, modified LDL caused the gene regulation characteristic of the stimulation of phagocytosis. Additionally, the knock-down effect of five master regulators, such as IL15, EIF2AK3, F2RL1, TSPYL2, and ANXA1, on intracellular lipid accumulation was tested. We knocked down these genes in primary macrophages derived from human monocytes. The addition of atherogenic naturally occurring LDL caused a significant accumulation of cholesterol in the control cells. The knock-down of the EIF2AK3 and IL15 genes completely prevented cholesterol accumulation in cultured macrophages. The knock-down of the ANXA1 gene caused a further decrease in cholesterol content in cultured macrophages. At the same time, knock-down of F2RL1 and TSPYL2 did not cause an effect. The results obtained allowed us to explain in which way the inflammatory response and the accumulation of cholesterol are related confirming our hypothesis of atherogenesis development based on the following viewpoints: LDL particles undergo atherogenic modifications that, in turn, accompanied by the formation of self-associates; large LDL associates stimulate phagocytosis; as a result of phagocytosis stimulation, pro-inflammatory molecules are secreted; these molecules cause or at least contribute to the accumulation of intracellular cholesterol. Therefore, it became obvious that the primary event in this sequence is not the accumulation of cholesterol but an inflammatory response.

scholarly journals Signaling Pathways and Key Genes Involved in Regulation of foam Cell Formation in Atherosclerosis

Cells ◽  
2020 ◽  
Vol 9 (3) ◽  
pp. 584 ◽  
Author(s):  
Anastasia V. Poznyak ◽  
Wei-Kai Wu ◽  
Alexandra A. Melnichenko ◽  
Reinhard Wetzker ◽  
Vasily Sukhorukov ◽  
...  
Keyword(s):  
Foam Cell ◽  
Low Density Lipoprotein ◽  
Cell Formation ◽  
Density Lipoprotein ◽  
Foam Cells ◽  
Foam Cell Formation ◽  
Beneficial Effects ◽  
Complex Process ◽  
Experimental Therapies ◽  
Inflammatory Drugs

Atherosclerosis is associated with acute cardiovascular conditions, such as ischemic heart disease, myocardial infarction, and stroke, and is the leading cause of morbidity and mortality worldwide. Our understanding of atherosclerosis and the processes triggering its initiation is constantly improving, and, during the last few decades, many pathological processes related to this disease have been investigated in detail. For example, atherosclerosis has been considered to be a chronic inflammation triggered by the injury of the arterial wall. However, recent works showed that atherogenesis is a more complex process involving not only the immune system, but also resident cells of the vessel wall, genetic factors, altered hemodynamics, and changes in lipid metabolism. In this review, we focus on foam cells that are crucial for atherosclerosis lesion formation. It has been demonstrated that the formation of foam cells is induced by modified low-density lipoprotein (LDL). The beneficial effects of the majority of therapeutic strategies with generalized action, such as the use of anti-inflammatory drugs or antioxidants, were not confirmed by clinical studies. However, the experimental therapies targeting certain stages of atherosclerosis, among which are lipid accumulation, were shown to be more effective. This emphasizes the relevance of future detailed investigation of atherogenesis and the importance of new therapies development.

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