TMT-based quantitative proteomic profiling of human monocyte-derived macrophages and foam cells

Abstract Background Cardiovascular diseases remain the leading cause of morbidity and mortality worldwide, most of which are caused by atherosclerosis. Discerning processes that participate in macrophage-to-foam cell formation are critical for understanding the basic mechanisms underlying atherosclerosis. To explore the molecular mechanisms of foam cell formation, differentially expressed proteins were identified. Methods Human peripheral blood mononuclear cells were stimulated with macrophage colony-stimulating factor, and obtained macrophages were transformed into foam cells by oxidized low-density lipoprotein. Tandem mass tag (TMT) labeling combined with mass spectrometry was performed to find associations between foam cell transformation and proteome profiles. Results Totally, 5146 quantifiable proteins were identified, among which 1515 and 182 differentially expressed proteins (DEPs) were found in macrophage/monocyte and foam cell/macrophage, respectively. Subcellular localization analysis revealed that downregulated DEPs of macrophages/monocytes were mostly located in the nucleus, whereas upregulated DEPs of foam cells/macrophages were mostly extracellular or located in the plasma membrane. Functional analysis of DEPs demonstrated that cholesterol metabolism-related proteins were upregulated in foam cells, whereas immune response-related proteins were downregulated in foam cells. The protein interaction network showed that the DEPs with the highest interaction scores between macrophages and foam cells were mainly concentrated in lysosomes and the endoplasmic reticulum. Conclusions Proteomics analysis suggested that cholesterol metabolism was upregulated, while the immune response was suppressed in foam cells. KEGG enrichment analysis and protein-protein interaction analysis indicated that DEPs located in the endoplasmic reticulum and lysosomes might be key drivers of foam cell formation. These data provide a basis for identifying the potential proteins associated with the molecular mechanism underlying macrophage transformation to foam cells.

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

International Journal of Molecular Sciences ◽

10.3390/ijms22052529 ◽

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.

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

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvb.37.suppl_1.57 ◽

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.

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

International Journal of Molecular Sciences ◽

10.3390/ijms21030817 ◽

2020 ◽

Vol 21 (3) ◽

pp. 817 ◽

Cited By ~ 5

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.

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

Cells ◽

10.3390/cells9030584 ◽

2020 ◽

Vol 9 (3) ◽

pp. 584 ◽

Cited By ~ 16

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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Prednisone and Its Active Metabolite Prednisolone Attenuate Lipid Accumulation in Macrophages

Journal of Cardiovascular Pharmacology and Therapeutics ◽

10.1177/1074248419883591 ◽

2019 ◽

Vol 25 (2) ◽

pp. 174-186

Author(s):

Helana Jeries ◽

Nina Volkova ◽

Claudia Grajeda-Iglesias ◽

Mahmoud Najjar ◽

Mira Rosenblat ◽

...

Keyword(s):

Lipid Metabolism ◽

Lipid Accumulation ◽

Cholesterol Metabolism ◽

Foam Cell ◽

Ex Vivo ◽

Cholesterol Biosynthesis ◽

Cell Formation ◽

Foam Cell Formation ◽

Macrophage Foam Cell ◽

Healthy Human

Background: Synthetic forms of glucocorticoids (GCs; eg, prednisone, prednisolone) are anti-inflammatory drugs that are widely used in clinical practice. The role of GCs in cardiovascular diseases, including atherosclerosis, is highly controversial, and their impact on macrophage foam cell formation is still unknown. We investigated the effects of prednisone and prednisolone on macrophage oxidative stress and lipid metabolism. Methods and Results: C57BL/6 mice were intraperitoneally injected with prednisone or prednisolone (5 mg/kg) for 4 weeks, followed by lipid metabolism analyses in the aorta and peritoneal macrophages. We also analyzed the effect of serum samples obtained from 9 healthy human volunteers before and after oral administration of prednisone (20 mg for 5 days) on J774A.1 macrophage atherogenicity. Finally, J774A.1 macrophages, human monocyte-derived macrophages, and fibroblasts were incubated with increasing concentrations (0-200 ng/mL) of prednisone or prednisolone, followed by determination of cellular oxidative status, and triglyceride and cholesterol metabolism. Prednisone and prednisolone treatment resulted in a significant reduction in triglyceride and cholesterol accumulation in macrophages, as observed in vivo, ex vivo, and in vitro. These effects were associated with GCs’ inhibitory effect on triglyceride- and cholesterol-biosynthesis rates, through downregulation of diacylglycerol acyltransferase 1 and HMG-CoA reductase expression. Glucocorticoid-induced reduction of cellular lipid accumulation was mediated by the GC receptors on the macrophages, because the GC-receptor antagonist (RU486) abolished these effects. In fibroblasts, unlike macrophages, GCs showed no effects. Conclusion: Prednisone and prednisolone exhibit antiatherogenic activity by protecting macrophages from lipid accumulation and foam cell formation.

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Foam Cell Macrophages in Tuberculosis

Frontiers in Immunology ◽

10.3389/fimmu.2021.775326 ◽

2021 ◽

Vol 12 ◽

Author(s):

Pooja Agarwal ◽

Siamon Gordon ◽

Fernando O. Martinez

Keyword(s):

Mycobacterium Tuberculosis ◽

Lipid Droplet ◽

Immune Cells ◽

Lipid Droplets ◽

Foam Cell ◽

Cell Formation ◽

Foam Cells ◽

Metabolic Changes ◽

Foam Cell Formation ◽

Lipid Contents

Mycobacterium tuberculosis infects primarily macrophages in the lungs. Infected macrophages are surrounded by other immune cells in well organised structures called granulomata. As part of the response to TB, a type of macrophage loaded with lipid droplets arises which we call Foam cell macrophages. They are macrophages filled with lipid laden droplets, which are synthesised in response to increased uptake of extracellular lipids, metabolic changes and infection itself. They share the appearance with atherosclerosis foam cells, but their lipid contents and roles are different. In fact, lipid droplets are immune and metabolic organelles with emerging roles in Tuberculosis. Here we discuss lipid droplet and foam cell formation, evidence regarding the inflammatory and immune properties of foam cells in TB, and address gaps in our knowledge to guide further research.

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Phage Display Preparation of Specific Polypeptides in Atherosclerotic Foam Cells

Applied Sciences ◽

10.3390/app12020562 ◽

2022 ◽

Vol 12 (2) ◽

pp. 562

Author(s):

Xiang Ji ◽

Dan Liu ◽

Feng Wu ◽

Yu Cen ◽

Lan Ma

Keyword(s):

Phage Display ◽

Foam Cell ◽

Early Stage ◽

Cell Formation ◽

Foam Cells ◽

Foam Cell Formation ◽

Atherosclerosis Progression ◽

Starting Point ◽

Common Causes ◽

Early Atherosclerosis

Atherosclerosis and related complications are the most common causes of death in modern societies. Macrophage-derived foam cells play critical roles in the initiation and progression of atherosclerosis. Effective, rapid, and instrument-independent detection in the early stage of chronic atherosclerosis progression could provide an opportunity for early intervention and treatment. Therefore, as a starting point, in this study, we aimed to isolate and prepare foam cell-specific polypeptides using a phage display platform. The six target polypeptides, which were acquired in this study, were evaluated by ELISA and showed strong specificity with foam cells. Streptavidin coupled quantum dots (QDs) were used as fluorescence developing agents, and images of biotin-modified polypeptides specifically binding with foam cells were clearly observed. The polypeptides obtained in this study could lay the foundation for developing a rapid detection kit for early atherosclerosis lesions and could provide new materials for research on the mechanisms of foam cell formation and the development of blocking drugs.

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Pro–angiogenic Induction of Myeloid Cells for Therapeutic Angiogenesis Can Favor MAPK p38–dependent Foam Cell Formation

Blood ◽

10.1182/blood.v116.21.4442.4442 ◽

2010 ◽

Vol 116 (21) ◽

pp. 4442-4442

Author(s):

Eva Rohde ◽

Katharina Schallmoser ◽

Andreas Reinisch ◽

Nicole A Hofmann ◽

Thomas Pfeifer ◽

...

Keyword(s):

Clinical Trials ◽

Progenitor Cells ◽

Foam Cell ◽

Cell Formation ◽

Hematopoietic Cells ◽

Cell Types ◽

Foam Cells ◽

Therapeutic Angiogenesis ◽

Foam Cell Formation ◽

Vascular Regeneration

Abstract Abstract 4442 Background: Clinical trials for therapeutic angiogenesis use blood- or marrow-derived transplants containing hematopoietic cells, endothelial progenitor cells (EPCs) and mesenchymal stem and progenitor cells (MSPCs) to support vascular regeneration. Recently concerns have emerged, as bone marrow-derived stem cell preparations also include these three cell types which probably may contribute to atherosclerosis. We therefore asked whether human myelomonocytic hematopoietic cells, EPCs or MSPCs after pro-angiogenic induction can accumulate lipid droplets (LDs) and develop into foam cells. Method: LD accumulation was quantified by flow cytometry, confocal microscopy and cholesterol measurement in each of the tested cell types. The impact of an initial three-day pro-angiogenic culture on subsequent foam cell formation was studied to mimic a relevant setting already being used in clinical trials. The phosphorylation state of intracellular signaling molecules in response to pro-angiogenic stimulation was determined to delineate the operative mechanisms and to establish a basis for interventional strategies. Result: Foam cells were formed by monocytes but neither by EPCs nor by MSPCs after pro-angiogenic induction. Mitogen-activated protein kinase (MAPK) p38 phosphorylation was enhanced in monocytes after pro-angiogenic stimulation. Kinase inhibition almost abrogated intracellular LD accumulation. Conclusion: These data suggest that hematopoietic cell preparations containing monocytes bear the risk of foam cell formation after pro-angiogenic induction. EPCs and MSPCs instead may drive vascular regeneration without atherogenesis aggravation. A thorough understanding of cell biology is necessary to develop new strategies combining pro-angiogenic and anti-atherogenic cellular effects during therapeutic angiogenesis. Disclosures: No relevant conflicts of interest to declare.

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