Hyperlipidaemia and IFNgamma/TNFalpha Synergism are associated with cholesterol crystal formation in Endothelial cells partly through modulation of Lysosomal pH and Cholesterol homeostasis.

BACKGROUND: Inflammation plays an important role in the development of cardiovascular disease (CVD). Patients with chronic inflammation diseases have high levels of inflammation and early fatal myocardial infarction due to early, unstable coronary plaques. Cholesterol crystals (CC) play a key role in atherogenesis. However, the underlying mechanisms of endothelial cell (EC)-derived CC formation are not well understood in chronic inflammation. METHODS: We utilized a combination of a mouse psoriasis model (K14-Rac1V12 mouse model) and human psoriasis patients to study the effect of inflammatory cytokines on CC formation in ECs. Lysosomal pH, alterations in lipid load and inflammatory proteins were evaluated as potential mechanisms linking inflammatory cytokines to CC formation. Coronary CT angiography was performed (n = 224) to characterize potential IFNγ and TNFα synergism on vascular diseases in vivo. FINDINGS: We detected CC presence in the aorta of K14-Rac1V12 mice on chow diet. IFNγ and TNFα were found to synergistically increase LDL-induced CC formation by almost 2-fold. There was an increase in lysosomal pH accompanied by a 28% loss in pH-dependent lysosomal signal and altered vATPaseV1E1 expression patterns. In parallel, we found that LDL+IFNγ/TNFα treatments increased free cholesterol content within EC and led to a decrease in SOAT-1 expression, an enzyme critically involved cholesterol homeostasis. Finally, the product of IFNγ and TNFα positively associated with early non-calcified coronary burden in patients with psoriasis (n = 224; ß = 0.28, p < 0.001). INTERPRETATION: Our results provide evidence that IFNγ and TNFα accelerate CC formation in endothelial cells in part by altering lysosomal pH and free cholesterol load. These changes promote early atherogenesis and contribute to understanding the burden of CVD in psoriasis. FUNDING: Funding was provided by the Intramural Research Program at NIH (NNM) and the National Psoriasis Foundation (NNM and YB).

Interleukin 10 promotes macrophage uptake of HDL and LDL by stimulating fluid-phase endocytosis.

OBJECTIVE: Highly elevated plasma levels of interleukin-10 (IL-10) are causally associated with "Disappearing HDL Syndrome" and low plasma LDL-cholesterol, but the underlying mechanism is poorly understood. Fluid-phase endocytosis, a process highly dependent on actin dynamics, enables cells to internalize relatively high amounts of extracellular fluids and solutes. We sought to investigate whether IL-10 induces lipoprotein uptake by fluid-phase endocytosis in macrophages. METHODS AND RESULTS: Macrophages (RAW264.7, Kupffer and human) were incubated with vehicle (PBS) or IL-10 (20 ng/ml) for 7 days. Uptake of HDL, LDL, and/or fluid-phase endocytosis probes (albumin-Alexa680®, 70 kDa FITC-Dextran and Lucifer Yellow, LY) was evaluated by FACS. Intracellular cofilin and phosphorylated cofilin (p-cofilin) levels were determined by immunoblotting. Macrophage uptake of lipoproteins and probes was non-saturable and increased after IL-10 incubation (p < 0.0001). Furthermore, pre-incubation with fluid-phase endocytosis inhibitors (LY294002, Latrunculin A, and Amiloride) significantly reduced uptake (p < 0.05). IL-10 increased the cofilin/p-cofilin ratio (p = 0.021), signifying increased cofilin activation and hence filamentous actin. Consistently, phalloidin staining revealed increased filamentous actin in macrophages after IL-10 treatment (p = 0.0018). Finally, RNA-seq analysis demonstrated enrichment of gene sets related to actin filament dynamics, membrane ruffle formation and endocytosis in IL-10-treated macrophages (p < 0.05). IL-10 did not alter mRNA levels of Ldlr, Vldlr, Scarb1, Cd36 or Lrp1. In primary human monocyte-derived macrophages and murine Kupffer cells, IL-10 incubation also increased uptake of lipoproteins, albumin and LY (p < 0.01). CONCLUSIONS: Interleukin-10 induces the uptake of HDL and LDL by fluid-phase endocytosis by increasing actin-filament rearrangement in macrophages, thus providing a plausible mechanism contributing to "Disappearing HDL Syndrome".

LCAT, ApoD, and ApoA1 Expression and Review of Cholesterol Deposition in the Cornea.

Lecithin:cholesterol acyltransferase (LCAT) is an enzyme secreted by the liver and circulates with high-density lipoprotein (HDL) in the blood. The enzyme esterifies plasma cholesterol and increases the capacity of HDL to carry and potentially remove cholesterol from tissues. Cholesterol accumulates within the extracellular connective tissue matrix of the cornea stroma in individuals with genetic deficiency of LCAT. LCAT can be activated by apolipoproteins (Apo) including ApoD and ApoA1. ApoA1 also mediates cellular synthesis of HDL. This study examined the expression of LCAT by epithelial cells, keratocytes, and endothelial cells, the cell types that comprise from anterior to posterior the three layers of the cornea. LCAT and ApoD were immunolocalized to all three cell types within the cornea, while ApoA1 was immunolocalized to keratocytes and endothelium but not epithelium. In situ hybridization was used to detect LCAT, ApoD, and ApoA1 mRNA to learn what cell types within the cornea synthesize these proteins. No corneal cells showed mRNA for ApoA1. Keratocytes and endothelium both showed ApoD mRNA, but epithelium did not. Epithelium and endothelium both showed LCAT mRNA, but despite the presence of LCAT protein in keratocytes, keratocytes did not show LCAT mRNA. RNA sequencing analysis of serum-cultured dedifferentiated keratocytes (commonly referred to as corneal stromal fibroblasts) revealed the presence of both LCAT and ApoD (but not ApoA1) mRNA, which was accompanied by their respective proteins detected by immunolabeling of the cultured keratocytes and Western blot analysis of keratocyte lysates. The results indicate that keratocytes in vivo show both ApoA1 and LCAT proteins, but do not synthesize these proteins. Rather, keratocytes in vivo must take up ApoA1 and LCAT from the corneal interstitial tissue fluid.

Ultramorphological analysis of plaque advancement and cholesterol crystal formation in Ldlr knockout mouse atherosclerosis.

BACKGOUND AND AIMS: The low-density lipoprotein receptor-deficient (Ldlr-/-) mouse has been utilized by cardiovascular researchers for more than two decades to study atherosclerosis. However, there has not yet been a systematic effort to document the ultrastructural changes that accompany the progression of atherosclerotic plaque in this model. METHODS: Employing several different staining and microscopic techniques, including immunohistochemistry, as well as electron and polarized microscopy, we analyzed atherosclerotic lesion development in Ldlr-/- mice fed an atherogenic diet over time. RESULTS: Lipid-like deposits occurred in the subendothelial space after only one week of atherogenic diet. At two weeks, cholesterol crystals (CC) formed and increased thereafter. Lipid, CC, vascular smooth muscles cells, and collagen progressively increased over time, while after 4 weeks, relative macrophage content decreased. Accelerated accumulation of plate- and needle-shaped CC accompanied plaque core necrosis. Lastly, CC were surrounded by cholesterol microdomains, which co-localized with CC through all stages of atherosclerosis, indicating that the cholesterol microdomains may be a source of CC. CONCLUSIONS: Here, we have documented, for the first time in a comprehensive way, atherosclerotic plaque morphology and composition from early to advanced stages in the Ldlr-/- mouse, one of the most commonly used animal models utilized in atherosclerosis research.

Modified LDL Particles Activate Inflammatory Pathways in Monocyte-derived Macrophages: Transcriptome Analysis.

BACKGROUND: A hallmark of atherosclerosis is its complex pathogenesis, which is dependent on altered cholesterol metabolism and inflammation. Both arms of pathogenesis involve myeloid cells. Monocytes migrating into the arterial walls interact with modified low-density lipoprotein (LDL) particles, accumulate cholesterol and convert into foam cells, which promote plaque formation and also contribute to inflammation by producing proinflammatory cytokines. A number of studies characterized transcriptomics of macrophages following interaction with modified LDL, and revealed alteration of the expression of genes responsible for inflammatory response and cholesterol metabolism. However, it is still unclear how these two processes are related to each other to contribute to atherosclerotic lesion formation. METHODS: We attempted to identify the main mater regulator genes in macrophages treated with atherogenic modified LDL using a bioinformatics approach. RESULTS: We found that most of the identified genes were involved in inflammation, and none of them was implicated in cholesterol metabolism. Among the key identified genes were interleukin (IL)-7, IL-7 receptor, IL- 15 and CXCL8. CONCLUSION: Our results indicate that activation of the inflammatory pathway is the primary response of the immune cells to modified LDL, while the lipid metabolism genes may be a secondary response triggered by inflammatory signalling.

HDL activates expression of genes stimulating cholesterol efflux in human monocyte-derived macrophages.

High density lipoproteins (HDL) are key components of reverse cholesterol transport pathway. HDL removes excessive cholesterol from peripheral cells, including macrophages, providing protection from cholesterol accumulation and conversion into foam cells, which is a key event in pathogenesis of atherosclerosis. The mechanism of cellular cholesterol efflux stimulation by HDL involves interaction with the ABCA1 lipid transporter and ensuing transfer of cholesterol to HDL particles. In this study, we looked for additional proteins contributing to HDL-dependent cholesterol efflux. Using RNAseq, we analyzed mRNAs induced by HDL in human monocyte-derived macrophages and identified three genes, fatty acid desaturase 1 (FADS1), insulin induced gene 1 (INSIG1), and the low-density lipoprotein receptor (LDLR), expression of which was significantly upregulated by HDL. We individually knocked down these genes in THP-1 cells using gene silencing by siRNA, and measured cellular cholesterol efflux to HDL. Knock down of FADS1 did not significantly change cholesterol efflux (p = 0.70), but knockdown of INSIG1 and LDLR resulted in highly significant reduction of the efflux to HDL (67% and 75% of control, respectively, p < 0.001). Importantly, the suppression of cholesterol efflux was independent of known effects of these genes on cellular cholesterol content, as cells were loaded with cholesterol using acetylated LDL. These results indicate that HDL particles stimulate expression of genes that enhance cellular cholesterol transfer to HDL.

Two polymorphic cholesterol monohydrate crystal structures form in macrophage culture models of atherosclerosis.

The formation of atherosclerotic plaques in the blood vessel walls is the result of LDL particle uptake, and consequently of cholesterol accumulation in macrophage cells. Excess cholesterol accumulation eventually results in cholesterol crystal deposition, the hallmark of mature atheromas. We followed the formation of cholesterol crystals in J774A.1 macrophage cells with time, during accumulation of LDL particles, using a previously developed correlative cryosoft X-ray tomography (cryo-SXT) and stochastic optical reconstruction microscopy (STORM) technique. We show, in the initial accumulation stages, formation of small quadrilateral crystal plates associated with the cell plasma membrane, which may subsequently assemble into large aggregates. These plates match crystals of the commonly observed cholesterol monohydrate triclinic structure. Large rod-like cholesterol crystals form at a later stage in intracellular locations. Using cryotransmission electron microscopy (cryo-TEM) and cryoelectron diffraction (cryo-ED), we show that the structure of the large elongated rods corresponds to that of monoclinic cholesterol monohydrate, a recently determined polymorph of the triclinic crystal structure. These monoclinic crystals form with an unusual hollow cylinder or helical architecture, which is preserved in the mature rod-like crystals. The rod-like morphology is akin to that observed in crystals isolated from atheromas. We suggest that the crystals in the atherosclerotic plaques preserve in their morphology the memory of the structure in which they were formed. The identification of the polymorph structure, besides explaining the different crystal morphologies, may serve to elucidate mechanisms of cholesterol segregation and precipitation in atherosclerotic plaques.

Macrophages Shed Excess Cholesterol in Unique Extracellular Structures Containing Cholesterol Microdomains.

OBJECTIVE: Cells use various mechanisms to maintain cellular cholesterol homeostasis including efflux of cholesterol from the cellular plasma membrane to cholesterol acceptors such as HDLs (high-density lipoproteins). Little is known about the transfer of cholesterol from cells into the extracellular matrix. Using a unique monoclonal antibody that detects ordered cholesterol arrays (ie, cholesterol micro[or nano]-domains), we previously identified that particles containing these cholesterol domains accumulate in the extracellular matrix during cholesterol enrichment of human monocyte-derived macrophages and are found in atherosclerotic lesions. In this study, we further investigate these deposited particles containing cholesterol microdomains and discover their unexpected morphology. APPROACH AND RESULTS: Although appearing spherical at the resolution of the conventional fluorescence microscope, super-resolution immunofluorescence and atomic force microscopy of in situ cholesterol microdomains, and immunoelectron microscopy of isolated cholesterol microdomains revealed that the microdomains are not vesicles or 3-dimensional crystals but rather appear as branching irregularly shaped deposits of varying size. These cholesterol microdomain-containing deposits are shed from the plasma membrane into the extracellular matrix. CONCLUSIONS: To date, research on cellular excretion of excess cholesterol has demonstrated cellular cholesterol efflux in the form of membranous vesicles and discoidal HDL particles released into the fluid-phase medium. Shedding of plasma membrane cholesterol microdomains provides an additional mechanism for cells such as macrophages to maintain plasma membrane cholesterol homeostasis. Furthermore, recognition that macrophages shed cholesterol microdomains into the extracellular matrix is important to our understanding of extracellular buildup of cholesterol in atherosclerosis.

Chronic skin inflammation accelerates macrophage cholesterol crystal formation and atherosclerosis.

Inflammation is critical to atherogenesis. Psoriasis is a chronic inflammatory skin disease that accelerates atherosclerosis in humans and provides a compelling model to understand potential pathways linking these diseases. A murine model capturing the vascular and metabolic diseases in psoriasis would accelerate our understanding and provide a platform to test emerging therapies. We aimed to characterize a new murine model of skin inflammation (Rac1V12) from a cardiovascular standpoint to identify novel atherosclerotic signaling pathways modulated in chronic skin inflammation. The RacV12 psoriasis mouse resembled the human disease state, including presence of systemic inflammation, dyslipidemia, and cardiometabolic dysfunction. Psoriasis macrophages had a proatherosclerotic phenotype with increased lipid uptake and foam cell formation, and also showed a 6-fold increase in cholesterol crystal formation. We generated a triple-genetic K14-RacV12-/+/Srb1-/-/ApoER61H/H mouse and confirmed psoriasis accelerates atherogenesis (~7-fold increase). Finally, we noted a 60% reduction in superoxide dismutase 2 (SOD2) expression in human psoriasis macrophages. When SOD2 activity was restored in macrophages, their proatherogenic phenotype reversed. We demonstrate that the K14-RacV12 murine model captures the cardiometabolic dysfunction and accelerates vascular disease observed in chronic inflammation and that skin inflammation induces a proatherosclerotic macrophage phenotype with impaired SOD2 function, which associated with accelerated atherogenesis.

Measurement of Aortic Cell Fluid-Phase Pinocytosis in vivo by Flow Cytometry.

OBJECTIVE: Fluid-phase pinocytosis is a receptor-independent mechanism of endocytosis that occurs in all mammalian cells and may be a mechanism for the uptake of LDL by macrophages. As there are currently no methods for the measurement of fluid-phase pinocytosis by individual aortic cells in vivo, we sought to identify a suitable method. METHODS: ApoE-/- mice were retro-orbitally injected with AngioSPARK fluorescent nanoparticles specifically designed to not interact with cells. After 24 h, mice were sacrificed, and the aortas were isolated and then digested to analyze aortic cell uptake of AngioSPARK by flow cytometry. RESULTS: CD11b-expressing aortic macrophages from mice injected with AngioSPARK showed high levels of fluid-phase pinocytosis compared to aortic cells not expressing CD11b (4,393.7 vs. 408.3 mean fluorescence intensity [MFI], respectively). CONCLUSION: This new technique allows for the measurement of fluid-phase pinocytosis by aortic cells in vivo, making it possible to examine the cell-signaling molecules and drugs that affect this process. Published by S. Karger AG, Basel.

Use of Primary Macrophages for Searching Novel Immunocorrectors.

In this mini-review, the role of macrophage phenotypes in atherogenesis is considered. Recent studies on distribution of M1 and M2 macrophages in different types of atherosclerotic lesions indicate that macrophages exhibit a high degree of plasticity of phenotype in response to various conditions in microenvironment. The effect of the accumulation of cholesterol, a key event in atherogenesis, on the macrophage phenotype is also discussed. The article presents the results of transcriptome analysis of cholesterol-loaded macrophages revealing genes involved in immune response whose expression rate has changed the most. It turned out that the interaction of macrophages with modified LDL leads to higher expression levels of pro-inflammatory marker TNF-α and antiinflammatory marker CCL18. Phenotypic profile of macrophage activation could be a good target for testing of novel anti-atherogenic immunocorrectors. A number of anti-atherogenic drugs were tested as potential immunocorrectors using primary macrophage-based model.

Development of Correlative Cryo-soft X-ray Tomography and Stochastic Reconstruction Microscopy. A Study of Cholesterol Crystal Early Formation in Cells.

We have developed a high resolution correlative method involving cryo-soft X-ray tomography (cryo-SXT) and stochastic optical reconstruction microscopy (STORM), which provides information in three dimensions on large cellular volumes at 70 nm resolution. Cryo-SXT morphologically identified and localized aggregations of carbon-rich materials. STORM identified specific markers on the desired epitopes, enabling colocalization between the identified objects, in this case cholesterol crystals, and the cellular environment. The samples were studied under ambient and cryogenic conditions without dehydration or heavy metal staining. The early events of cholesterol crystal development were investigated in relation to atherosclerosis, using as model macrophage cell cultures enriched with LDL particles. Atherosclerotic plaques build up in arteries in a slow process involving cholesterol crystal accumulation. Cholesterol crystal deposition is a crucial stage in the pathological cascade. Our results show that cholesterol crystals can be identified and imaged at a very early stage on the cell plasma membrane and in intracellular locations. This technique can in principle be applied to other biological samples where specific molecular identification is required in conjunction with high resolution 3D-imaging.

ABCA1 (ATP-Binding Cassette Transporter A1) Mediates ApoA-I (Apolipoprotein A-I) and ApoA-I Mimetic Peptide Mobilization of Extracellular Cholesterol Microdomains Deposited by Macrophages.

OBJECTIVE: We examined the function of ABCA1 (ATP-binding cassette transporter A1) in ApoA-I (apolipoprotein A-I) mobilization of cholesterol microdomains deposited into the extracellular matrix by cholesterol-enriched macrophages. We have also determined whether an ApoA-I mimetic peptide without and with complexing to sphingomyelin can mobilize macrophage-deposited cholesterol microdomains. APPROACH AND RESULTS: Extracellular cholesterol microdomains deposited by cholesterol-enriched macrophages were detected with a monoclonal antibody, 58B1. ApoA-I and an ApoA-I mimetic peptide 5A mobilized cholesterol microdomains deposited by ABCA1+/+ macrophages but not by ABCA1-/- macrophages. In contrast, ApoA-I mimetic peptide 5A complexed with sphingomyelin could mobilize cholesterol microdomains deposited by ABCA1-/- macrophages. CONCLUSIONS: Our findings show that a unique pool of extracellular cholesterol microdomains deposited by macrophages can be mobilized by both ApoA-I and an ApoA-I mimetic peptide but that mobilization depends on macrophage ABCA1. It is known that ABCA1 complexes ApoA-I and ApoA-I mimetic peptide with phospholipid, a cholesterol-solubilizing agent, explaining the requirement for ABCA1 in extracellular cholesterol microdomain mobilization. Importantly, ApoA-I mimetic peptide already complexed with phospholipid can mobilize macrophage-deposited extracellular cholesterol microdomains even in the absence of ABCA1.

Culture of Macrophage Colony-stimulating Factor Differentiated Human Monocyte-derived Macrophages.

A protocol is presented for cell culture of macrophage colony-stimulating factor (M-CSF) differentiated human monocyte-derived macrophages. For initiation of experiments, fresh or frozen monocytes are cultured in flasks for 1 week with M-CSF to induce their differentiation into macrophages. Then, the macrophages can be harvested and seeded into culture wells at required cell densities for carrying out experiments. The use of defined numbers of macrophages rather than defined numbers of monocytes to initiate macrophage cultures for experiments yields macrophage cultures in which the desired cell density can be more consistently attained. Use of cryopreserved monocytes reduces dependency on donor availability and produces more homogeneous macrophage cultures.

ABCA1 contributes to macrophage deposition of extracellular cholesterol.

We previously reported that cholesterol-enriched macrophages excrete cholesterol into the extracellular matrix. A monoclonal antibody that detects cholesterol microdomains labels the deposited extracellular particles. Macro-phage deposition of extracellular cholesterol depends, in part, on ABCG1, and this cholesterol can be mobilized by HDL components of the reverse cholesterol transport process. The objective of the current study was to determine whether ABCA1 also contributes to macrophage deposition of extracellular cholesterol. ABCA1 functioned in extracellular cholesterol deposition. The liver X receptor agonist, TO901317 (TO9), an ABCA1-inducing factor, restored cholesterol deposition that was absent in cholesterol-enriched ABCG1(-/-) mouse macrophages. In addition, the ABCA1 inhibitor, probucol, blocked the increment in cholesterol deposited by TO9-treated wild-type macrophages, and completely inhibited deposition from TO9-treated ABCG1(-/-) macrophages. Lastly, ABCA1(-/-) macrophages deposited much less extracellular cholesterol than wild-type macrophages. These findings demonstrate a novel function of ABCA1 in contributing to macrophage export of cholesterol into the extracellular matrix.

Endotoxin contamination of apolipoprotein A-I: effect on macrophage proliferation--a cautionary tale.

This technical report addresses the problem of endotoxin contamination of apolipoprotein reagents. Using a bromodeoxyuridine incorporation cell proliferation assay, we observed that human plasma ApoA-I as low as 1 µg/ml resulted in a >90% inhibition in macrophage proliferation. However, not all ApoA-I from different sources showed this effect. We considered the possibility that endotoxin contamination of the apolipoproteins contributed to the differential inhibition of macrophage cell proliferation. Endotoxin alone very potently inhibited macrophage proliferation (0.1 ng/ml inhibited macrophage proliferation>90%). Measurement of endotoxin levels in the apolipoprotein products, including an analysis of free versus total endotoxin, the latter which included endotoxin that was masked due to binding to protein, suggested that free endotoxin mediated inhibition of macrophage proliferation. Despite the use of an advanced endotoxin removal procedure and agents commonly used to inhibit endotoxin action, the potency of endotoxin precluded successful elimination of endotoxin effect. Our findings show that endotoxin contamination can significantly influence apparent apolipoprotein-mediated cell effects (or effects of any other biological products), especially when these products are tested on highly endotoxin-sensitive cells, such as macrophages.

ABCG1-mediated generation of extracellular cholesterol microdomains.

Previous studies have demonstrated that the ATP-binding cassette transporters (ABC)A1 and ABCG1 function in many aspects of cholesterol efflux from macrophages. In this current study, we continued our investigation of extracellular cholesterol microdomains that form during enrichment of macrophages with cholesterol. Human monocyte-derived macrophages and mouse bone marrow-derived macrophages, differentiated with macrophage colony-stimulating factor (M-CSF) or granulocyte macrophage colony-stimulation factor (GM-CSF), were incubated with acetylated LDL (AcLDL) to allow for cholesterol enrichment and processing. We utilized an anti-cholesterol microdomain monoclonal antibody to reveal pools of unesterified cholesterol, which were found both in the extracellular matrix and associated with the cell surface, that we show function in reverse cholesterol transport. Coincubation of AcLDL with 50 µg/ml apoA-I eliminated all extracellular and cell surface-associated cholesterol microdomains, while coincubation with the same concentration of HDL only removed extracellular matrix-associated cholesterol microdomains. Only at an HDL concentration of 200 µg/ml did HDL eliminate the cholesterol microdomains that were cell-surface associated. The deposition of cholesterol microdomains was inhibited by probucol, but it was increased by the liver X receptor (LXR) agonist TO901317, which upregulates ABCA1 and ABCG1. Extracellular cholesterol microdomains did not develop when ABCG1-deficient mouse bone marrow-derived macrophages were enriched with cholesterol. Our findings show that generation of extracellular cholesterol microdomains is mediated by ABCG1 and that reverse cholesterol transport occurs not only at the cell surface but also within the extracellular space.

The TERE1 protein interacts with mitochondrial TBL2: regulation of trans-membrane potential, ROS/RNS and SXR target genes.

We originally discovered TERE1 as a potential tumor suppressor protein based upon reduced expression in bladder and prostate cancer specimens and growth inhibition of tumor cell lines/xenografts upon ectopic expression. Analysis of TERE1 (aka UBIAD1) has shown it is a prenyltransferase enzyme in the natural bio-synthetic pathways for both vitamin K-2 and COQ10 production and exhibits multiple subcellular localizations including mitochondria, endoplasmic reticulum, and golgi. Vitamin K-2 is involved in mitochondrial electron transport, SXR nuclear hormone receptor signaling and redox cycling: together these functions may form the basis for tumor suppressor function. To gain further insight into mechanisms of growth suppression and enzymatic regulation of TERE1 we isolated TERE1 associated proteins and identified the WD40 repeat, mitochondrial protein TBL2. We examined whether disease specific mutations in TERE1 affected interactions with TBL2 and the role of each protein in altering mitochondrial function, ROS/RNS production and SXR target gene regulation. Biochemical binding assays demonstrated a direct, high affinity interaction between TERE1 and TBL2 proteins; TERE1 was localized to both mitochondrial and non-mitochondrial membranes whereas TBL2 was predominantly mitochondrial; multiple independent single amino acid substitutions in TERE1 which cause a human hereditary corneal disease reduced binding to TBL2 strongly suggesting the relevance of this interaction. Ectopic TERE1 expression elevated mitochondrial trans-membrane potential, oxidative stress, NO production, and activated SXR targets. A TERE1-TBL2 complex likely functions in oxidative/nitrosative stress, lipid metabolism, and SXR signaling pathways in its role as a tumor suppressor.

Fluid-phase pinocytosis of native low density lipoprotein promotes murine M-CSF differentiated macrophage foam cell formation.

During atherosclerosis, low-density lipoprotein (LDL)-derived cholesterol accumulates in macrophages to form foam cells. Macrophage uptake of LDL promotes foam cell formation but the mechanism mediating this process is not clear. The present study investigates the mechanism of LDL uptake for macrophage colony-stimulating factor (M-CSF)-differentiated murine bone marrow-derived macrophages. LDL receptor-null (LDLR-/-) macrophages incubated with LDL showed non-saturable accumulation of cholesterol that did not down-regulate for the 24 h examined. Incubation of LDLR-/- macrophages with increasing concentrations of (125)I-LDL showed non-saturable macrophage LDL uptake. A 20-fold excess of unlabeled LDL had no effect on (125)I-LDL uptake by wild-type macrophages and genetic deletion of the macrophage scavenger receptors CD36 and SRA did not affect (125)I-LDL uptake, showing that LDL uptake occurred by fluid-phase pinocytosis independently of receptors. Cholesterol accumulation was inhibited approximately 50% in wild-type and LDLR-/- mice treated with LY294002 or wortmannin, inhibitors of all classes of phosphoinositide 3-kinases (PI3K). Time-lapse, phase-contrast microscopy showed that macropinocytosis, an important fluid-phase uptake pathway in macrophages, was blocked almost completely by PI3K inhibition with wortmannin. Pharmacological inhibition of the class I PI3K isoforms alpha, beta, gamma or delta did not affect macrophage LDL-derived cholesterol accumulation or macropinocytosis. Furthermore, macrophages from mice expressing kinase-dead class I PI3K beta, gamma or delta isoforms showed no decrease in cholesterol accumulation or macropinocytosis when compared with wild-type macrophages. Thus, non-class I PI3K isoforms mediated macropinocytosis in these macrophages. Further characterization of the components necessary for LDL uptake, cholesterol accumulation, and macropinocytosis identified dynamin, microtubules, actin, and vacuolar type H(+)-ATPase as contributing to uptake. However, Pak1, Rac1, and Src-family kinases, which mediate fluid-phase pinocytosis in certain other cell types, were unnecessary. In conclusion, our findings provide evidence that targeting those components mediating macrophage macropinocytosis with inhibitors may be an effective strategy to limit macrophage accumulation of LDL-derived cholesterol in arteries.

Murine bone marrow-derived macrophages differentiated with GM-CSF become foam cells by PI3Kγ-dependent fluid-phase pinocytosis of native LDL.

Accumulation of cholesterol by macrophage uptake of LDL is a key event in the formation of atherosclerotic plaques. Previous research has shown that granulocyte-macrophage colony-stimulating factor (GM-CSF) is present in atherosclerotic plaques and promotes aortic lipid accumulation. However, it has not been determined whether murine GM-CSF-differentiated macrophages take up LDL to become foam cells. GM-CSF-differentiated macrophages from LDL receptor-null mice were incubated with LDL, resulting in massive macrophage cholesterol accumulation. Incubation of LDL receptor-null or wild-type macrophages with increasing concentrations of ¹²5I-LDL showed nonsaturable macrophage LDL uptake that was linearly related to the amount of LDL added, indicating that LDL uptake was mediated by fluid-phase pinocytosis. Previous studies suggest that phosphoinositide 3-kinases (PI3K) mediate macrophage fluid-phase pinocytosis, although the isoform mediating this process has not been determined. Because PI3Kγ is known to promote aortic lipid accumulation, we investigated its role in mediating macrophage fluid-phase pinocytosis of LDL. Wild-type macrophages incubated with LDL and the PI3Kγ inhibitor AS605240 or PI3Kγ-null macrophages incubated with LDL showed an ∼50% reduction in LDL uptake and cholesterol accumulation compared with wild-type macrophages incubated with LDL only. These results show that GM-CSF-differentiated murine macrophages become foam cells by fluid-phase pinocytosis of LDL and identify PI3Kγ as contributing to this process.