Examining atherosclerotic lesions in three dimensions at the nanometer scale with cryo-FIB-SEM.

We employed in a correlative manner an unconventional combination of methods, comprising cathodoluminescence, cryo-scanning electron microscopy (SEM), and cryo-focused ion beam (FIB)-SEM, to examine the volumes of thousands of cubed micrometers from rabbit atherosclerotic tissues, maintained in close-to-native conditions, with a resolution of tens of nanometers. Data from three different intralesional regions, at the media-lesion interface, in the core, and toward the lumen, were analyzed following segmentation and volume or surface representation. The media-lesion interface region is rich in cells and lipid droplets, whereas the core region is markedly richer in crystals and has lower cell density. In the three regions, thin crystals appear to be associated with intracellular or extracellular lipid droplets and multilamellar bodies. Large crystals are independently positioned in the tissue, not associated with specific cellular components. This extensive evidence strongly supports the idea that the lipid droplet surfaces and the outer membranes of multilamellar bodies play a role in cholesterol crystal nucleation and growth and that crystal formation occurs, in part, inside cells. The correlative combination of methods that allowed the direct examination of cholesterol crystals and lipid deposits in the atherosclerotic lesions may be similarly used for high-resolution examination of other tissues containing pathological or physiological cholesterol deposits.

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.

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.

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.

Macrophage sortilin promotes LDL uptake, foam cell formation, and atherosclerosis.

RATIONALE: Noncoding gene variants at the SORT1 locus are strongly associated with low-density lipoprotein cholesterol (LDL-C) levels, as well as with coronary artery disease. SORT1 encodes a protein called sortilin, and hepatic sortilin modulates LDL metabolism by targeting apolipoprotein B-containing lipoproteins to the lysosome. Sortilin is also expressed in macrophages, but its role in macrophage uptake of LDL and in atherosclerosis independent of plasma LDL-C levels is unknown. OBJECTIVE: To determine the effect of macrophage sortilin expression on LDL uptake, foam cell formation, and atherosclerosis. METHODS AND RESULTS: We crossed Sort1(-/-) mice onto a humanized Apobec1(-/-); hAPOB transgenic background and determined that Sort1 deficiency on this background had no effect on plasma LDL-C levels but dramatically reduced atherosclerosis in the aorta and aortic root. To test whether this effect was a result of macrophage sortilin deficiency, we transplanted Sort1(-/-);LDLR(-/-) or Sort1(+/+);LDLR(-/-) bone marrow into Ldlr(-/-) mice and observed a similar reduction in atherosclerosis in mice lacking hematopoetic sortilin without an effect on plasma LDL-C levels. In an effort to determine the mechanism by which hematopoetic sortilin deficiency reduced atherosclerosis, we found no effect of sortilin deficiency on macrophage recruitment or lipopolysaccharide-induced cytokine release in vivo. In contrast, sortilin-deficient macrophages had significantly reduced uptake of native LDL ex vivo and reduced foam cell formation in vivo, whereas sortilin overexpression in macrophages resulted in increased LDL uptake and foam cell formation. CONCLUSIONS: Macrophage sortilin deficiency protects against atherosclerosis by reducing macrophage uptake of LDL. Sortilin-mediated uptake of native LDL into macrophages may be an important mechanism of foam cell formation and contributor to atherosclerosis development.

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.

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 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.

Increased plasma cholesterol esterification by LCAT reduces diet-induced atherosclerosis in SR-BI knockout mice.

LCAT, a plasma enzyme that esterifies cholesterol, has been proposed to play an antiatherogenic role, but animal and epidemiologic studies have yielded conflicting results. To gain insight into LCAT and the role of free cholesterol (FC) in atherosclerosis, we examined the effect of LCAT over- and underexpression in diet-induced atherosclerosis in scavenger receptor class B member I-deficient [Scarab(-/-)] mice, which have a secondary defect in cholesterol esterification. Scarab(-/-)×LCAT-null [Lcat(-/-)] mice had a decrease in HDL-cholesterol and a high plasma ratio of FC/total cholesterol (TC) (0.88 ± 0.033) and a marked increase in VLDL-cholesterol (VLDL-C) on a high-fat diet. Scarab(-/-)×LCAT-transgenic (Tg) mice had lower levels of VLDL-C and a normal plasma FC/TC ratio (0.28 ± 0.005). Plasma from Scarab(-/-)×LCAT-Tg mice also showed an increase in cholesterol esterification during in vitro cholesterol efflux, but increased esterification did not appear to affect the overall rate of cholesterol efflux or hepatic uptake of cholesterol. Scarab(-/-)×LCAT-Tg mice also displayed a 51% decrease in aortic sinus atherosclerosis compared with Scarab(-/-) mice (P < 0.05). In summary, we demonstrate that increased cholesterol esterification by LCAT is atheroprotective, most likely through its ability to increase HDL levels and decrease pro-atherogenic apoB-containing lipoprotein particles.

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 UBIAD1 prenyltransferase links menaquinone-4 [corrected] synthesis to cholesterol metabolic enzymes.

Schnyder corneal dystrophy (SCD) is an autosomal dominant disease characterized by germline variants in UBIAD1 introducing missense alterations leading to deposition of cholesterol in the cornea, progressive opacification, and loss of visual acuity. UBIAD1 was recently shown to synthesize menaquinone-4 (MK-4, vitamin K(2) ), but causal mechanisms of SCD are unknown. We report a novel c.864G>A UBIAD1 mutation altering glycine 177 to glutamic acid (p.G177E) in six SCD families, including four families from Finland who share a likely founder mutation. We observed reduced MK-4 synthesis by UBIAD1 altered by SCD mutations p.N102S, p.G177R/E, and p.D112N, and molecular models showed p.G177-mutant UBIAD1 disrupted transmembrane helices and active site residues. We show UBIAD1 interacts with HMGCR and SOAT1, enzymes catalyzing cholesterol synthesis and storage, respectively, using yeast two-hybrid screening and immunoprecipitation. Docking simulations indicate cholesterol binds to UBIAD1 in the substrate-binding cleft and substrate-binding overlaps with GGPP binding, an MK-4 substrate, suggesting potential competition between these metabolites. Impaired MK-4 synthesis is a biochemical defect identified in SCD suggesting UBIAD1 links vitamin K and cholesterol metabolism through physical contact between enzymes and metabolites. Our data suggest a role for endogenous MK-4 in maintaining cornea health and visual acuity.

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.

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.

Receptor-independent fluid-phase pinocytosis mechanisms for induction of foam cell formation with native low-density lipoprotein particles.

PURPOSE OF REVIEW: Because early findings indicated that native low-density lipoprotein (LDL) did not substantially increase macrophage cholesterol content during in-vitro incubations, investigators presumed that LDL must be modified in some way to trigger its uptake by the macrophage. The purpose of this review is to discuss recent findings showing that native unmodified LDL can induce massive macrophage cholesterol accumulation mimicking macrophage foam cell formation that occurs within atherosclerotic plaques. RECENT FINDINGS: Macrophages that show high rates of fluid-phase pinocytosis also show similar high rates of uptake of native unmodified LDL through nonreceptor mediated uptake within both macropinosomes and micropinosomes. Nonsaturable fluid-phase uptake of LDL by macrophages converts the macrophages into foam cells. Different macrophage phenotypes demonstrate either constitutive fluid-phase pinocytosis or inducible fluid-phase pinocytosis. Fluid-phase pinocytosis has been demonstrated by macrophages within mouse atherosclerotic plaques indicating that this pathway contributes to plaque macrophage cholesterol accumulation. SUMMARY: Contrary to what has been believed previously, macrophages can take up large amounts of native unmodified LDL by receptor-independent, fluid-phase pinocytosis converting these macrophages into foam cells. Thus, targeting macrophage fluid-phase pinocytosis should be considered when investigating strategies to limit macrophage cholesterol accumulation in atherosclerotic plaques.

Evaluation of transduction efficiency in macrophage colony-stimulating factor differentiated human macrophages using HIV-1 based lentiviral vectors.

BACKGROUND: Monocyte-derived macrophages contribute to atherosclerotic plaque formation. Therefore, manipulating macrophage function could have significant therapeutic value. The objective of this study was to determine transduction efficiency of two HIV-based lentiviral vector configurations as delivery systems for the transduction of primary human blood monocyte-derived macrophages. RESULTS: Human blood monocytes were transduced using two VSV-G pseudotyped HIV-1 based lentiviral vectors containing EGFP expression driven by either native HIV-LTR (VRX494) or EF1α promoters (VRX1090). Lentiviral vectors were added to cultured macrophages at different times and multiplicities of infection (MOI). Transduction efficiency was assessed using fluorescence microscopy and flow cytometry. Macrophages transduced between 2 and 120 hours after culturing showed the highest transduction efficiency at 2-hours transduction time. Subsequently, cells were transduced 2 hours after culturing at various vector concentrations (MOIs of 5, 10, 25 and 50) to determine the amount of lentiviral vector particles required to maximally transduce human monocyte-derived macrophages. On day 7, all transduced cultures showed EGFP-positive cells by microscopy. Flow cytometric analysis showed with all MOIs a peak shift corresponding to the presence of EGFP-positive cells. For VRX494, transduction efficiency was maximal at an MOI of 25 to 50 and ranged between 58 and 67%. For VRX1090, transduction efficiency was maximal at an MOI of 10 and ranged between 80 and 90%. Thus, transductions performed with VRX1090 showed a higher number of EGFP-positive cells than VRX494. CONCLUSIONS: This report shows that VSV-G pseudotyped HIV-based lentiviral vectors can efficiently transduce human blood monocyte-derived macrophages early during differentiation using low particle numbers that do not interfere with differentiation of monocytes into macrophages.

Passage of low-density lipoproteins through Bruch's membrane and choroid.

Plasma lipoproteins are thought to transport cholesterol, vitamins and carotenoids to the retinal pigment epithelium (RPE) for ultimate use by the photoreceptors. However, to reach the RPE, these lipoprotein particles must cross Bruch's membrane. We examined the reflection coefficient of Bruch's membrane (BrM) to low-density lipoprotein (LDL). Bruch's membrane and choroid were removed from 47 bovine eyes. Specimens were placed in a Ussing chamber and perfused with phosphate-buffered saline (PBS) with (31 specimens) or without (16 specimens) fluorescent low-density lipoproteins (DiI-LDL). The hydraulic conductivity of the tissue was determined for both calf and cow eyes. In the perfusions with DiI-LDL, the fluorescence intensity emitted by DiI-LDL in the efflux was measured and the reflection coefficient of BrM/choroid preparations to DiI-LDL determined. Leakage tests were done to confirm tissue integrity. Several specimens were examined using scanning electron microscopy (SEM) to examine tissue integrity before and after perfusion. Leak testing confirmed that BrM was intact both before and after perfusion. The average hydraulic conductivity of BrM/choroid perfusion of calf eyes with PBS alone was 1.42 ± 0.55 × 10(-9) m/s/Pa (mean ± SD, n = 11). The average hydraulic conductivity of the cow eyes was 4.94 ± 1.48 × 10(-10) m/s/Pa (n = 5), nearly a 3-fold decrease with age. While the flow rate remained constant during the PBS perfusions, it decreased as a function of time during perfusion with DiI-LDLs. Our major finding was of fluorescence in the effluent collected in all perfusions with DiI-LDLs, demonstrating passage of LDL through the tissue. The average reflection coefficient of calf BrM/choroid preparations to DiI-LDL was 0.58 ± 0.25 (n = 23); a similar distribution of reflection coefficients was seen in tissue from cow eyes (0.51 ± 0.33, n = 8). Our data suggested that the DiI-LDL was modestly hindered and/or captured by the tissue. This might explain the progressive decrease of hydraulic conductivity with continued perfusion of DiI-LDL.

Native low-density lipoprotein uptake by macrophage colony-stimulating factor-differentiated human macrophages is mediated by macropinocytosis and micropinocytosis.

OBJECTIVE: To examine the pinocytotic pathways mediating native low-density lipoprotein (LDL) uptake by human macrophage colony-stimulating factor-differentiated macrophages (the predominant macrophage phenotype in human atherosclerotic plaques). METHODS AND RESULTS: We identified the kinase inhibitor SU6656 and the Rho GTPase inhibitor toxin B as inhibitors of macrophage fluid-phase pinocytosis of LDL. Assessment of macropinocytosis by time-lapse microscopy revealed that both drugs almost completely inhibited macropinocytosis, although LDL uptake and cholesterol accumulation by macrophages were only partially inhibited (approximately 40%) by these agents. Therefore, we investigated the role of micropinocytosis in mediating LDL uptake in macrophages and identified bafilomycin A1 as an additional partial inhibitor (approximately 40%) of macrophage LDL uptake that targeted micropinocytosis. When macrophages were incubated with both bafilomycin A1 and SU6656, inhibition of LDL uptake was additive (reaching 80%), showing that these inhibitors target different pathways. Microscopic analysis of fluid-phase uptake pathways in these macrophages confirmed that LDL uptake occurs through both macropinocytosis and micropinocytosis. CONCLUSIONS: Our findings show that human macrophage colony-stimulating factor-differentiated macrophages take up native LDL by macropinocytosis and micropinocytosis, underscoring the importance of both pathways in mediating LDL uptake by these cells.

Extracellular cholesterol-rich microdomains generated by human macrophages and their potential function in reverse cholesterol transport.

Previous studies have shown that cholesterol in atherosclerotic plaques is present in both intracellular and extracellular forms. In the current study, we investigated a mechanism for extracellular cholesterol accumulation and examined the capacity of this pool of cholesterol to be removed by cholesterol acceptors, a step in reverse cholesterol transport. Human monocyte-derived macrophages differentiated with macrophage-colony stimulating factor were incubated with acetylated LDL to allow cholesterol enrichment and processing. These macrophages were subsequently labeled with a monoclonal antibody that specifically detects ordered cholesterol arrays, revealing the presence of unesterified cholesterol-rich microdomains on the cell surfaces and in the extracellular matrix. Similar unesterified cholesterol-rich microdomains were present in human atherosclerotic plaques. Actin microfilaments functioned in microdomain deposition or maintenance, and Src family kinases regulated transfer of these microdomains from the cell surface onto the extracellular matrix. Mediators of reverse cholesterol transport, apolipoprotein A-I (apoA-I), and HDL were capable of removing these extracellular un-esterified cholesterol-rich microdomains. However, apoA-I removed the microdomains only when macrophages were present. ApoA-I removal of microdomains was blocked by glyburide and inhibitor of ATP-binding cassette transporter A1 (ABCA1) function. In summary, cultures of cholesterol-enriched human monocyte-derived macrophages generate extracellular unesterified cholesterol-rich microdomains, which can subsequently be removed by cholesterol acceptors and therefore potentially function in reverse cholesterol transport.

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".