Retention Of Lipoproteins Research Articles

Serum Amyloid A, but Not C-Reactive Protein, Stimulates Vascular Proteoglycan Synthesis in a Pro-Atherogenic Manner

Inflammatory markers serum amyloid A (SAA) and C-reactive protein (CRP) are predictive of cardiac disease and are proposed to play causal roles in the development of atherosclerosis, in which the retention of lipoproteins by vascular wall proteoglycans is critical. The purpose of this study was to determine whether SAA and/or CRP alters vascular proteoglycan synthesis and lipoprotein retention in a pro-atherogenic manner. Vascular smooth muscle cells were stimulated with either SAA or CRP (1 to 100 mg/L) and proteoglycans were then isolated and characterized. SAA, but not CRP, increased proteoglycan sulfate incorporation by 50 to 100% in a dose-dependent manner (P < 0.0001), increased glycosaminoglycan chain length, and increased low-density lipoprotein (LDL) binding affinity (K(d), 29 microg/ml LDL versus 90 microg/ml LDL for SAA versus control proteoglycans; P < 0.005). Furthermore, SAA up-regulated biglycan via the induction of endogenous transforming growth factor (TGF)-beta. To determine whether SAA stimulated proteoglycan synthesis in vivo, ApoE(-/-) mice were injected with an adenovirus expressing human SAA-1, a null virus, or saline. Mice that received adenovirus expressing SAA had increased TGF-beta concentrations in plasma and increased aortic biglycan content compared with mice that received either null virus or saline. Thus, SAA alters vascular proteoglycans in a pro-atherogenic manner via the stimulation of TGF-beta and may play a causal role in the development of atherosclerosis.

Hyperelongated biglycan: the surreptitious initiator of atherosclerosis

To outline a role for the dermatan sulfate proteoglycan biglycan and specifically its growth factor modified form having elongated glycosaminoglycan chains as being a primary initiator of atherosclerosis. Antiatherosclerotic therapies have mostly targeted epidemiologically identified, experimentally confirmed risk factors. The efficacy of such therapies is less than optimal, and rates of cardiovascular disease remain stubbornly high. A variety of targets have been actively pursued, but as yet no new therapy has emerged that specifically targets the vessel wall. One area concerns the role of proteoglycans in the trapping of atherogenic lipoproteins as an early and initiating step in atherogenesis. On the basis of studies in human coronary arteries, the prime proteoglycan for lipoprotein retention is biglycan. The glycosaminoglycan chains on biglycan are subject to regulation that yields several structural changes, but most prominently elongation of the chains to form 'hyperelongated biglycan'. Multiple animal studies and a recent human disorder study have demonstrated the colocalization of atherogenic lipoproteins with biglycan in atherosclerotic lesions. Moreover, in the human atherosclerosis, the deposition of lipid appears to precede the chronic inflammatory response typical of atherosclerotic lesions. The process of biglycan-associated glycosaminoglycan elongation represents a novel potential therapeutic target worthy of full investigation for the prevention of atherosclerosis.

Apolipoprotein CIII and Atherosclerosis

The prevailing concept of mechanisms responsible for the development of atherosclerotic lesions largely focuses on the accumulation and retention of low-density lipoproteins in the arterial intima and their subsequent oxidative modification. This oxidation leads to activation of the endothelium, and particularly, expression of adhesion molecules that mediate leukocyte adherence and chemokines which initiate the inflammation reaction that is widely accepted as being responsible for the development and progression of atherosclerotic lesions.1,2 There is also a strong body of evidence to indicate that elevated triacylglycerides (triglycerides) are an independent risk factor for atherosclerosis.3–5 Article p 731 One mechanism that can contribute to elevated triglycerides involves apolipoprotein CIII (apoCIII). apoCIII is a small protein that resides on the surface of very-low-density lipoproteins (VLDLs), low-density lipoproteins, chylomicrons, and high-density lipoproteins (Figure). It exists as multiple species, as either a nonglycosylated isoform (apoCIIIo) or a glycosylated isoform (apoCIII1 or apoCIII2); all three isoforms have similar plasma half-lives and probably have very similar physiological functions. Increased apoCIII production is a characteristic feature of patients with hypertriglyceridemia,6 and plasma apoCIII levels have been positively correlated with plasma triacylglycerol concentrations and also have been associated with severity of hypertriglyceridemia.7 Elevated plasma apoCIII concentration and, specifically, accumulation of apoCIII in triacylglycerol-rich lipoproteins is casually related to hypertriglyceridemia in patients with metabolic syndrome and has also been associated with insulin resistance.8 apoCIII is a major regulator of lipolysis, as it noncompetitively inhibits endothelial-bound lipoprotein lipase, the enzyme that hydrolyzes triacylglycerols in …

Effect of Sphingomyelinase-Mediated Generation of Ceramide on Aggregation of Low-Density Lipoprotein

This study addresses the response-to-retention hypothesis, which states that the subendothelial retention of atherogenic lipoproteins is the necessary and sufficient condition for the initiation of atherosclerosis. Here we focus on the relationship between the generation of ceramide in the low-density lipoprotein (LDL) phospholipid monolayer and the resulting aggregation of LDL particles. This study provides the first measurement of neutral, Mg (2+)-dependent Sphingomyelinase (Smase)-mediated ceramide formation from LDL-sphingomyelin and does so for a range of enzyme concentrations (0-0.22 units Smase/mL). The kinetics of ceramide generation was measured using a fluorescence assay for the above enzyme concentrations with a fixed substrate concentration (0.33 mg LDL/mL). The kinetics of LDL aggregate formation was measured by dynamic light scattering (DLS, method of cumulants) for identical enzyme concentrations. Ceramide concentration profiles were fit with a modification of the Michaelis-Menten model ( k a = 1.11 x 10 (-1) microM (-1) min (-1), k -a = 6.54 x 10 (2) microM (-1) min (-1), k 1 = 3.33 x 10 (1) microM (-1) min (-1), k -1 = 1.41 x 10 (-2) min (-1), k cat = 8.05 x 10 (1) min (-1), K M = 2.418 microM, k deact = 4.66 x 10 (-2) microM (-1) min (-1)) that accounts for the effects of enzyme attachment to the LDL monolayer and for deactivation of Smase due to product inhibition. LDL aggregation is described by a mass action model as explained in previous studies. A key result of this work is the finding that LDL aggregate size depends directly on ceramide concentration and is independent of enzyme concentration. This study demonstrates how principles of colloid science are relevant to important biomedical problems.

Acid Sphingomyelinase Promotes Lipoprotein Retention Within Early Atheromata and Accelerates Lesion Progression

The key initial step in atherogenesis is the subendothelial retention of apolipoprotein B-containing lipoproteins. Acid sphingomyelinase (acid SMase), an enzyme present extracellularly within the arterial wall, strongly enhances lipoprotein retention in model systems in vitro, and retained lipoproteins in human plaques are enriched in ceramide, a product of SMase. We now sought to test a direct causative role for acid SMase in lipoprotein retention and atherogenesis in vivo. We studied atherogenesis and lipoprotein retention in Asm(-/-) versus Asm(+/+) mice on the Apoe(-/-) and Ldlr(-/-) backgrounds. Asm(-/-);Apoe(-/-) mice had a approximately 40% to 50% decrease in early foam cell aortic root lesion area compared with Asm(+/+);Apoe(-/-) mice (P<0.05) despite no difference in plasma cholesterol or lipoproteins. To assay lipoprotein retention in vivo, the two groups of mice were injected with fluorescently labeled Apoe(-/-) lipoproteins. Early foam cell lesions of Asm(-/-);Apoe(-/-) mice showed a striking 87% reduction in lipoprotein trapping (P<0.0001) compared with Asm(+/+);Apoe(-/-) lesions. Similar results were obtained with Ldlr(-/-) mice, including an 81% reduction in lipoprotein retention within Asm(-/-);Ldlr(-/-) lesions compared with Asm(+/+);Ldlr(-/-) lesions (P<0.0005). These findings support a causal role for acid SMase in lipoprotein retention and lesion progression and provides further support for the response-to-retention model of atherogenesis.

Heparan Sulfate in Perlecan Promotes Mouse Atherosclerosis

Heparan sulfate (HS) has been proposed to be antiatherogenic through inhibition of lipoprotein retention, inflammation, and smooth muscle cell proliferation. Perlecan is the predominant HS proteoglycan in the artery wall. Here, we investigated the role of perlecan HS chains using apoE null (ApoE0) mice that were cross-bred with mice expressing HS-deficient perlecan ( Hspg2 Δ3/Δ3 ). Morphometry of cross-sections from aortic roots and en face preparations of whole aortas revealed a significant decrease in lesion formation in ApoE0/ Hspg2 Δ3/Δ3 mice at both 15 and 33 weeks. In vitro, binding of labeled mouse triglyceride-rich lipoproteins and human LDL to total extracellular matrix, as well as to purified proteoglycans, prepared from ApoE0/ Hspg2 Δ3/Δ3 smooth muscle cells was reduced. In vivo, at 20 minutes influx of human 125 I-LDL or mouse triglyceride-rich lipoproteins into the aortic wall was increased in ApoE0/ Hspg2 Δ3/Δ3 mice compared to ApoE0 mice. However, at 72 hours accumulation of 125 I-LDL was similar in ApoE0/ Hspg2 Δ3/Δ3 and ApoE0 mice. Immunohistochemistry of lesions from ApoE0/ Hspg2 Δ3/Δ3 mice showed decreased staining for apoB and increased smooth muscle α-actin content, whereas accumulation of CD68-positive inflammatory cells was unchanged. We conclude that the perlecan HS chains are proatherogenic in mice, possibly through increased lipoprotein retention, altered vascular permeability, or other mechanisms. The ability of HS to inhibit smooth muscle cell growth may also influence development as well as instability of lesions.

Heparan Sulfate in Perlecan Promotes Mouse Atherosclerosis: Roles in Lipid Permeability, Lipid Retention, and Smooth Muscle Cell Proliferation

Heparan sulfate (HS) has been proposed to be antiatherogenic through inhibition of lipoprotein retention, inflammation, and smooth muscle cell proliferation. Perlecan is the predominant HS proteoglycan in the artery wall. Here, we investigated the role of perlecan HS chains using apoE null (ApoE0) mice that were cross-bred with mice expressing HS-deficient perlecan (Hspg2(Delta3/Delta3)). Morphometry of cross-sections from aortic roots and en face preparations of whole aortas revealed a significant decrease in lesion formation in ApoE0/Hspg2(Delta3/Delta3) mice at both 15 and 33 weeks. In vitro, binding of labeled mouse triglyceride-rich lipoproteins and human LDL to total extracellular matrix, as well as to purified proteoglycans, prepared from ApoE0/Hspg2(Delta3/Delta3) smooth muscle cells was reduced. In vivo, at 20 minutes influx of human (125)I-LDL or mouse triglyceride-rich lipoproteins into the aortic wall was increased in ApoE0/Hspg2(Delta3/Delta3) mice compared to ApoE0 mice. However, at 72 hours accumulation of (125)I-LDL was similar in ApoE0/Hspg2(Delta3/Delta3) and ApoE0 mice. Immunohistochemistry of lesions from ApoE0/Hspg2(Delta3/Delta3) mice showed decreased staining for apoB and increased smooth muscle alpha-actin content, whereas accumulation of CD68-positive inflammatory cells was unchanged. We conclude that the perlecan HS chains are proatherogenic in mice, possibly through increased lipoprotein retention, altered vascular permeability, or other mechanisms. The ability of HS to inhibit smooth muscle cell growth may also influence development as well as instability of lesions.

Proteoglycan mediated lipoprotein retention: A mechanism of diabetic atherosclerosis

The response to retention hypothesis outlines the initial stages of atherosclerotic lesion formation. The central theme of the hypothesis is that proteoglycan mediated lipoprotein retention plays a critical step in the initiation of atherosclerosis development. Recent research using human arterial specimens, transgenic mouse models and molecular biology techniques have added to our understanding of atherosclerosis development, and provided experimental data in support of the response to retention hypothesis. In this review we summarize the recent data, in particular that which addresses mechanisms by which diabetes can accelerate atherosclerosis formation, with a focus on proteoglycan-mediated LDL retention.

Thrombin regulates vascular smooth muscle cell proteoglycan synthesis via PAR-1 and multiple downstream signalling pathways

Introduction Atherosclerosis is the underlying pathological process of most cardiovascular disease. Thrombin is a serine protease which can activate protease activated receptors (PAR) on vascular smooth muscle cells (VSMC) to elicit cellular responses that can contribute to the pathogenesis of atherosclerosis. Human atherosclerosis commences with the binding and retention of lipoproteins by the glycosaminoglycan (GAG) chains of chondroitin/dermatan sulfate proteoglycans. The potential effects of thrombin on the synthesis and structure of CS/DS proteoglycans produced by VSMCs was investigated. Materials and methods VSMCs were derived from human internal mammary arteries. Proteoglycan synthesis was assessed by [ 35S]sulfate and [ 3H]glucosamine incorporation. Proteoglycan size was assessed by SDS-PAGE and size exclusion chromatography. Results and conclusion Thrombin caused a dose-dependent increase in [ 35S]sulfate and [ 3H]glucosamine incorporation with maximum effects of approximately 150% at the highest doses tested. This increase was associated with increased size of biglycan and decorin assessed by SDS-PAGE. Chemically cleaved glycosaminoglycan (GAG) chains analyzed by SDS-PAGE and size exclusion chromatography were larger for proteoglycans from thrombin treated cells. VSMCs synthesize small GAGs when provided with exogenous xyloside and thrombin treatment also increased the size of the secreted xyloside GAGs. The effect of thrombin was not mimicked by the catalytically inactive FPRCK-HCT and was blocked in a concentration- dependent manner by the PAR-1 antagonist, JNJ5177049. Inhibition of PK C with GF 109203X resulted in concentration dependent but partial inhibition of [ 35S]sulfate incorporation accompanied by a reduction in the size of biglycan and decorin. Epidermal growth factor (EGF) stimulated [ 35S]sulfate incorporation and increased proteoglycan size and this was completely blocked by the EGF receptor tyrosine kinase inhibitor AG1478. AG1478 partially (32%, p < 0.01) blocked the effect of thrombin. Thrombin treatment of VSMCs increased the proportion of disaccharides sulfated at the 6 position of the GalNAc residues. Thus, thrombin has actions on VSMCs which increase the length and modify the sulfation pattern of GAG chains on proteoglycans in a manner that would enhance the binding of LDL. If manifest in vivo, this effect on proteoglycan synthesis and structure represents a new biochemical mechanism through which thrombin contributes to the development of atherosclerosis.

Role of the N- and C-Terminal Domains in Binding of Apolipoprotein E Isoforms to Heparan Sulfate and Dermatan Sulfate: A Surface Plasmon Resonance Study

The ability of apolipoprotein E (apoE) to bind to cell-surface glycosaminoglycans (GAGs) is important for lipoprotein remnant catabolism. Using surface plasmon resonance, we previously showed that the binding of apoE to heparin is a two-step process; the initial binding involves fast electrostatic interaction, followed by a slower hydrophobic interaction. Here we examined the contributions of the N- and C-terminal domains to each step of the binding of apoE isoforms to heparan sulfate (HS) and dermatan sulfate (DS). ApoE3 bound to less sulfated HS and DS with a decreased favorable free energy of binding in the first step compared to heparin, indicating that the degree of sulfation has a major effect on the electrostatic interaction of GAGs with apoE. Mutation of a key Lys residue in the N-terminal heparin binding site of apoE significantly affected this electrostatic interaction. Progressive truncation of the C-terminal alpha-helical regions which favors the monomeric form of apoE3 greatly weakened the ability of apoE3 to bind to HS, with a much reduced favorable free energy of binding of the first step, suggesting that the C-terminal domain contributes to the GAG binding of apoE by the oligomerization effect. In agreement with this, dimerization of the apoE3 N-terminal fragment via disulfide linkage restored the electrostatic interaction of apoE with HS. Significantly, apoE4 exhibited much stronger binding to HS and DS than apoE2 or apoE3 in both lipid-free and lipidated states, perhaps resulting from enhanced electrostatic interaction through the N-terminal domain. This isoform difference in GAG binding of apoE may be physiologically significant such as in the retention of apoE-containing lipoproteins in the arterial wall.

Atherosclerosis regression

Atherosclerosis follows the deposition, retention, and oxidative modification of lipoproteins, especially low-density lipoprotein (LDL) in the walls of large arteries. Uptake of oxidized LDL results in the formation of macrophage foam cells. Proliferation of vascular smooth muscle cells and secretion of extracellular matrix contribute "fibrous" components of the plaque, whereas ongoing accumulation of lipid and inflammatory cell debris forms the necrotic lipid core of the mature atherosclerotic plaque. Both the size and composition of plaques determine the clinical course. In particular, a large lipid core, thin fibrous cap, dense inflammatory cell infiltrate, and proteolytic enzyme activity are associated with adverse risk. Atherosclerosis has often been considered a relentlessly progressive disease. However, new imaging techniques that can quantify plaque burden and provide insights into some of the specific plaque components have allowed regression to be mapped for the first time. In this article, drugs targeting atherosclerosis that have potential or proven benefit in atherosclerosis regression are discussed.

Contribution of Macromolecular Structure to the Retention of Low-Density Lipoprotein at Arterial Branch Points

Extracellular deposition of low-density lipoprotein (LDL) in the arterial wall is an essential early step in atherosclerosis. This process preferentially occurs at arterial branch points, reflecting a regional variation in lipoprotein-arterial wall interactions. In this study, we characterized the submicron microstructure of arterial wall collagen and elastin to evaluate its potential role in regional LDL deposition. With 2-photon microscopy, we used the intrinsic optical properties of collagen and elastin to determine the arterial wall macromolecular microstructure in fresh porcine and murine arteries. This optical approach generated unique nondestructive en face 3-dimensional views of the wall. The collagen/elastin microstructure was found to vary with the topology of the arterial bed. A nearly confluent elastin surface layer was present throughout but was missing at atherosclerosis-susceptible branch points, exposing dense collagen-proteoglycan complexes. In LDL binding studies, this luminal elastin layer limited LDL penetration, whereas its absence at the branches resulted in extensive LDL binding. Furthermore, LDL colocalized with proteoglycans with a sigmoidal dose dependence (inflection point, approximately 130 mg LDL/dL). Ionic strength and competing anions studies were consistent with the initial interaction of LDL with proteoglycans to be electrostatic in nature. This optical sectioning approach provided a robust 3-dimensional collagen/elastin microstructure of the arterial wall in fresh samples. At atherosclerosis-susceptible vascular branch points, the absence of a luminal elastin barrier and the presence of a dense collagen/proteoglycan matrix contribute to increased retention of LDL.

Autoimmunity in atherosclerosis: a protective response losing control?

Atherosclerosis is a chronic inflammatory disease characterized by accumulation of oxidized lipoproteins, increased cell death and hypertrophic degeneration of the arterial intima. The disease process is associated with local formation of modified self antigens that are targeted by both innate and adaptive immune responses. Although it remains to be firmly established it is likely that these autoimmune responses initially have a beneficial effect facilitating the removal of potentially harmful rest products from oxidized LDL and dying cells. However, studies performed on hypercholesterolaemic mice deficient in different components of the immune system uniformly suggest that the net effect of immune activation is pro-atherogenic and that atherosclerosis, at least to some extent, should be regarded as an autoimmune disease. These observations point to the possibility of developing new treatments for atherosclerosis based on modulation of immune responses against plaque antigens, an approach presently tested clinically for several other chronic inflammatory diseases with autoimmune components. Pilot studies in animals have provided promising results for both parental and oral vaccines based on oxidized LDL antigens. The time when this concept is ready for clinical testing is rapidly approaching but it will be important not to underestimate the difficulties that will be encountered in transferring the promising results from experimental animals into humans.

Early atherosclerosis in humans: role of diffuse intimal thickening and extracellular matrix proteoglycans

This review attempts to define the early events that lead to lesions of human atherosclerosis based on careful morphological studies in human autopsy specimens. In contrast to most small laboratory animals, diffuse intimal thickening (DIT) is present in human arteries before atherosclerosis develops, particularly in the atherosclerosis-prone arteries such as coronary arteries and abdominal aorta. In the earliest stage of atherosclerosis, lipids deposit eccentrically in the deep layer of DIT to form Type I lesions. These layers are enriched in extracellular matrix (ECM) proteoglycans such as biglycan. Following lipid deposition, macrophages appear in these regions and foam cells are observed (Type II lesions). Such observations support the 'response-to-retention' hypothesis that states that a principle early event in the pathogenesis of human atherosclerosis is the trapping and retention of lipoproteins by ECM proteoglycans followed by infiltration and accumulation of macrophages.

Diabetes and Arterial Extracellular Matrix Changes in a Porcine Model of Atherosclerosis

Patients with diabetes are at substantially increased risk for atherosclerosis and clinical cardiovascular events. Because arterial extracellular matrix contains several molecules, including biglycan, versican, hyaluronan, and elastin, that may affect plaque lipid retention and stability, we determined whether diabetes affects plaque content of these molecules in a porcine model of hyperlipidemia and diabetes. Coronary artery sections were studied from non-diabetic normolipidemic (n=11, N-NL), diabetic normolipidemic (n=10, DM-NL), non-diabetic hyperlipidemic (n=16, N-HL), and diabetic hyperlipidemic (n=15, DM-HL) animals. Hyaluronan, biglycan, versican, and apolipoprotein B (apoB) were detected with monospecific peptides or antisera, and elastin with Movat's pentachrome stain, and contents of each were quantified by computer-assisted morphometry. In the hyperlipidemic groups, diabetes was associated with a 4-fold increase in intimal area, with strong correlations between intimal area and immunostained areas for hyaluronan (R(2) = 0.83, p<0.0001), biglycan (R(2) = 0.72, p<0.0001), and apoB (R(2) = 0.23, p=0.0069). In contrast, median (interquartile range) intimal elastin content was significantly lower with diabetes [N-HL: 5.2% (2.4-8.2%) vs DM-HL: 1.5% (0.5-4.2%), p=0.01], and there was a strong negative correlation between intimal total and elastin areas (Spearman r = -0.62, p=0.001). In this porcine model, diabetes was associated with multiple extracellular matrix changes that have been associated with increased lesion instability, greater atherogenic lipoprotein retention, and accelerated atherogenesis.

Subendothelial Lipoprotein Retention as the Initiating Process in Atherosclerosis

The key initiating process in atherogenesis is the subendothelial retention of apolipoprotein B-containing lipoproteins. Local biological responses to these retained lipoproteins, including a chronic and maladaptive macrophage- and T-cell-dominated inflammatory response, promote subsequent lesion development. The most effective therapy against atherothrombotic cardiovascular disease to date--low density lipoprotein-lowering drugs--is based on the principle that decreasing circulating apolipoprotein B lipoproteins decreases the probability that they will enter and be retained in the subendothelium. Ongoing improvements in this area include more aggressive lowering of low-density lipoprotein and other atherogenic lipoproteins in the plasma and initiation of low-density lipoprotein-lowering therapy at an earlier age in at-risk individuals. Potential future therapeutic approaches include attempts to block the interaction of apolipoprotein B lipoproteins with the specific subendothelial matrix molecules that mediate retention and to interfere with accessory molecules within the arterial wall that promote retention such as lipoprotein lipase, secretory sphingomyelinase, and secretory phospholipase A2. Although not the primary focus of this review, therapeutic strategies that target the proatherogenic responses to retained lipoproteins and that promote the removal of atherogenic components of retained lipoproteins also hold promise. The finding that certain human populations of individuals who maintain lifelong low plasma levels of apolipoprotein B lipoproteins have an approximately 90% decreased risk of coronary artery disease gives hope that our further understanding of the pathogenesis of this leading killer could lead to its eradication.

2007 George L. Duff Memorial Lecture—The Immune Response in Atherosclerosis

Immune responses participate in every phase of atherosclerosis. Indeed, atherosclerosis can be viewed as an immune/inflammatory response to lipoprotein retention in the artery wall. There is increasing evidence that both adaptive and innate immunity tightly regulate the atherosclerotic process. Specific antigens and pathogen-like molecular patterns initiate the 2 aspects of immunity by ligating T- and B-cell receptors and pattern recognition receptors, respectively. Effector responses of vascular immune reactions include macrophage activation, cellular immunity, antibody formation, and vascular inflammation. Whereas experimental studies in gene-targeted models have identified major roles for innate immunity and Th1 responses in plaque initiation and progression, clinical, epidemiological and genetic studies suggest that plaque activation, rupture, and atherothrombosis also depend on immune reactions. This lecture will focus on the role of immune mechanisms in the formation and activation of atherosclerotic plaques.

Adipocyte differentiation-related protein is induced by LRP1-mediated aggregated LDL internalization in human vascular smooth muscle cells and macrophages

Aggregated LDL (agLDL) is internalized by LDL receptor-related protein (LRP1) in vascular smooth muscle cells (VSMCs) and human monocyte-derived macrophages (HMDMs). AgLDL is, therefore, a potent inducer of massive intracellular cholesteryl ester accumulation in lipid droplets. The adipocyte differentiation-related protein (ADRP) has been found on the surface of lipid droplets. The objectives of this work were to analyze whether agLDL uptake modulates ADRP expression levels and whether the effect of agLDL internalization on ADRP expression depends on LRP1 in human VSMCs and HMDMs. AgLDL strongly upregulates ADRP mRNA (real-time PCR) and protein expression (Western blot) in human VSMCs (mRNA: by 3.06-fold; protein: 8.58-fold) and HMDMs (mRNA: by 3.5-fold; protein: by 3.71-fold). Treatment of VSMCs and HMDMs with small anti-LRP1-interfering RNA (siRNA-LRP1) leads to specific inhibition of LRP1 expression. siRNA-LRP1 treatment significantly reduced agLDL-induced ADRP overexpression in HMDMs (by 69%) and in VSMCs (by 53%). Immunohystochemical studies evidence a colocolocalization between ADRP/macrophages and ADRP/VSMCs in advanced lipid-enriched atherosclerotic plaques. These results demonstrate that agLDL-LRP1 engagement induces ADRP overexpression in both HMDMs and human VSMCs and that ADRP is highly expressed in advanced lipid-enriched human atherosclerotic plaques. Therefore, LRP1-mediated agLDL uptake might play a pivotal role in vascular foam cell formation.

Retention of Low-Density Lipoprotein in Atherosclerotic Lesions of the Mouse

Direct binding of apolipoprotein (apo)B-containing lipoproteins to proteoglycans is the initiating event in atherosclerosis, but the processes involved at later stages of development are unclear. Here, we investigated the importance of the apoB-proteoglycan interaction in the development of atherosclerosis over time and investigated the role of lipoprotein lipase (LPL) to facilitate low-density lipoprotein (LDL) retention at later stages of development. Atherosclerosis was analyzed in apoB transgenic mice expressing LDL with normal (control LDL) or reduced proteoglycan-binding (RK3359-3369SA LDL) activity after an atherogenic diet for 0 to 40 weeks. The initiation of atherosclerosis was delayed in mice expressing RK3359-3369SA LDL, but they eventually developed the same level of atherosclerosis as mice expressing control LDL. Retention studies in vivo showed that although higher levels of 131I-tyramine cellobiose-labeled control LDL (131I-TC-LDL) were retained in nonatherosclerotic aortae compared with RK3359-3369SA 131I-TC-LDL, the retention was significantly higher and there was no difference between the groups in atherosclerotic aortae. Lower levels of control 125I-TC-LDL and RK3359-3369SA 125I-TC-LDL were retained in atherosclerotic aortae from ldlr-/- mice transplanted with lpl-/- compared with lpl+/+ bone marrow. Uptake of control LDL or RK3359-3369SA LDL into macrophages with specific expression of human catalytically active or inactive LPL was increased compared with control macrophages. Furthermore, transgenic mice expressing catalytically active or inactive LPL developed the same extent of atherosclerosis. Thus, retention of LDL in the artery wall is initiated by direct LDL-proteoglycan binding but shifts to indirect binding with bridging molecules such as LPL.

Expansive remodeling in venous bypass grafts: Novel implications for vein graft disease

Objective To date, intimal hyperplasia has been regarded as the principle mechanism responsible for subsequent vein graft disease. Lumen remodeling has not been previously considered as an additional mechanism. The objectives of this study were to determine changes in lumen remodeling in arterialized vein grafts, the accompanying cellular and extracellular matrix events contributing to remodeling, and the effects of a high cholesterol diet. Methods and results Reversed jugular vein-to-common carotid artery interposition grafts were constructed in 70 normocholesterolemic and 11 hypercholesterolemic male New Zealand white rabbits. The lumen area initially remained unchanged between 1 and 4 weeks but significantly increased by 40% at 12 weeks. This phase of expansive positive remodeling was accompanied by significantly increased cell apoptosis, collagen synthesis (1.7-fold), collagen content (3.7-fold), gelatinase (MMP-2 and MMP-9) levels and decreased tissue inhibitor of metalloproteinase (TIMP) levels. Expansive remodeling temporally corresponded to high macrophage infiltration and increased low density lipoprotein (LDL) retention (fourfold) in the vein grafts. A high cholesterol diet stimulated early macrophage infiltration and increased MMP-12 (metalloelastase) levels, which was associated with earlier onset of expansive remodeling. Conclusion Expansive lumenal remodeling is a novel mechanism of vein graft response to the arterial circulation, which is accelerated by a high cholesterol diet.