Insights from Murine Studies on the Site Specificity of Atherosclerosis.

Atherosclerosis is an inflammatory reaction that develops at specific regions within the artery wall and at specific sites of the arterial tree over a varying time frame in response to a variety of risk factors. The mechanisms that account for the interaction of systemic factors and atherosclerosis-susceptible regions of the arterial tree to mediate this site-specific development of atherosclerosis are not clear. The dynamics of blood flow has a major influence on where in the arterial tree atherosclerosis develops, priming the site for interactions with atherosclerotic risk factors and inducing cellular and molecular participants in atherogenesis. But how this accounts for lesion development at various locations along the vascular tree across differing time frames still requires additional study. Currently, murine models are favored for the experimental study of atherogenesis and provide the most insight into the mechanisms that may contribute to the development of atherosclerosis. Based largely on these studies, in this review, we discuss the role of hemodynamic shear stress, SR-B1, and other factors that may contribute to the site-specific development of atherosclerosis.

Atherosclerosis: cell biology and lipoproteins.

PURPOSE OF REVIEW: Lipoproteins have significant role in both the promotion and prevention of atherosclerosis. This brief review will focus on recent reports on relationship between HDL and HDL subclasses and their composition and function, the role of apoC-III in metabolism of triglyceride-rich lipoproteins, the impact of Lipoprotein (a) (Lp(a)) on endothelial cells, and the mechanism of uptake of aggregated LDL by macrophages. RECENT FINDINGS: The complexity of the protein and lipid content of murine and human HDL and their relationship to its cholesterol efflux capacity have been examined. HDL has also been shown to have both antiatherogenic and proatherogenic properties. The relationship between apoC-III and LPL activity, apoprotein E mediated clearance of triglyceride-rich lipoproteins and the potential importance of apoC-III in the increased risk of cardiovascular disease in type 1 diabetics has been investigated. Oxidized phospholipid in Lp(a) promotes endothelial cells inflammatory and glycolytic responses. TLR4 participates in the uptake of aggregated LDL to contribute to foam cell formation. SUMMARY: These studies contribute to our mechanistic understanding of how lipoproteins contribute to atherogenesis and identify potential therapeutic targets.

Proceedings of the Ninth HDL (High-Density Lipoprotein) Workshop: Focus on Cardiovascular Disease.

The HDL (high-density lipoprotein) Workshop was established in 2009 as a forum for candid discussions among academic basic scientists, clinical investigators, and industry researchers about the role of HDL in cardiovascular disease. This ninth HDL Workshop was held on May 16 to 17, 2019 in Boston, MA, and included outstanding oral presentations from established and emerging investigators. The Workshop featured 5 sessions with topics that tackled the role of HDL in the vasculature, its structural complexity, its role in health and disease states, and its interaction with the intestinal microbiome. The highlight of the program was awarding the Jack Oram Award to the distinguished professor emeritus G.S. Getz from the University of Chicago. The tenth HDL Workshop will be held on May 2020 in Chicago and will continue the focus on intellectually stimulating presentations by established and emerging investigators on novel roles of HDL in cardiovascular and noncardiovascular health and disease states.

Invariant Natural Killer T-Cells and Total CD1d Restricted Cells Differentially Influence Lipid Metabolism and Atherosclerosis in Low Density Receptor Deficient Mice.

Natural killer T (NKT) cells are a distinct subset of lymphocytes that bridge the innate and adaptive immune response and can be divided into type I invariant NKT cells (iNKT) and type II NKT cells. The objective of this study is to examine the effects of NKT cell on lipid metabolism and the initiation and progression of atherosclerosis in LDL receptor deficient (LDLR-/-) mice. Mice were fed an atherogenic diet for 4 or 8 weeks and plasma lipids, lipoproteins, and atherosclerosis were measured. The selective absence of iNKT cells in Jα18-/-LDLR-/- mice led to an increase in plasma cholesterol levels in female mice. Transgenic Vα14tg/LDLR-/- mice with elevated numbers of iNKT cells had increased late atherosclerosis of the innominate artery, though absence of either iNKT cells or all NKT cells and other CD1d expressing cells had varying effects on atherosclerotic lesion burden in the ascending aortic arch and aortic root. These studies not only highlight the potential modulatory role played by NKT cells in atherosclerosis and lipid metabolism, but also raise the possibility that divergent roles may be played by iNKT and CD1d restricted cells such as type II NKT cells or other CD1d expressing cells.

Apoprotein E and Reverse Cholesterol Transport.

Apoprotein E (apoE) is a multifunctional protein. Its best-characterized function is as a ligand for low-density lipoprotein (LDL) receptor family members to mediate the clearance of apoB-containing atherogenic lipoproteins. Among its other functions, apoE is involved in cholesterol efflux, especially from cholesterol-loaded macrophage foam cells and other atherosclerosis-relevant cells, and in reverse cholesterol transport. Reverse cholesterol transport is a mechanism by which excess cellular cholesterol is transported via lipoproteins in the plasma to the liver where it can be excreted from the body in the feces. This process is thought to have a role in the attenuation of atherosclerosis. This review summarizes studies on the role of apoE in cellular cholesterol efflux and reverse cholesterol transport and discusses the identification of apoE mimetic peptides that may promote these pathways.

Deficiency of Invariant Natural Killer T Cells Does Not Protect Against Obesity but Exacerbates Atherosclerosis in Ldlr-/- Mice.

Obesity is a chronic inflammatory state characterized by altered levels of adipose tissue immune cell populations. Natural killer T (NKT) cells are CD1d restricted lymphocyte subsets that recognize lipid antigens whose level decreases in obese adipose tissue. However, studies in mice with deficiency or increased levels of NKT cells have yielded contradictory results, so the exact role of these cells in obesity and adipose tissue inflammation is not yet established. We previously showed that Ldlr-/- mice with excess invariant NKT (iNKT) cells demonstrate significant weight gain, adiposity, metabolic abnormalities, and atherosclerosis. The current study evaluates the effects of NKT cell deficiency on obesity, associated metabolic changes, and atherosclerosis in Jα18-/-Ldlr-/- (lacking iNKT cells) and Cd1d-/-Ldlr-/- (lacking invariant and type II NKT cells) mice, and control mice were fed an obesogenic diet (high fat, sucrose, cholesterol) for 16 weeks. Contrary to expectations, Ja18-/-Ldlr-/- mice gained significantly more weight than Ldlr-/- or Cd1d-/-Ldlr-/- mice, developed hypertriglyceridemia, and had worsened adipose tissue inflammation. All the mice developed insulin resistance and hepatic triglyceride accumulation. Ja18-/-Ldlr-/- mice also had increased atherosclerotic lesion area. Our findings suggest that iNKT cells exacerbates the metabolic, inflammatory, and atherosclerotic features of diet-induced obesity. Further work is required to unravel the paradox of an apparently similar effect of iNKT cell surplus and depletion on obesity.

Genetic control of apoprotein A-I and atheroprotection: some insights from inbred strains of mice.

PURPOSE OF REVIEW: Previous epidemiological studies and studies in experimental animals have provided strong evidence for the atheroprotective effect of HDL and its major apoprotein, apolipoprotein A-I (apoA-I). Identification of genetic loci associating apoA-I/HDL with cardiovascular disease is needed to establish a causal relationship. RECENT FINDINGS: Pharmacological interventions to increase apoA-I or HDL cholesterol levels in humans are not associated with reduction in atherosclerosis. Genome wide association study (GWAS) studies in humans and hybrid mouse diversity panel (HMDP) studies looking for genetic variants associated with apoA-I or HDL cholesterol levels with cardiovascular disease and atherosclerosis have not provided strong evidence for their atheroprotective function. SUMMARY: These findings indicate that GWAS and HMDP studies identifying possible genetic determinants of HDL and apoA-I function are needed.

Do the Apoe-/- and Ldlr-/- Mice Yield the Same Insight on Atherogenesis?

Murine models of atherosclerosis are useful for investigating the environmental and genetic influences on lesion formation and composition. Apoe(-/-) and Ldlr(-/-) mice are the 2 most extensively used models. The models differ in important ways with respect to the precise mechanism by which their absence enhances atherosclerosis, including differences in plasma lipoproteins. The majority of the gene function studies have utilized only 1 model, with the results being generalized to atherogenic mechanisms. In only a relatively few cases have studies been conducted in both atherogenic murine models. This review will discuss important differences between the 2 atherogenic models and will point out studies that have been performed in the 2 models where results are comparable and those where different results were obtained.

Enzymatically Modified Low-Density Lipoprotein Promotes Foam Cell Formation in Smooth Muscle Cells via Macropinocytosis and Enhances Receptor-Mediated Uptake of Oxidized Low-Density Lipoprotein.

OBJECTIVE: Enzyme-modified nonoxidized low-density lipoprotein (ELDL) is present in human atherosclerotic lesions. Our objective is to understand the mechanisms of ELDL uptake and its effects on vascular smooth muscle cells (SMC). APPROACH AND RESULTS: Transformation of murine aortic SMCs into foam cells in response to ELDL was analyzed. ELDL, but not acetylated or oxidized LDL, was potent in inducing SMC foam cell formation. Inhibitors of macropinocytosis (LY294002, wortmannin, amiloride) attenuated ELDL uptake. In contrast, inhibitors of receptor-mediated endocytosis (dynasore, sucrose) and inhibitor of caveolae-/lipid raft-mediated endocytosis (filipin) had no effect on ELDL uptake in SMC, suggesting that macropinocytosis is the main mechanism of ELDL uptake by SMC. Receptor for advanced glycation end products (RAGE) is not obligatory for ELDL-induced SMC foam cell formation, but primes SMC for the uptake of oxidized LDL in a RAGE-dependent manner. ELDL increased intracellular reactive oxygen species, cytosolic calcium, and expression of lectin-like oxidized LDL receptor-1 in wild-type SMC but not in RAGE(-/-) SMC. The macropinocytotic uptake of ELDL is regulated predominantly by intracellular calcium because ELDL uptake was completely inhibited by pretreatment with the calcium channel inhibitor lacidipine in wild-type and RAGE(-/-) SMC. This is in contrast to pretreatment with PI3 kinase inhibitors which completely prevented ELDL uptake in RAGE(-/-) SMC, but only partially in wild-type SMC. CONCLUSIONS: ELDL is highly potent in inducing foam cells in murine SMC. ELDL endocytosis is mediated by calcium-dependent macropinocytosis. Priming SMC with ELDL enhances the uptake of oxidized LDL.

Serum amyloid A and atherosclerosis.

PURPOSE OF REVIEW: Atherosclerosis is a chronic inflammation associated with increased expression of the acute phase isoforms of serum amyloid A (SAA) and in humans is a plasma biomarker for future cardiovascular events. However, whether SAA is only a biomarker or participates in the development of cardiovascular disease is not well characterized. The purpose of this review is to summarize putative functions of SAA relevant to atherogenesis and in-vivo murine studies that directly examine the effect of SAA on atherosclerosis. RECENT FINDINGS: Modulation of the expression of SAA1 and/or SAA2 in murine models of atherosclerosis suggests that SAA promotes early atherogenesis. SAA secreted from bone-marrow-derived cells contributes to this antiatherogenic phenotype. SAA also promotes angiotensin-induced abdominal aneurysm in atherogenic mouse models. The reduction in atherosclerosis may be due, at least in part, to remodeling of the acute phase HDL to reduce its capacity to promote cholesterol efflux and reduce its anti-inflammatory ability. SUMMARY: SAA is more than a marker of cardiovascular disease and is a participant in the early atherogenic process.

ApoE knockout and knockin mice: the history of their contribution to the understanding of atherogenesis.

ApoE is a multifunctional protein that is expressed by many cell types that influences many aspects of cardiovascular physiology. In humans, there are three major allelic variants that differentially influence lipoprotein metabolism and risk for the development of atherosclerosis. Apoe-deficient mice and human apoE isoform knockin mice, as well as hypomorphic Apoe mice, have significantly contributed to our understanding of the role of apoE in lipoprotein metabolism, monocyte/macrophage biology, and atherosclerosis. This brief history of these mouse models will highlight their contribution to the understanding of the role of apoE in these processes. These Apoe(-/-) mice have also been extensively utilized as an atherosensitive platform upon which to assess the impact of modulator genes on the development and regression of atherosclerosis.

Proteomic analysis of HDL from inbred mouse strains implicates APOE associated with HDL in reduced cholesterol efflux capacity via the ABCA1 pathway.

Cholesterol efflux capacity associates strongly and negatively with the incidence and prevalence of human CVD. We investigated the relationships of HDL's size and protein cargo with its cholesterol efflux capacity using APOB-depleted serum and HDLs isolated from five inbred mouse strains with different susceptibilities to atherosclerosis. Like humans, mouse HDL carried >70 proteins linked to lipid metabolism, the acute-phase response, proteinase inhibition, and the immune system. HDL's content of specific proteins strongly correlated with its size and cholesterol efflux capacity, suggesting that its protein cargo regulates its function. Cholesterol efflux capacity with macrophages strongly and positively correlated with retinol binding protein 4 (RBP4) and PLTP, but not APOA1. In contrast, ABCA1-specific cholesterol efflux correlated strongly with HDL's content of APOA1, APOC3, and APOD, but not RBP4 and PLTP. Unexpectedly, APOE had a strong negative correlation with ABCA1-specific cholesterol efflux capacity. Moreover, the ABCA1-specific cholesterol efflux capacity of HDL isolated from APOE-deficient mice was significantly greater than that of HDL from wild-type mice. Our observations demonstrate that the HDL-associated APOE regulates HDL's ABCA1-specific cholesterol efflux capacity. These findings may be clinically relevant because HDL's APOE content associates with CVD risk and ABCA1 deficiency promotes unregulated cholesterol accumulation in human macrophages.

Cutting edge: Dexamethasone potentiates the responses of both regulatory T cells and B-1 cells to antigen immunization in the ApoE(-/-) mouse model of atherosclerosis.

The immunosuppressant dexamethasone was shown to preferentially deplete CD4+ effector T cells while sparing regulatory T cells (Tregs) in vivo. In the current study, we show that it also preferentially depletes B-2 cells while sparing B-1 cells. In the ApoE(-/-) mouse model of atherosclerosis, in which both Tregs and B-1 cells are thought to play an atheroprotective role, we show that HSP60-targeted immunization in the presence of dexamethasone raises Ag-reactive Tregs and B-1 cells concomitantly and reduces the severity of atherosclerosis. These results indicate that dexamethasone is an adjuvant that potentiates both the Treg and B-1 responses to immunogens. This study shows that B-1 cells with a specificity for a disease-relevant Ag can be raised in vivo by immunization.

Atherogenic lipids and macrophage subsets.

PURPOSE OF REVIEW: Macrophage foam cells are important cells in the vascular wall that contribute to the inflammation associated with atherosclerotic lesions. Recent studies have demonstrated the heterogeneity of macrophages in lesions. In this review, advances in our understanding of the formation of foam cells by macrophage subsets in atherosclerotic plaques will be discussed. RECENT FINDINGS: Macrophage subsets develop in response to the microenvironment in the arterial wall. The uptake of lipoproteins, particularly oxidized LDL, has been considered the major mechanism of foam cell formation. However, native and aggregated LDL can also be taken up by macrophages and M2 macrophages have been shown to be efficient in the uptake of apoptotic cells that can contribute lipids to the cells. The ability of the macrophage subsets to respond to bioactive lipids in the artery wall to either promote macrophage subset polarization and/or to promote foam cell formation is only beginning to be understood. SUMMARY: Although we are beginning to appreciate the heterogeneity of macrophages present in atherosclerotic plaques, further work is required to fully understand the molecular basis for the differential ability of macrophage subsets to form foam cells and to respond to bioactive lipids.

Selective suppression of adipose tissue apoE expression impacts systemic metabolic phenotype and adipose tissue inflammation.

apoE is a multi-functional protein expressed in several cell types and in several organs. It is highly expressed in adipose tissue, where it is important for modulating adipocyte lipid flux and gene expression in isolated adipocytes. In order to investigate a potential systemic role for apoE that is produced in adipose tissue, mice were generated with selective suppression of adipose tissue apoE expression and normal circulating apoE levels. These mice had less adipose tissue with smaller adipocytes containing fewer lipids, but no change in adipocyte number compared with control mice. Adipocyte TG synthesis in the presence of apoE-containing VLDL was markedly impaired. Adipocyte caveolin and leptin gene expression were reduced, but adiponectin, PGC-1, and CPT-1 gene expression were increased. Mice with selective suppression of adipose tissue apoE had lower fasting lipid, insulin, and glucose levels, and glucose and insulin tolerance tests were consistent with increased insulin sensitivity. Lipid storage in muscle, heart, and liver was significantly reduced. Adipose tissue macrophage inflammatory activation was markedly diminished with suppression of adipose tissue apoE expression. Our results establish a novel effect of adipose tissue apoE expression, distinct from circulating apoE, on systemic substrate metabolism and adipose tissue inflammatory state.

Alginic acid cell entrapment: a novel method for measuring in vivo macrophage cholesterol homeostasis.

Macrophage conversion to atherosclerotic foam cells is partly due to the balance of uptake and efflux of cholesterol. Cholesterol efflux from cells by HDL and its apoproteins for subsequent hepatic elimination is known as reverse cholesterol transport. Numerous methods have been developed to measure in vivo macrophage cholesterol efflux. Most methods do not allow for macrophage recovery for analysis of changes in cellular cholesterol status. We describe a novel method for measuring cellular cholesterol balance using the in vivo entrapment of macrophages in alginate, which retains incorporated cells while being permeable to lipoproteins. Recipient mice were injected subcutaneously with CaCl2 forming a bubble into which a macrophage/alginate suspension was injected, entrapping the macrophages. Cells were recovered after 24 h. Cellular free and esterified cholesterol mass were determined enzymatically and normalized to cellular protein. Both normal and cholesterol loaded macrophages undergo measureable changes in cell cholesterol when injected into WT and apoA-I-, LDL-receptor-, or apoE-deficient mice. Cellular cholesterol balance is dependent on initial cellular cholesterol status, macrophage cholesterol transporter expression, and apolipoprotein deficiency. Alginate entrapment allows for the in vivo measurement of macrophage cholesterol homeostasis and is a novel platform for investigating the role of genetics and therapeutic interventions in atherogenesis.

Apolipoprotein A-I protection against atherosclerosis is dependent on genetic background.

OBJECTIVE: Inbred mouse strains have different susceptibilities to experimental atherosclerosis. The C57BL/6 strain is among the most sensitive and has, therefore, been the most widely used in atherosclerosis studies, whereas many strains are resistant. The FVB/N strain is highly resistant to atherosclerosis on the apolipoprotein E (apoE)- and low-density lipoprotein (LDL) receptor-deficient backgrounds. High-density lipoprotein and its major apoprotein, apoA-I, have been shown to be protective against atherogenesis on the C57BL/6 background. We here examine the influence of genetic background on the atheroprotective nature of apoA-I. APPROACH AND RESULTS: ApoE-deficient/apoA-I-deficient mice were generated in the C57BL/6 and FVB/N strains from apoE-deficient mice. After 6 to 10 weeks on a Western-type diet, plasma lipids and atherosclerotic lesion size were assessed. Macrophage recruitment, cholesterol regulation, and blood monocyte levels were examined as potential mechanisms driving lesion size differences. FVB/N knockout mice had higher plasma very-LDL/LDL cholesterol than their C57BL/6 counterparts. ApoA-I deficiency decreased very-LDL/LDL cholesterol in C57BL/6 mice but not in FVB/N mice. FVB/N single and double knockout mice had less lesion than C57BL/6 6 to 10 weeks on diet. ApoA-I deficiency augmented lesion development only in C57BL/6 mice. Macrophage recruitment to thioglycollate-treated peritoneum and diet-induced blood monocyte levels reflected the pattern of lesion development among the 4 genotypes. ApoA-I deficiency increased macrophage cholesterol content only in C57BL/6. FVB/N plasma was a better acceptor for macrophage cholesterol efflux than C57BL/6. CONCLUSIONS: ApoA-I is atheroprotective only in certain genetic contexts. In the C57BL/6 context, but not FVB/N, apoA-I decreases inflammatory macrophage recruitment and monocytosis, contributors to lesion formation.

Dexamethasone promotes tolerance in vivo by enriching CD11clo CD40lo tolerogenic macrophages.

We previously showed that antigen immunization in the presence of the immunosuppressant dexamethasone (a strategy we termed "suppressed immunization") could tolerize established recall responses of T cells. However, the mechanism by which dexamethasone acts as a tolerogenic adjuvant has remained unclear. In the present study, we show that dexamethasone enriches CD11c(lo) CD40(lo) macrophages in a dose-dependent manner in the spleen and peripheral lymph nodes of mice by depleting all other CD11c(+) CD40(+) cells including dendritic cells. The enriched macrophages display a distinct MHC class II (MHC II)(lo) CD86(hi) phenotype. Upon activation by antigen in vivo, CD11c(lo) CD40(lo) macrophages upregulate IL-10, a classic marker for tolerogenic antigen-presenting cells, and elicit a serum IL-10 response. When presenting antigen in vivo, these cells do not elicit recall responses from memory T cells, but rather stimulate the expansion of antigen-specific regulatory T cells. Moreover, the depletion of CD11c(lo) CD40(lo) macrophages during suppressed immunization diminishes the tolerogenic efficacy of the treatment. These results indicate that dexamethasone acts as a tolerogenic adjuvant partly by enriching the CD11c(lo) CD40(lo) tolerogenic macrophages.