James X. Rong

The endoplasmic reticulum is the site of cholesterol-induced cytotoxicity in macrophages

Excess cellular cholesterol induces apoptosis in macrophages, an event likely to promote progression of atherosclerosis. The cellular mechanism of cholesterol-induced apoptosis is unknown but had previously been thought to involve the plasma membrane. Here we report that the unfolded protein response (UPR) in the endoplasmic reticulum is activated in cholesterol-loaded macrophages, resulting in expression of the cell death effector CHOP. Cholesterol loading depletes endoplasmic reticulum calcium stores, an event known to induce the UPR. Furthermore, endoplasmic reticulum calcium depletion, the UPR, caspase-3 activation and apoptosis are markedly inhibited by selective inhibition of cholesterol trafficking to the endoplasmic reticulum, and Chop−/− macrophages are protected from cholesterol-induced apoptosis. We propose that cholesterol trafficking to endoplasmic reticulum membranes, resulting in activation of the CHOP arm of the UPR, is the key signalling step in cholesterol-induced apoptosis in macrophages.

Transdifferentiation of mouse aortic smooth muscle cells to a macrophage-like state after cholesterol loading

Mouse aortic smooth muscle cells (SMCs) were loaded for 72 h with cholesterol by using cholesterol:methyl-β-cyclodextrin complexes, leading to ≈2-fold and ≈10-fold increases in the contents of total cholesterol and cholesteryl ester, respectively. Foam-cell formation was demonstrated by accumulation of intracellular, Oil Red O-stained lipid droplets. Immunostaining showed decreased protein levels of smooth muscle α-actin and α-tropomyosin and increased levels of macrophage markers CD68 and Mac-2 antigen. Quantitative real-time RT-PCR revealed that after cholesterol loading, the expression of SMC-related genes α-actin, α-tropomyosin, myosin heavy chain, and calponin H1 decreased (to 11.5 ± 0.5%, 29.3 ± 1.4%, 23.8 ± 1.4%, and 3.8 ± 0.5% of unloaded cells, respectively; P < 0.05 for all), whereas expression of macrophage-related genes CD68, Mac-2, and ABCA1 mRNA increased (to 709 ± 84%, 330 ± 11%, and 207 ± 13% of unloaded cells, respectively; P < 0.05 for all), thereby demonstrating that the protein changes were regulated at the mRNA level. Furthermore, these changes were accompanied by a gain in macrophage-like function as assessed by phagocytotic activity. Expression of vascular cell adhesion molecule 1 and monocyte chemoattractant protein 1, known responders to inflammation, were not changed. In conclusion, cholesterol loading of SMC causes phenotypic changes regulated at the mRNA level that result in a transdifferentiation to a macrophage-like state. This finding suggests that not all foam cells in lesions may have a macrophage origin, despite what is indicated by immunostaining for macrophage-related markers. Furthermore, inflammatory changes in foam cells observed in vivo may not be simple consequences of cholesterol accumulation.

HDL promotes rapid atherosclerosis regression in mice and alters inflammatory properties of plaque monocyte-derived cells

HDL cholesterol (HDL-C) plasma levels are inversely related to cardiovascular disease risk. Previous studies have shown in animals and humans that HDL promotes regression of atherosclerosis. We hypothesized that this was related to an ability to promote the loss of monocyte-derived cells (CD68+, primarily macrophages and macrophage foam cells) from plaques. To test this hypothesis, we used an established model of atherosclerosis regression in which plaque-bearing aortic arches from apolipoprotein E-deficient (apoE−/−) mice (low HDL-C, high non–HDL-C) were transplanted into recipient mice with differing levels of HDL-C and non–HDL-C: C57BL6 mice (normal HDL-C, low non–HDL-C), apoAI−/− mice (low HDL-C, low non–HDL-C), or apoE−/− mice transgenic for human apoAI (hAI/apoE−/−; normal HDL-C, high non–HDL-C). Remarkably, despite persistent elevated non–HDL-C in hAI/apoE−/− recipients, plaque CD68+ cell content decreased by >50% by 1 wk after transplantation, whereas there was little change in apoAI−/− recipient mice despite hypolipidemia. The decreased content of plaque CD68+ cells after HDL-C normalization was associated with their emigration and induction of their chemokine receptor CCR7. Furthermore, in CD68+ cells laser-captured from the plaques, normalization of HDL-C led to decreased expression of inflammatory factors and enrichment of markers of the M2 (tissue repair) macrophage state. Again, none of these beneficial changes were observed in the apoAI−/− recipients, suggesting a major requirement for reverse cholesterol transport for the beneficial effects of HDL. Overall, these results establish HDL as a regulator in vivo of the migratory and inflammatory properties of monocyte-derived cells in mouse atherosclerotic plaques, and highlight the phenotypic plasticity of these cells.

Reversal of Hyperlipidemia With a Genetic Switch Favorably Affects the Content and Inflammatory State of Macrophages in Atherosclerotic Plaques

Background— We previously showed that the progression of atherosclerosis in the Reversa mouse (Ldlr−/−Apob100/100Mttpfl/fl Mx1Cre+/+) was arrested when the hyperlipidemia was normalized by inactivating the gene for microsomal triglyceride transfer protein. Here, we tested whether atherosclerosis would regress if the lipid levels were reduced after advanced plaques formed. Methods and Results— Reversa mice were fed an atherogenic diet for 16 weeks. Plasma lipid levels were then reduced. Within 2 weeks, this reduction led to decreased monocyte-derived (CD68+) cells in atherosclerotic plaques and was associated with emigration of these cells out of plaques. In addition, the fall in lipid levels was accompanied by lower plaque lipid content and by reduced expression in plaque CD68+ cells of inflammatory genes and higher expression of genes for markers of antiinflammatory M2 macrophages. Plaque composition was affected more than plaque size, with the decreased content of lipid and CD68+ cells balanced by a higher content of collagen. When the reduced lipid level was combined with the administration of pioglitazone to simulate the clinical aggressive lipid management and proliferator-activated receptor-&ggr; agonist treatment, the rate of depletion of plaque CD68+ cells was unaffected, but there was a further increase in their expression of antiinflammatory macrophage markers. Conclusion— The Reversa mouse is a new model of atherosclerosis regression. After lipid lowering, favorable changes in plaque composition were independent of changes in size. In addition, plaque CD68+ cells became less inflammatory, an effect enhanced by treatment with pioglitazone.

Laser capture microdissection analysis of gene expression in macrophages from atherosclerotic lesions of apolipoprotein E-deficient mice

Macrophage foam cells are integral in the development of atherosclerotic lesions. Gene expression analysis of lesional macrophage foam cells is complicated by the cellular heterogeneity of atherosclerotic plaque and the presence of lesions of various degrees of severity. To overcome these limitations, we tested the ability of laser capture microdissection (LCM) and real-time quantitative reverse transcription PCR to selectively analyze RNA from lesional macrophages of apolipoprotein E (apoE)-deficient mice. Proximal aortic tissue sections were immunostained for macrophagespecific CD68/macrosialin by a rapid (≈15-min) protocol. Alternating sections from each animal were used to isolate RNA either from entire sections (analogous to isolation from whole tissue) or by LCM selection of CD68-positive cells. We measured the mRNA levels of CD68, a macrophage-specific marker, α-actin, a smooth muscle cell marker, and cyclophilin A, a control gene. Compared with whole sections, CD68 mRNA levels were greatly enriched (33.6-fold) in the laser-captured lesional macrophages. In contrast to whole sections, LCM-derived RNA had undetectable levels of α-actin. To illustrate the ability of this method to measure changes in lesional macrophage gene expression, we injected 100 μg of lipopolysaccharide i.p. into apoE-deficient mice and detected in laser-captured lesional macrophages increased mRNA expression for vascular cell adhesion molecule-1, intercellular cell adhesion molecule-1, and monocyte chemoattractant protein-1 (11.9-, 32.5-, and 31.0-fold, respectively). By selectively enriching foam cell RNA, LCM provides a powerful approach to study the in situ expression and regulation of atherosclerosis-related genes. This approach will allow the study of macrophage gene expression under various conditions of plaque formation, regression, and response to genetic and environmental perturbations.

Eliminating Atherogenesis in Mice by Switching Off Hepatic Lipoprotein Secretion

Background—LDL receptor–deficient “apolipoprotein (apo)-B100–only” mice (Ldlr−/−Apob100/100 have elevated LDL cholesterol levels on a chow diet and develop severe aortic atherosclerosis. We hypothesized that both the hypercholesterolemia and the susceptibility to atherosclerosis could be eliminated by switching off hepatic lipoprotein production. Methods and Results—We bred Ldlr−/−Apob100/100 mice that were homozygous for a conditional allele for Mttp (the gene for microsomal triglyceride transfer protein) and the inducible Mx1-Cre transgene. In these animals, which we called “Reversa mice,” the hypercholesterolemia could be reversed, without modifying the diet or initiating a hypolipidemic drug, by the transient induction of Cre expression in the liver. After Cre induction, hepatic Mttp expression was virtually eliminated (as judged by quantitative real-time PCR), hepatic lipoprotein secretion was abolished (as judged by electron microscopy), and LDLs were virtually eliminated from the plasma. Intestinal lipoprotein production was unaffected. In mice fed a chow diet, Cre induction reduced plasma cholesterol levels from 233.9±46.0 to 37.2±6.5 mg/dL. In mice fed a high-fat diet, cholesterol levels fell from 525.7±32.2 to 100.6±14.3 mg/dL. The elimination of hepatic lipoprotein production completely prevented both the development of atherosclerosis and the changes in gene expression that accompany atherogenesis. Conclusions—We developed mice in which hypercholesterolemia can be reversed with a genetic switch. These mice will be useful for understanding gene-expression changes that accompany the reversal of hypercholesterolemia and atherosclerosis.

High-Density Lipoproteins Retard the Progression of Atherosclerosis and Favorably Remodel Lesions Without Suppressing Indices of Inflammation or Oxidation

Objective—Protective properties of high-density lipoproteins (HDL) may include reverse cholesterol transport and suppression of oxidation and inflammation. These were investigated in vivo, as were the effects of HDL on the characteristics of atherosclerotic lesions. Methods and Results—Male apolipoprotein E knockout (apoE−/−) and apoE−/− mice expressing human apolipoprotein AI (hAI/apoE−/−) were studied up to 20 weeks after commencing a high-fat diet. Plasma HDL cholesterol was twice as high in hAI/apoE−/− mice. Over time, aortic root lesion area remained less in hAI/apoE−/− mice, although plaques became complex. In advanced lesions, plaque lipid was higher in apoE−/− mice, whereas plaque collagen and alpha actin were increased in hAI/apoE−/− mice. In nonlesional aorta, mRNA abundance for pro-inflammatory proteins (vascular cell adhesion molecule [VCAM]-1, intercellular adhesion molecule-1 [ICAM-1], monocyte chemoattractant protein-1 [MCP-1]) increased between 4 and 16 weeks in apoE−/− (but not wild-type) mice, and were not reduced by elevated HDL. Autoantibodies to malondialdehyde low-density lipoprotein (LDL) increased progressively in apoE−/− mice, with similar results in hAI/apoE−/− mice. Conclusions—HDL retarded plaque size progression despite greatly elevated plasma cholesterol. This effect was over a wide range of lesion severity. Expression of hAI reduced plaque lipid and increased the proportion of plaque occupied by collagen and smooth muscle cells, but did not affect indicators of inflammation or LDL oxidation.

Brief Report: Increased Apoptosis in Advanced Atherosclerotic Lesions of Apoe−/− Mice Lacking Macrophage Bcl-2

Objective—Macrophage apoptosis plays important roles in atherosclerosis. Bcl-2 is a key cell survival molecule, but its role in macrophage apoptosis in atherosclerosis is not known. The goal herein was to determine the effect of macrophage-targeted deletion of Bcl-2 on macrophage apoptosis in atherosclerotic lesions of Apoe−/− mice. Methods and Results—Bcl2flox-LysMCre mice were created as a model of macrophage Bcl-2 deficiency. Macrophages from these mice were more susceptible to apoptosis than those from control Bcl2WT-LysMCre mice. The mice were bred onto the Apoe−/− background and fed a Western-type diet for 4 or 10 weeks. Apoptotic cells were equally very rare in the lesions of both groups of the 4-week-diet mice, and there was no difference in lesion area. However, Bcl2flox-LysMCre;Apoe−/− plaques from the 10-week-diet protocol had a 40% to 45% increase in apoptotic cells and, in female mice, a ≈25% increase in plaque necrosis (P<0.05) compared with Bcl2WT-LysMCre lesions. Conclusions—Macrophage Bcl-2 plays a protective role against macrophage apoptosis specifically in advanced atherosclerotic lesions of Apoe−/− mice.

Lysophosphatidylcholine stimulates monocyte chemoattractant protein-1 gene expression in rat aortic smooth muscle cells

Objective—Monocyte chemoattractant protein (MCP)-1 is a proatherogenic factor that is responsible for ≈60% of plaque macrophages in mouse models of atherosclerosis. We investigated whether lysophosphatidylcholine (LPC), enriched in oxidized low density lipoprotein, can modulate the expression of MCP-1 in arterial wall cells. Methods and Results—LPC induced a 3-fold increase in MCP-1 mRNA in rat vascular smooth muscle cells (VSMCs) in a time- and dose-dependent manner. Nuclear runon analysis showed that this increase was attributable to increased MCP-1 gene transcription. There was a 2-fold increase in MCP-1 protein in the conditioned media of cells treated with LPC. LPC-associated increases of MCP-1 mRNA and protein were similar to those produced by platelet-derived growth factor-BB, a known inducer of MCP-1. Analyses of the MCP-1 promoter in transiently transfected VSMCs indicated an LPC-responsive element(s) between base pairs −146 and −261 (relative to transcription initiation). Further studies suggested that LPC-induced MCP-1 expression partially involves mitogen-activated protein kinase/extracellular signal–regulated kinase, a tyrosine kinase(s), and (to a lesser extent) protein kinase C but not the activation of the platelet-derived growth factor receptor. Conclusions—LPC stimulates MCP-1 expression at the transcriptional level in VSMCs, suggesting a molecular mechanism by which LPC contributes to the atherogenicity of oxidized low density lipoprotein.

Cholesterol Oxidation Products Induce Vascular Foam Cell Lesion Formation in Hypercholesterolemic New Zealand White Rabbits

Circulating cholesterol oxidation products (ChOx) have long been implicated in the etiology of early atherosclerosis; however, direct in vivo evidence elucidating their role in atherogenesis is only recently becoming available. This study investigated ChOx effects on vascular lesion formation in New Zealand White rabbits under controlled hypercholesterolemic conditions. By closely monitoring plasma cholesterol levels and adjusting dietary cholesterol intake during a 78-day period, total plasma cholesterol exposures (cumulative plasma cholesterol levels over time) were controlled between 27 000 and 34 000 mg/dLxday (final plasma cholesterol concentration, 467+/-77 mg/mL), representing a threshold range for sudanophilic lesion formation in the aorta. Twenty injections of a ChOx mixture (70 mg per injection) were made bearing an oxysterol composition similar to that found in circulating oxidatively modified low density lipoprotein. At sacrifice, the ChOx-injected rabbits (n=5) had (1) significantly higher plasma ChOx levels, (2) significantly increased cholesterol content in the aortas, mainly as esterified cholesterol, and (3) significantly greater sudanophilic lesion size and frequency in the aortas compared with vehicle-injected control rabbits (n=5). The aortic cholesterol content and extent of sudanophilic lesion area were correlated significantly with total plasma ChOx exposure (P<0.003 and P<0.0001, respectively) but not with total cholesterol exposure. The results indicate that for moderate experimental hypercholesterolemia, a situation more relevant to physiological hypercholesterolemia in humans, circulating ChOx may play an important role in inducing formation of early atherosclerotic lesions. Because ChOx are often present in cholesterol-containing diets, foam cell lesion formation induced by ChOx rather than cholesterol cannot be overlooked.