Yankun Li

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.

Cholesterol-induced macrophage apoptosis requires ER stress pathways and engagement of the type A scavenger receptor.

Macrophage death in advanced atherosclerosis promotes necrosis and plaque destabilization. A likely cause of macrophage death is accumulation of free cholesterol (FC) in the ER, leading to activation of the unfolded protein response (UPR) and C/EBP homologous protein (CHOP)–induced apoptosis. Here we show that p38 MAPK signaling is necessary for CHOP induction and apoptosis. Additionally, two other signaling pathways must cooperate with p38-CHOP to effect apoptosis. One involves the type A scavenger receptor (SRA). As evidence, FC loading by non-SRA mechanisms activates p38 and CHOP, but not apoptosis unless the SRA is engaged. The other pathway involves c-Jun NH2-terminal kinase (JNK)2, which is activated by cholesterol trafficking to the ER, but is independent of CHOP. Thus, FC-induced apoptosis requires cholesterol trafficking to the ER, which triggers p38-CHOP and JNK2, and engagement of the SRA. These findings have important implications for understanding how the UPR, MAPKs, and the SRA might conspire to cause macrophage death, lesional necrosis, and plaque destabilization in advanced atherosclerotic lesions.

Cholesterol-induced Apoptotic Macrophages Elicit an Inflammatory Response in Phagocytes, Which Is Partially Attenuated by the Mer Receptor

Macrophage apoptosis and the ability of phagocytes to clear these apoptotic cells are important processes in advanced atherosclerosis. Phagocytic clearance not only disposes of dead cells but usually elicits an anti-inflammatory response. To study this process in a model of advanced lesional macrophage death, macrophages rendered apoptotic by free cholesterol loading (FC-AMs) were incubated briefly with fresh macrophages (“phagocytes”). FC-AMs were promptly ingested by the phagocytes, which was dependent upon actin polymerization and the phagocyte Mer receptor. Surprisingly, this brief exposure to FC-AMs triggered a modest proinflammatory response in the phagocytes: tumor necrosis factor-α (TNF-α) and interleukin (IL)-1β were induced, whereas the levels of transforming growth factor-β and IL-10 were not increased. This response required cell contact between the FC-AMs and phagocytes but not FC-AM ingestion. TNF-α and IL-1β induction required one or more proteins on the FC-AM surface and was dependent on signaling through extracellular signal-regulated kinase-1/2 mitogen-activated protein kinase and nuclear factor-κB in the phagocytes. TNF-α production was markedly greater when Mer-defective phagocytes were used, indicating that Mer attenuated the inflammatory response. Interestingly, a more typical anti-inflammatory response was elicited when phagocytes were exposed to macrophages rendered apoptotic by oxidized low density lipoprotein or UV radiation. Thus, the proinflammatory milieu of advanced atherosclerotic lesions may be promoted, or at least not dampened, by contact between FC-induced apoptotic macrophages and neighboring phagocytes prior to apoptotic cell ingestion.

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.

Sitosterol-containing Lipoproteins Trigger Free Sterol-induced Caspase-independent Death in ACAT-competent Macrophages

Sitosterolemia is a disease characterized by very high levels of sitosterol and other plant sterols and premature atherothrombotic vascular disease. One theory holds that plant sterols can directly promote atherosclerosis, but the mechanism is not known. Unesterified, or “free,” cholesterol (FC) is a potent inducer of macrophage death, which causes plaque necrosis, a precursor to atherothrombosis. FC-induced macrophage death, however, requires dysfunction of the sterol esterifying enzyme acyl-coenzyme A-cholesterol acyltransferase (ACAT), which likely occurs slowly during lesion progression. In contrast, plant sterols are relatively poorly esterified by ACAT, and so they may cause macrophage death and plaque necrosis in an accelerated manner. In support of this hypothesis, we show here that macrophages incubated with sitosterol-containing lipoproteins accumulate free sterols and undergo death in the absence of an ACAT inhibitor. As with FC loading, sitosterol-induced macrophage death requires sterol trafficking to the endoplasmic reticulum, and sitosterol-enriched endoplasmic reticulum membranes show evidence of membrane protein dysfunction. However, whereas FC induces caspase-dependent apoptosis through activation of the unfolded protein response and JNK, sitosterol-induced death is caspase-independent and involves neither the unfolded protein response nor JNK. Rather, cell death shows signs of necroptosis and autophagy and is suppressed by inhibitors of both processes. These data establish two new concepts. First, a relatively subtle change in sterol structure fundamentally alters the type of death program triggered in macrophages. Understanding the basis of this alteration should provide new insights into the molecular basis of death pathway signaling. Second, sitosterol-induced macrophage death does not require ACAT dysfunction and so may occur in an accelerated fashion. Pending future in vivo studies, this concept may provide at least one mechanism for accelerated plaque necrosis and atherothrombotic disease in patients with sitosterolemia.

Pivotal Advance: Macrophages become resistant to cholesterol‐induced death after phagocytosis of apoptotic cells

One of the most important functions of macrophages is the phagocytosis of apoptotic cells (ACs). ACs deliver large amounts membrane‐derived cholesterol to phagocytes, which, if not handled properly, can be cytotoxic. In atherosclerosis, where the ACs are cholesterol‐loaded, this situation is exaggerated, because the ACs deliver both endogenous membrane cholesterol and stored lipoprotein‐derived cholesterol. To examine how phagocytes handle this very large amount of cholesterol, we incubated macrophage phagocytes with cholesterol‐loaded ACs. Our results show that the phagocytes call into play a number of cellular responses to protect them from cholesterol‐induced cytotoxicity. First, through efficient trafficking of the internalized AC‐derived cholesterol to acyl‐CoA:cholesterol acyltransferase (ACAT) in the endoplasmic reticulum, phagocytes efficiently esterify the cholesterol and thus prevent its toxic effects. However, the phagocytes show no signs of cytotoxicity even when ACAT is rendered dysfunctional, as might occur in advanced atherosclerotic lesions. Under these conditions, the phagocytes remain viable through massive efflux of AC‐derived cholesterol. Remarkably, these phagocytes still show a survival response even when high cholesterol levels are maintained in the post‐phagocytosis period by subsequent incubation with atherogenic lipoproteins, as also may occur in atheromata. In this case, death in phagocytes is prevented by activation of survival pathways involving PI‐3 kinase/Akt and NF‐κB. Thus, macrophages that have ingested ACs successfully employ three survival mechanisms—cholesterol esterification, massive cholesterol efflux, and cell‐survival signaling. These findings have implications for macrophage physiology in both AC clearance and atherosclerotic plaque progression.

Attenuated Free Cholesterol Loading-induced Apoptosis but Preserved Phospholipid Composition of Peritoneal Macrophages from Mice That Do Not Express Group VIA Phospholipase A2

Mouse macrophages undergo ER stress and apoptosis upon free cholesterol loading (FCL). We recently generated iPLA2β-null mice, and here we demonstrate that iPLA2β-null macrophages have reduced sensitivity to FCL-induced apoptosis, although they and wild-type (WT) cells exhibit similar increases in the transcriptional regulator CHOP. iPLA2β-null macrophages are also less sensitive to apoptosis induced by the sarcoplasmic reticulum Ca2+-ATPase inhibitor thapsigargin and the scavenger receptor A ligand fucoidan, and restoring iPLA2βexpression with recombinant adenovirus increases apoptosis toward WT levels. WT and iPLA2β-null macrophages incorporate [3H]arachidonic acid ([3H]AA]) into glycerophosphocholine lipids equally rapidly and exhibit identical zymosan-induced, cPLA2α-catalyzed [3H]AA release. In contrast, although WT macrophages exhibit robust [3H]AA release upon FCL, this is attenuated in iPLA2β-null macrophages and increases toward WT levels upon restoring iPLA2β expression. Recent reports indicate that iPLA2β modulates mitochondrial cytochrome c release, and we find that thapsigargin and fucoidan induce mitochondrial phospholipid loss and cytochrome c release into WT macrophage cytosol and that these events are blunted in iPLA2β-null cells. Immunoblotting studies indicate that iPLA2β associates with mitochondria in macrophages subjected to ER stress. AA incorporation into glycerophosphocholine lipids is unimpaired in iPLA2β-null macrophages upon electrospray ionization-tandem mass spectrometry analyses, and their complex lipid composition is similar to WT cells. These findings suggest that iPLA2β participates in ER stress-induced macrophage apoptosis caused by FCL or thapsigargin but that deletion of iPLA2β does not impair macrophage arachidonate incorporation or phospholipid composition.

Cholesteryl ester loading of mouse peritoneal macrophages is associated with changes in the expression or modification of specific cellular proteins, including increase in an alpha-enolase isoform.

This report explores the hypothesis that massive cholesteryl ester (CE) accumulation in macrophages, such as that occurring in atheroma foam cells, results in changes in the expression or modification of specific cellular proteins. Two-dimensional (2-D) gel electrophoretic patterns of metabolically labeled cellular proteins from mouse peritoneal macrophages that were loaded with CE (through incubation with acetylated low density lipoprotein [acetyl-LDL] for 4 days) were compared with those of control macrophages. Densitometric analysis of 2-D gel autoradiograms from the cell lysates revealed statistically significant changes in seven cellular proteins (five decreases and two increases). The changes in protein expression (foam cell versus control) ranged from a 458 +/- 164% (p < 0.001) increase to a 35 +/- 34% (p < 0.001) decrease (n = 11). Incubation of macrophages with beta-very low density lipoprotein, which also increased the CE content of macrophages (albeit to a lesser extent than acetyl-LDL), resulted in changes in five of the seven proteins. In contrast, incubation of cells with LDL, fucoidan, or latex beads, none of which caused CE accumulation, did not lead to significant changes in four of these five proteins. One of these four proteins, which increased fourfold to fivefold in foam cells (M(r) = 49,000; isoelectric point of 6.8), was purified by preparative 2-D gel electrophoresis. Internal amino acid sequence of cyanogen bromide fragments of this protein as well as Western blot analysis identified this protein as an isoform of alpha-enolase. The increased expression of this alpha-enolase isoform, which was seen as early as day 2 of acetyl-LDL incubation of the macrophages, was diminished by including an inhibitor of cholesterol esterification during the acetyl-LDL incubation period. In conclusion, macrophage foam cell formation is associated with distinct changes in protein expression, including a marked increase in an isoform of alpha-enolase, suggesting a specific biological adaptation to CE loading.

The inflammatory cytokine response of cholesterol‐enriched macrophages is dampened by stimulated pinocytosis

Two features of advanced atherosclerotic lesions are large numbers of macrophages and a heightened state of inflammation. Some of the macrophages appear to be enriched with free cholesterol (FCMφs), and we have shown that this process induces the synthesis and secretion of inflammatory cytokines, including TNF‐α and IL‐6. However, lesions contain many other macrophages that are not FC‐enriched (non‐FCMφs). Therefore, we sought to understand how the interaction of these two populations of macrophages would influence the inflammatory response. We show here that non‐FCMφs possess a robust ability to deplete TNF‐α and IL‐6 secreted by FCMφs. The mechanism involves enhanced pinocytic uptake and lysosomal degradation of the FCMφ‐secreted cytokines by the non‐FCMφs. The FCMφs contribute directly to this process by secreting pinocytosis‐stimulatory factors that act on non‐FCMφs but not on the FCMφs themselves. One of these pinocytosis‐stimulatory factors is M‐CSF, which is induced by a process involving cholesterol trafficking to the endoplasmic reticulum and signaling through PI‐3K and ERK MAPK pathways. However, one or more other FCMφ‐secreted factors are also required for stimulating pinocytosis in non‐FCMφs. Thus, FCMφs secrete inflammatory cytokines as well as factors that promote the eventual pinocytosis and degradation of these cytokines by neighboring macrophages. This process may normally serve to prevent prolonged or disseminated effects of inflammatory cytokines during inflammation. Moreover, possible perturbation of stimulated pinocytosis during the progression of advanced atherosclerosis may contribute to the heightened inflammatory state of these lesions.