Atherogenic Lipids and Lipoproteins Trigger CD36-TLR2-Dependent Apoptosis in Macrophages Undergoing Endoplasmic Reticulum Stress

Macrophage apoptosis is an important feature of atherosclerotic plaque development. Research directed at understanding the functional consequences of macrophage death in atherosclerosis has revealed opposing roles for apoptosis in atherosclerotic plaque progression. In early lesions, macrophage apoptosis limits lesion cellularity and suppresses plaque progression. In advanced lesions, macrophages apoptosis promotes the development of the necrotic core, a key factor in rendering plaques vulnerable to disruption and in acute lumenal thrombosis. The first section of this review will examine the role of phagocytic clearance of apoptotic macrophages, a process known as efferocytosis, in the dichotomous roles of macrophage apoptosis in early vs. advanced lesions. The second section will focus on the molecular and cellular mechanisms that are thought to govern macrophage death during atherosclerosis. Of particular interest is the complex and coordinated role that the endoplasmic reticulum (ER) stress pathway and pattern recognition receptors (PRRs) may play in triggering macrophage apoptosis.

Macrophage apoptosis is a critical process in the formation of necrotic cores in vulnerable atherosclerotic plaques. In vitro and in vivo data suggest that macrophage apoptosis in advanced atheromata may be triggered by a combination of endoplasmic reticulum stress and engagement of the type A scavenger receptor, which together induce death through a rise in cytosolic calcium and activation of toll-like receptor-4. Using both primary peritoneal macrophages and studies in advanced atheromata in vivo, we introduce signal transducer and activator of transcription-1 (STAT1) as a critical and necessary component of endoplasmic reticulum stress/type A scavenger receptor-induced macrophage apoptosis. We show that STAT1 is serine phosphorylated in macrophages subjected to type A scavenger receptor ligands and endoplasmic reticulum stress in a manner requiring cytosolic calcium, calcium/calmodulin-dependent protein kinase II, and toll-like receptor-4. Remarkably, apoptosis was inhibited by approximately 80% to 90% (P<0.05) by STAT1 deficiency or calcium/calmodulin-dependent protein kinase II inhibition. In vivo, nuclear Ser-P-STAT1 was found in macrophage-rich regions of advanced murine and human atheromata. Most important, macrophage apoptosis was decreased by 61% (P=0.034) and plaque necrosis by 34% (P=0.02) in the plaques of fat-fed low density lipoprotein receptor null Ldlr-/- mice transplanted with Stat1-/- bone marrow. STAT1 is critical for endoplasmic reticulum stress/type A scavenger receptor-induced apoptosis in primary tissue macrophages and in macrophage apoptosis in advanced atheromata. These findings suggest a potentially important role for STAT1-mediated macrophage apoptosis in atherosclerotic plaque progression.

Plaque necrosis in advanced atheromata, which triggers acute atherothrombotic vascular events, is caused by the apoptosis of lesional macrophages coupled with defective phagocytic clearance of the dead cells. The central enabling event in macrophage apoptosis relevant to advanced atherosclerosis is the unfolded protein response (UPR), an endoplasmic reticulum (ER) stress pathway. The UPR effector CHOP (GADD153) amplifies release of ER Ca(2+) stores, which activates a central integrator of apoptosis signaling, calcium/calmodulin-dependent protein kinase II (CaMKII). CaMKII, in turn, leads to activation of pro-apoptotic STAT1, induction of the death receptor Fas, and stimulation of the mitochondria-cytochrome c pathway of apoptosis. While these pathways are necessary for apoptosis, apoptosis occurs only when the cells are also exposed to one or more additional "hits." These hits amplify pro-apoptotic pathways and/or suppress compensatory cell-survival pathways. A second hit relevant to atherosclerosis is activation of pattern recognition receptors (PRRs), such as scavenger and toll-like receptors. In vivo relevance is suggested by the fact that advanced human lesions express markers of UPR activation that correlate closely with the degree of plaque vulnerability and macrophage apoptosis. Moreover, studies with genetically altered mice have shown that ER stress and PRR activation are causative for advanced lesional macrophage apoptosis and plaque necrosis. In summary, a key cellular event in the conversion of benign to vulnerable atherosclerotic plaques is ER stress-induced macrophage apoptosis. Further understanding of the mechanisms and consequences of this event may lead to novel therapies directed at preventing the clinical progression of atheromata.

A Role of RIP3-Mediated Macrophage Necrosis in Atherosclerosis Development

Macrophages are important in the host's immune defense against pathogens. However, recent evidence has demonstrated that macrophages are also involved in the development of disease, including cancer. Therefore, it is important to regulate apoptosis in tumor‑related macrophages for effective cancer treatment. In the present study, the effect of endoplasmic reticulum (ER) stress on apoptosis induction was examined in human monocytic cell‑derived macrophages. Radiation therapy in cancer results in irradiating macrophages as well as cancer cells in the tumor microenvironment. Since ER stress has been demonstrated to sensitize cancer cells to radiation, it was hypothesized that ER stress may induce a similar effect in macrophages. Therefore, the effect of combination treatment with ER stress inducers and ionizing radiation on macrophage apoptosis was examined. Treatment of macrophages with ER stress inducers thapsigargin and tunicamycin, enhanced unfolded protein responses, including phosphorylation of eukaryotic initiation factor 2‑α and increased expression of binding immunoglobulin protein. Furthermore, treatment with thapsigargin and tunicamycin induced apoptosis in macrophages compared with untreated cells, although ionizing radiation did not. The thapsigargin-induced apoptosis in macrophages was demonstrated to be caspase‑3‑dependent. Finally, combination treatment with thapsigargin and ionizing radiation, did not result in any significant change in macrophage apoptosis. The present study demonstrated that ER stress regulated apoptosis in radioresistant macrophages and that ionizing radiation had no added effect on ER stress‑induced apoptosis in macrophages.

Reduced Apoptosis and Plaque Necrosis in Advanced Atherosclerotic Lesions of Apoe−/− and Ldlr−/− Mice Lacking CHOP

Colon Cancer Cell-Derived High Mobility Group 1/Amphoterin Induces Growth Inhibition and Apoptosis in Macrophages

Palmitic acid (PA) upregulates oxidized LDL receptor-1 (LOX-1), a scavenger receptor responsible for uptake of oxidized LDL (oxLDL), and enhances oxLDL uptake in macrophages. However, the precise underlying mechanism remains to be elucidated. PA is known to induce endoplasmic reticulum (ER) stress in various cell types. Therefore, we investigated whether ER stress is involved in PA-induced LOX-1 upregulation. PA induced ER stress, as determined by phosphorylation of PERK, eIF2α, and JNK, as well as induction of CHOP in macrophage-like THP-1 cells. Inhibitors [4-phenylbutyric acid (PBA), sodium tauroursodeoxycholate (TUDCA), and salubrinal] and small interfering RNA (siRNA) for the ER stress response decreased PA-induced LOX-1 upregulation. Thapsigargin, an ER stress inducer, upregulated LOX-1, which was decreased by PBA and TUDCA. We next examined whether unsaturated FAs could counteract the effect of PA. Both oleic acid (OA) and linoleic acid (LA) suppressed PA-induced LOX-1. Activation of the ER stress response observed in the PA-treated cells was markedly attenuated when the cells were cotreated with OA or LA. In addition, OA and LA suppressed thapsigargin-induced LOX-1 upregulation with reduced activation of ER stress markers. Our results indicate that activation of ER stress is involved in PA-induced LOX-1 upregulation in macrophages, and that OA and LA inhibit LOX-1 induction through suppression of ER stress.

Macrophage death in advanced atherosclerosis causes plaque necrosis, which promotes plaque rupture and acute atherothrombotic vascular events. Of interest, plaque necrosis and atherothrombotic disease are markedly increased in diabetes and metabolic syndrome. We discovered a novel 'multi-hit' macrophage apoptosis pathway that appears to be highly relevant to advanced atherosclerosis. The elements of the pathway include: (a) activation of the unfolded protein response (UPR) by cholesterol overloading of the endoplasmic reticulum or by other UPR activators known to exist in atheromata; and (b) pro-apoptotic signalling involving the type A scavenger receptor (SRA). The downstream apoptosis effectors include CHOP (GADD153) for the UPR and JNK for SRA signalling. Remarkably, components of this pathway are enhanced in macrophages with defective insulin signalling, including UPR activation and SRA expression. As a result, insulin-resistant macrophages show increased susceptibility to apoptosis when exposed to UPR activators and SRA ligands. Moreover, the advanced lesions of atherosclerosis-prone mice reconstituted with insulin-resistant macrophages show increased macrophage apoptosis and plaque necrosis. Based on these findings, we propose that one mechanism of increased plaque necrosis and atherothrombotic vascular disease in insulin resistant syndromes is up-regulation of a two-hit signal transduction pathway involved in advanced lesional macrophage death.

MafB and the role of macrophage apoptosis in atherosclerosis: A time to kill, a time to heal

Conditions that interfere with proper functioning of the Endoplasmic Reticulum (ER), such as the accumulation of misfolded proteins, lead to activation of the Unfolded Protein Response (UPR). Initially the UPR is protective, serving to restore ER homeostasis by reducing protein load and increasing the expression of protein folding chaperones. However, in certain cases when stress is prolonged or severe, the UPR can alternatively trigger apoptosis. Accordingly, ER stress-induced apoptosis has been implicated in the pathogenesis of several diseases such as diabetes, cancer, neurodegenerative disease, and atherosclerosis (1). In the case of advanced atherosclerosis, a number of recent studies in humans and experimental animals, indicate that the UPR plays a pivotal role in the progression of disease. Advanced atherosclerotic plaques that are prone to rupture are characterized by a large necrotic core and thinning of the plaque fibrous cap, which separates thrombogenic intraplaque matrix from the overlying circulation. Macrophage apoptosis is a prominent feature of advanced atherosclerotic plaques. In the absence of efficient apoptotic cell clearance by phagocytosis, increased apoptosis contributes to necrotic core expansion. Both mechanistic studies in vitro and advanced plaque causation studies in vivo have shown that ER stress plays a role in advanced lesional macrophage apoptosis (2). Indeed, Myoishi et al. have shown a close correlation among UPR-effector C/EBP-homologous protein (CHOP) expression, apoptosis, and plaque vulnerability in human coronary artery lesions (3). There are many potential causes of ER stress in atherosclerotic plaques, including oxysterols, particularly 7-ketocholesterol; oxidized phospholipids; unesterified cholesterol; oxidant stress; saturated fatty acids; and hypoxia. Moreover, relevant to the epidemic of insulin resistance-driven coronary artery disease, insulin resistance is a potent inducer of the UPR in insulin-resistant macrophages (4). Previous studies from our group have shown that the key initiating event in the apoptosis cascade in ER-stressed macrophages is release of calcium from ER stores into the cytosol. Certain ER stressors like unesterifed cholesterol promotes this calcium release through inhibition of the ER calcium reuptake pump, sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA) (5). Additionally, we found that UPR itself amplifies this calcium release by activating inositol-3-phosphate receptor (IP3R) via CHOP-induced ER oxidase-1α (ERO1α) (6). In our recent study, we have revealed a novel mechanism where a calcium responsive kinase called calcium/calmodulin-dependent protein kinase II (CaMKII) is activated through ER-released calcium and this in turn serves as a unifying link between ER stress and the downstream apoptotic pathways (7). The physiological release of calcium from the ER serves important signaling roles in maintaining normal cellular function. Calcium transfer from the ER to mitochondria contributes to a number of physiological events, notably cellular bioenergetics. However, after prolonged ER stress conditions, excess calcium release from ER and consequent calcium increase in the cytosol and in the mitochondrial matrix has been shown be involved in mitochondrial pathways of apoptosis (8). In our recent study (7), we found that CaMKII is essential for excess calcium uptake by the mitochondria, which in turn leads to outer mitochondrial membrane permeabilization and release of cytochrome c. Moreover, induction of the cell-surface death receptor Fas by ER stress in macrophages involves activation of CaMKII and c-jun amino terminal kinase (JNK) (Fig. 1). Consistent with these in vitro studies, our in vivo experiments also showed that CaMKII is important in macrophage apoptosis and loss of mitochondrial membrane potential induced by systemic ER stress. Figure 1 Scheme of CaMKII-mediated events lending to ER stress induced macrophage apoptosis. ER stress doplotes the calcium stores within the ER lumon. Calcium subsequently accumulates in the cytoplasm and activates CuMKII. CuMKII enables apoptosis through JNK-mediated ... The molecular mechanisms involved in increased calcium transfer from the ER to the mitochondria under ER stress is not completely understood. Growing evidence indicates that calcium uptake into mitochondria is controlled by specific proteins residing at specific contact points between the ER and mitochondria known as mitochondrial-associated membranes (MAMs) (9) and by a calcium uniporter that has been identified through physiologic and pharmacologic means (10) but has not yet been cloned. In this context, it will be important to determine weather CaMKII is affecting these two processes in order to facilitate calcium uptake into the mitochondria. Given that CaMKII acts as an upstream molecule regulating multiple apoptosis pathways, targeting CaMKII inhibition may have critical implications for the diseases related to ER stress-induced cell death, including that occurring in advanced atherosclerosis.

Forkhead transcription factors (FoxOs) promote apoptosis of insulin-resistant macrophages during cholesterol-induced endoplasmic reticulum stress.

Endoplasmic reticulum (ER) stress is induced by the accumulation of unfolded and misfolded proteins in the ER. Although apoptosis induced by ER stress has been implicated in several aging-associated diseases, such as atherosclerosis, it is unclear how aging modifies ER stress response in macrophages. To decipher this relationship, we assessed apoptosis in macrophages isolated from young (1.5-2months) and aged (16-18months) mice and exposed the cells to the ER stress inducer tunicamycin. We found that aged macrophages exhibited more apoptosis than young macrophages, which was accompanied by reduced activation of phosphorylated inositol-requiring enzyme-1 (p-IRE1α), one of the three key ER stress signal transducers. Reduced gene expression of x-box binding protein 1 (XBP1), a downstream effector of IRE1α, enhanced p-IRE1α levels and reduced apoptosis in aged, but not young macrophages treated with tunicamycin. These findings delineate a novel, age-dependent interaction by which macrophages undergo apoptosis upon ER stress, and suggest an important protective role of IRE1α in aging-associated ER stress-induced apoptosis. This novel pathway may not only be important in our understanding of longevity, but may also have important implications for pathogenesis and potential treatment of aging-associated diseases in general.

Macrophage apoptosis is a prominent characteristic of advanced atherosclerotic plaques and leads to plaque destabilization. Certain studies have confirmed that influenza virusA(IVA) infection is related to acute myocardial infarction(AMI). However, it remains unknown as to whether this phenomenon is associated with Toll-like receptor(TLR)7, since single-stranded RNA (ssRNA) of IVA is a natural ligand of TLR7. Thus, in the present study, THP-1‑derived macrophages were infected with IVA or treated with imiquimod(IMQ) in the presence or absence of pre-treatment with oxidized low-density lipoprotein (oxLDL). The macrophages were pre-treated with oxLDL(5µg/ml) for 24h to mimic high lipid conditions. Cell viability and apoptosis were detected by 3-(4,5-dimethylthiazol-2-y-1)‑2,5-diphenyl-2H-tetrazolium bromide(MTT) assay and flow cytometry, respectively. Our results revealed that TLR7 played an important role in macrophage apoptosis and cytokine secretion. Both IVA infection and IMQ treatment increased TLR7 expression, as well as the secretion of pro-inflammatory cytokines [interleukin (IL)-6, monocyte chemotactic protein (MCP)-1] and apoptosis. However, this increase in cytokine secretion occurred independently of cell apoptosis. oxLDL had potential synergistic pro-apoptotic effects combined with TLR7 activation. To determine whether endoplasmic reticulum(ER) stress plays a role in cell apoptosis, the mRNA and protein expression of known markers of ER stress [glucose-regulated protein(GRP)78 and C/EBP homologous protein(CHOP)] was detected by reverse transcription PCR(RT-PCR), quantitative reverse transcription PCR(qRT-PCR) and western blot analysis. Our results revealed that apoptosis aggravated ER stress, as shown by the overexpression of the pro-apoptotic sensor, CHOP. In conclusion, our study demonstrates the converging role of oxLDL pre-treatment, IVA infection and IMQ in ER stress-induced cell apoptosis.

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