A Role of RIP3-Mediated Macrophage Necrosis in Atherosclerosis Development

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1 Cell Reports Article A Role of RIP3-Mediated Macrophage Necrosis in Atherosclerosis Development Juan Lin, 1,6 Hanjie Li, 1,6 Min Yang, 2 Junming Ren, 1 Zhe Huang, 1 Felicia Han, 1 Jian Huang, 3 Jianhui Ma, 1 Duanwu Zhang, 1 Zhirong Zhang, 1 Jianfeng Wu, 1 Deli Huang, 1 Muzhen Qiao, 1 Guanghui Jin, 4 Qiao Wu, 1 Yinghui Huang, 5 Jie Du, 2, * and Jiahuai Han 1, * 1 State Key Laboratory of Cellular Stress Biology and School of Life Sciences, Xiamen University, Xiamen, Fujian, China 2 Beijing Institute of Heart, Lung and Blood Vessel Diseases, Beijing Anzhen Hospital, Capital Medical University, Beijing 129, China 3 The First Affiliated Hospital of Xiamen University, Xiamen, Fujian, China 4 Department of Basic Medical Sciences, Medical College, Xiamen University, Xiamen, Fujian, China 5 China-Japan Union Hospital, Jilin University, Changchun, Jilin, China 6 These authors contribute equally to this work *Correspondence: jiedu@yahoo.com (J.D.), jhan@scripps.edu (J.H.) SUMMARY Necrotic death of macrophages has long been known to be present in atherosclerotic lesions but has not been studied. We examined the role of receptor interacting protein (RIP) 3, a mediator of necrotic cell death, in atherosclerosis and found that RIP3 / ;Ldlr / mice were no different from RIP3 +/+ ;Ldlr / mice in early atherosclerosis but had significant reduction in advanced atherosclerotic lesions. Similar results were observed in Apoe / background mice. Bone marrow transplantation revealed that loss of RIP3 expression from bone-marrow-derived cells is responsible for the reduced disease progression. While no difference was found in apoptosis between RIP3 / ;Ldlr / and RIP3 +/+ ;Ldlr / mice, electron microscopy revealed a significant reduction of macrophage primary necrosis in the advanced lesions of RIP3 / mice. In vitro cellular studies showed that RIP3 deletion had no effect on oxidized low-density lipoprotein (LDL)-induced macrophage apoptosis, but prevented macrophage primary necrosis occurring in response to oxidized LDL under caspase inhibition or RIP3 overexpression conditions. RIP3-dependent necrosis is not postapoptotic, and the increased primary necrosis in advanced atherosclerotic lesions most likely resulted from the increase of RIP3 expression. Our data demonstrate that primary necrosis of macrophages is proatherogenic during advanced atherosclerosis development. INTRODUCTION Macrophages have been well known to play a key role in atherosclerosis development (Moore and Tabas, 211; Tabas, 21). During the initial stages of atherosclerosis, blood-borne monocytes are recruited into the subendothelial space of the blood vessel and differentiate into macrophages. Under nonresolving inflammatory conditions, monocytes continue to enter plaques and differentiate into macrophages as atherosclerotic lesions progress. Macrophages contribute to size-independent changes in plaque morphology, notably formation of the necrotic core and thinning of the fibrous cap, which characterize the vulnerable plaque (Moore and Tabas, 211; Tabas, 21). In response to lesional cytotoxic stimuli, macrophages usually undergo cell death. Apoptosis of macrophages has been studied in atherosclerosis development, which can either inhibit or promote the development of atherosclerosis, depending on the stage of atherogenesis (Tabas, 21). In early lesions where efferocytosis is effective, macrophage apoptosis can thwart the progression of atherosclerosis. In contrast, when the clearance of apoptotic macrophages is ineffective in advanced lesions, the apoptotic macrophages are speculated to undergo postapoptotic necrosis and coalesce over time into a key feature of vulnerable plaques, the necrotic core. Necrotic core formation in these advanced atheromata is thought to promote plaque disruption and, ultimately, acute atherothrombotic vascular disease (Tabas, 21). Morphological analysis of human samples with a transmission electron microscope shows that there are signs of macrophage necrosis in atherosclerotic lesions (Bauriedel et al., 1998; Crisby et al., 1997; Hegyi et al., 1996). Perhaps due to the fact that necrosis has long been considered as an accidental and uncontrolled mode of cell death, atherosclerosis-associated necrotic death of macrophages has been believed to be mostly composed of postapoptotic events, though morphological analyses suggest the presence of primary necrosis in the lesions (Bauriedel et al., 1998; Crisby et al., 1997; Hegyi et al., 1996). One early study showed that prolonged free cholesterol loading can lead to macrophage necrosis; this in vitro phenomenon was proposed to be involved in macrophage necrosis in advanced atherosclerosis (Tabas et al., 1996). A recent study also showed that macrophages incubated with lipoproteins containing plant sterol can undergo death in a caspase-independent way with signs of necrosis and autophagy, which provides a possible mechanism for accelerated plaque necrosis in patients with sitosterolemia 2 Cell Reports 3, 2 21, January 31, 213 ª213 The Authors

2 (high levels of sitosterol and other plant sterol) (Bao et al., 26). Those pioneering studies imply that primary necrosis of macrophage could play a role in atherosclerosis. However, to date there has not been any report to unambiguously address whether primary cellular necrosis occurs in atherosclerosis and whether it plays a role in plaque progression. Recently, significant progress has been made in the study of programmed necrosis (also called necroptosis) (Han et al., 211). A protein kinase, receptor interacting protein 3 (RIP3), was identified as a key regulator in mediating necrosis and switching apoptosis to necrosis (Cho et al., 29; He et al., 29; Zhang et al., 29). Formation of necrosome complex containing RIP3, RIP1, caspase-8, and other proteins is essential for necrosis to proceed. Without RIP3, a similar signaling complex triggers apoptotic cell death. The intermodulation between apoptosis and necrosis is bidirectional, because caspase-8 also has an inhibitory effect on RIP3-mediated necrosis (Kaiser et al., 211; Oberst et al., 211). The necrosis and apoptosis pathways appear to compete with each other and can switch between each other depending on the balance between caspase-8 and RIP3 (Han et al., 211). RIP3-dependent necrosis has now been shown to be a common cell-death pathway involved in cytokine-, virus-, and genotoxic stress-induced cell death and has also been demonstrated to be involved in a variety of pathological conditions, including the antiviral response (Cho et al., 29), acute pancreatitis (He et al., 29; Zhang et al., 29), photoreceptor loss (Trichonas et al., 21), embryonic lethality (Kaiser et al., 211), and intestine inflammation (Welz et al., 211). In this study, we evaluated the role of RIP3-dependent necrosis in mouse models of atherosclerosis. We found that deletion of RIP3 did not influence the development of early atherosclerosis, but significantly reduced the size of plaques in advanced atherosclerotic lesions. RIP3 has no role in macrophage apoptosis, but its deletion significantly reduced primary necrosis of macrophages in the lesions of advanced atherosclerosis. Necrotic macrophages are more difficult to remove through efferocytosis than apoptotic macrophages, and thus are more proinflammatory and proatherogenic. Our data suggest that increase of RIP3 expression in advanced atherosclerotic lesions promotes primary necrosis of macrophages, and demonstrates that RIP3-dependent necrosis plays an important role in the development of advanced atherosclerosis. RESULTS RIP3 Deficiency in Bone-Marrow-Derived Cells Reduces Advanced Atherosclerotic Lesions Genetic deletion of RIP3 in mice has no detectable effect in normal mouse development (Newton et al., 24). Because of the essential role of RIP3 in necrotic cell death, RIP3 knockout mice can be used to determine whether necrotic cell death plays any role in the development of atherosclerosis. To test the role of RIP3 in atherosclerosis, RIP3 / mice were bred into atherosclerosis-prone Ldlr / and Apoe / backgrounds. The RIP3 +/+ ;Ldlr / and RIP3 / ;Ldlr / mice were then fed a Western-type diet for either 8 or 16 weeks. Analysis of aortic lesion areas revealed no difference between the RIP3 +/+ ;Ldlr / and RIP3 / ;Ldlr / mice fed with a Western-type diet for 8 weeks (Figure 1A), but there was a significant difference between RIP3 +/+ ;Ldlr / and RIP3 / ;Ldlr / mice after the mice were fed a Western-type diet for 16 weeks (p <.5) (Figure 1B). It appears that RIP3 has little or no involvement in the early development of atherosclerosis, but has a promoting effect on advanced atherosclerosis. Further analysis was performed for advanced atherosclerotic lesions at the aortic root. Quantitative analysis of representative images revealed that lesion areas were much smaller in RIP3 / ;Ldlr / mice compared with RIP3 +/+ ;Ldlr / mice (p <.5) (Figure 1C). Analysis was also performed on features of the necrotic core, a key determinant of plaque vulnerability, which revealed a clear decrease in plaque necrosis in the RIP3 / lesions. Quantification of this parameter for the entire cohort of mice indicated a 6% decrease in total necrotic areas in the lesions of RIP3 / ;Ldlr / mice (p <.5) (Figure 1D). In addition to plaque lesion areas and necrotic areas, other important vulnerable plaque features are disruption of collagen, fibrous cap thinning, and elastin depletion. Although no significant difference in elastin break numbers of plaque lesions was observed between 16 week Western-diet-fed RIP3 +/+ ;Ldlr / and RIP3 / ;Ldlr / mice (Figure S1A), RIP3 deficiency significantly reduces the degradation of collagen and thinning of the fibrous cap (Figures S1B and S1C). Collectively, these results together with the reduced lesion areas and necrotic areas in RIP3 / ;Ldlr / mice demonstrate that RIP3 deficiency prevents atherosclerosis development. To investigate the possible cell types involved, bone marrow transplantation was carried out with lethally irradiated female RIP3 +/+ ;Ldlr / mice as recipients. Those mice were transplanted with bone marrow from either RIP3 +/+ ;Ldlr / or RIP3 / ;Ldlr / mice. Mice transplanted with RIP3 / ;Ldlr / bone marrow showed less lesion areas than the mice transplanted with RIP3 +/+ ;Ldlr / bone marrow after being fed with a Western-type diet for 16 weeks (Figure 2), suggesting that RIP3 deficiency in macrophages plays a role in reducing atherosclerotic lesions. Since body weight and plasma lipid levels are associated with the development of atherosclerosis, we compared body weight and plasma lipid levels between RIP3 +/+ ; Ldlr / and RIP3 / ;Ldlr / mice after being fat-fed for 16 weeks. As shown in Figure S1D, there was no difference between RIP3 +/+ and RIP3 / groups in body weight, total cholesterol (TC), low-density lipoprotein cholesterol (LDLC), high-density lipoprotein cholesterol (HDLC), and triglyceride (TG). RIP3 +/+ ;Apoe / and RIP3 / ;Apoe / mice were also used to study the role of RIP3 in atherosclerosis and the results obtained were very similar: decreased aortic lesion areas, decreased atherosclerotic lesions, and necrotic areas at the aortic root in RIP3 / mice (Figures S2A S2C), and no difference in body weight, TC, LDLC, HDLC, and TG between RIP3 +/+ and RIP3 / mice (Figure S2D). RIP3 Deficiency Reduces Primary Necrosis of Macrophages in Advanced Atherosclerotic Lesions but Has No Effect on Macrophage Apoptosis It is known that macrophage death plays a key role in atherosclerosis and that both apoptosis and necrosis of macrophages are Cell Reports 3, 2 21, January 31, 213 ª213 The Authors 21

3 A C D X X X X Lesion area (%) X NS B Total lesion area (μm ) Total necrotic area (μm ) Lesion area (%) *P<.5 *P< *P<.5 Figure 1. RIP3 / ;Ldlr / Mice Have Reduced Lesion and Necrotic Area Compared to RIP3 +/+ ;Ldlr / Mice after Being Fed a Western-type Diet for 16 Weeks (A) RIP3 +/+ ;Ldlr / (n = 7) and RIP3 / ;Ldlr / (n = 7) female mice fed a Western-type diet for 8 weeks. Representative images of pinned Sudan- IV-stained aortas and quantification of the surface area occupied by the lesions (percent area occupied by the lesions) are shown. Horizontal lines within the data sets represent means. Each symbol represents a measurement from a single mouse. NS, no significant difference. (B) RIP3 +/+ ;Ldlr / (n = 14) and RIP3 / ;Ldlr / (n = 16) female mice fed a Western-type diet for 16 weeks. Representative images of pinned Sudan-IV-stained aortas and quantification of the surface area occupied by the lesions (percent area occupied by the lesions) are shown. Horizontal lines within the data sets represent means. Each symbol represents a measurement from a single mouse. *p <.5. (C) The images show representative sections of aortic roots from the mice described in (B) stained with hematoxylin and eosin (H&E). Bar scales, 5 mm. Quantitative analyses of atherosclerotic lesion size in H&E-stained aortic root sections are shown. Data are expressed as mean ± SEM. *p <.5. (D) Representative sections of H&E-stained aortic roots from each group (X indicates necrotic area). Bar scales, 1 mm. The bar graph shows quantification of anuclear, afibrotic, and eosin-negative necrotic areas. Data are expressed as mean ± SEM. *p <.5. See also Figures S1 and S2. present in atherosclerotic lesions (Bauriedel et al., 1998; Crisby et al., 1997; Hegyi et al., 1996; Moore and Tabas, 211; Tabas, 21). Because RIP3 is essential for cell necrosis and the bone marrow transplantation results suggest that RIP3 deficiency in macrophages reduces lesion development (Figure 2), we sought to determine whether RIP3 knockout has any effect on macrophage apoptosis or necrosis. Because macrophage apoptosis and necrosis in atherosclerotic plaques can be identified morphologically under a transmission electron microscope (Bauriedel et al., 1998; Crisby et al., 1997; Hegyi et al., 1996), apoptotic and necrotic macrophages within atherosclerotic intimal areas at the aortic roots from RIP3 +/+ ;Ldlr / mice and RIP3 / ;Ldlr / mice were analyzed (Figure S3). Apoptotic macrophages in lesional intima are characterized by cell and nuclear shrinkage, chromatin condensation, and presence of apoptotic bodies, whereas necrotic macrophages show plasma membrane rupture with no apoptotic nuclear changes. Three pairs of 8-week fat-fed and four pairs of 16-week fat-fed RIP3 +/+ ;Ldlr / and RIP3 / ;Ldlr / mice were used to measure macrophage apoptosis and necrosis. Atherosclerotic plaques of aortic roots from each mouse were analyzed using electron microscopy. As shown in the left panel of Figure 3A, macrophage death in 8-week fat-fed mice is predominantly apoptotic, and there is no difference between RIP3 +/+ and RIP3 / mice in either apoptotic or necrotic macrophage death. In contrast, although the numbers of apoptotic macrophages in 16-week fat-fed RIP3 +/+ and RIP3 / mice were similar, RIP3 deletion was found to result in about a 5% decrease in necrotic macrophages in the atherosclerosis plaques (Figure 3A, right panel). To further confirm that RIP3 deficiency had no effect on macrophage apoptosis in the atherosclerotic lesions of 16- week fat-fed mice, activated caspase-3 was immunostained in serial sections from the proximal aorta, and no difference was found between RIP3 +/+ ;Ldlr / and RIP3 / ;Ldlr / mice (Figure 3B) or between RIP3 +/+ ;Apoe / and RIP3 / ;Apoe / mice (Figure S2E). Similarly, TUNEL staining of the lesions from RIP3 +/+ ;Ldlr / and RIP3 / ;Ldlr / mice showed no difference between the two groups of mice (Figure 3C). Collectively, the data demonstrate that RIP3 does not play any role in macrophage apoptosis, but promotes macrophage necrosis in advanced lesions. RIP3-Dependent Macrophage Necrosis Is Not a Postapoptotic Form of Cell Death The correlation between reduced lesion sizes and decreased macrophage necrosis in RIP3-deficient mice suggests that RIP3-dependent macrophage necrosis may promote the development of advanced atherosclerosis. Therefore, in vitro experiments were carried out to further characterize the feature of RIP3-dependent macrophage death. Translocation of 22 Cell Reports 3, 2 21, January 31, 213 ª213 The Authors

4 Donor Recipient Lesion area (%) phosphatidylserine from the inner part of the plasma membrane to the outer layer is an early event in apoptosis, which can be detected by annexin V staining. Treatment of RIP3 +/+ and RIP3 / macrophages with oxidized low-density lipoprotein (oxldl) for 12 hr led to the appearance of about 1% annexin V-positive cells in both RIP3 +/+ and RIP3 / cells (Figure 4A, upper panel). Uptake of propidium iodide (PI) is an indicator of a loss in cellmembrane integrity. The 12 hr oxldl treatment did not lead to the appearance of detectable PI-positive cells in either RIP3 +/+ or RIP3 / macrophages (Figure 4A, lower panel). The annexin V-positive and PI-negative cells should be apoptotic cells. These data indicated that oxldl induces macrophage apoptosis and that RIP3 is not involved in oxldl-induced apoptosis of macrophages. Similarly, it was found that RIP3 deficiency did not have any effect on macrophage apoptosis induced by other proatherogenic apoptotic stimuli (data not shown), including 7-KC, 25-HOC, and oxldl plus SIN-1, which generates peroxynitrite and induces endoplasmic reticulum stress in cells (Thorp et al., 29). Because inhibition of caspases in fibroblasts and other cells blocks apoptosis and simultaneously enhances RIP3-dependent necrosis (Zhang et al., 29), the possible effect of caspase inhibition on oxldl-induced macrophage death was evaluated. Treatment of macrophages with pan-caspase inhibitor zvad alone had no effect on cell viability (Figure 4A). zvad did not reduce but rather dramatically increased oxldl-induced annexin V-positive cells in RIP3 +/+ macrophages (Figure 4A, upper panel). These annexin V-positive cells were different from those *P<.5 Figure 2. Lethally Irradiated RIP3 +/+ ;Ldlr / Mice Transplanted with RIP3 / ;Ldlr / Bone Marrow Have Reduced Lesion Area as Compared with mice Transplanted with RIP3 +/+ ;Ldlr / Bone Marrow Eight-week-old female RIP3 +/+ ;Ldlr / mice were lethally irradiated and transplanted with female RIP3 +/+ ;Ldlr / (n = 8) or RIP3 / ;Ldlr / (n = 8) bone marrow. Eight weeks posttransplantation, the two groups were fed with a Western diet for 16 weeks. Representative images of pinned Sudan-IVstained aortas and quantifications of the surface areas occupied by the lesions (percent area occupied by the lesions) are shown. Horizontal lines within the data sets represent means. Each symbol represents a measurement from a single mouse. *p <.5. with oxldl treatment alone, because they were also PI positive (Figure 4A, lower panel), indicating that oxldl-induced macrophage necrosis occurred under caspase inhibition. Electron microscopy analysis revealed a dramatic increase in macrophage death with the morphology of primary necrosis (Figure 4B). In addition, rapid loss of plasma membrane integrity was also detected through the release of radiolabeled materials from the cells (Figure 4C) in RIP3 +/+ macrophages treated with oxldl in the presence of zvad. Therefore, caspase inhibition switches oxldl-induced macrophage death from apoptosis to necrosis. Notably, necrotic cell death was completely abolished in RIP3- deficient macrophages (Figures 4A 4C). Similarly, caspase inhibition also enhanced RIP3-dependent macrophage necrosis induced by 7-KC and 25-HOC (data not shown). Because necrotic cell death could be a postapoptotic event, we next examined the dynamic relationship between oxldlinduced apoptosis and RIP3-dependent necrosis. Sixty hours of oxldl treatment caused 1% of cells to become annexin V positive (Figure 4D, upper panel). PI-positive cells were barely detectable before 6 hr, but appeared at a later time (Figure 4D, upper panel). These PI-positive cells most likely resulted from postapoptotic necrosis. There was no difference between RIP3 +/+ and RIP3 / macrophages in oxldl-induced apoptosis and postapoptotic necrosis (Figure 4D, upper panel). We then analyzed the time course of cell death in oxldl-plus-zvadtreated macrophages. OxLDL plus zvad led to quick death of RIP3 +/+ macrophages with a necrotic phenotype of annexin V and PI double-positive staining (Figure 4D, lower panel). This type of cell death depends on RIP3 because it was blocked when RIP3 was deleted in macrophages (Figure 4D, lower panel). To confirm that oxldl-plus-zvad-induced macrophage death is necrosis, active caspase-3 was stained and TUNEL assay was used to determine whether these apoptosis markers were absent in oxldl-plus-zvad-treated macrophages. Sixteen hours of oxldl plus zvad treatment resulted in annexin V and PI double-positive staining in almost all RIP3 +/+ macrophages, but not RIP3 / cells (Figures S4Ac and S4Ad). No caspase-3 activation or TUNEL-positive signals were detected in these cells (Figures S4Bc and S4Cc), indicating that those cells did not undergo apoptosis. In contrast, caspase-3 activation and TUNEL-positive signals were detected in both RIP3 +/+ and RIP3 / macrophages treated solely with oxldl for 72 hr (Figures S4Ae, S4Af, S4Be, S4Bf, S4Ce, and S4Cf), indicating that oxldl-induced cell death is apoptosis. Collectively, these data demonstrate that RIP3 is not involved in apoptosis or postapoptotic necrosis, but rather plays a key role in the primary necrosis of macrophages. These in vitro data are consistent with the data obtained by analyzing atherosclerotic lesions, which showed a decrease in macrophage primary necrosis in RIP3 / mice (Figure 3A). RIP3 Deficiency Reduces Lesional Inflammation and Macrophage Infiltration in Advanced Lesions Because inflammation in lesions plays an important role in the acceleration of atherosclerosis (Barish et al., 28; Lee et al., 23; Moore and Tabas, 211; Tabas, 21), we tested whether RIP3 deficiency affects the inflammatory responses or the efferocytosis of macrophages, both of which can regulate Cell Reports 3, 2 21, January 31, 213 ª213 The Authors 23

5 A B Percentage of total macrophge NS Apoptosis % activated caspase3 positive cells Early lesion NS NS Necrosis C Percentage of total macrophge NS Apoptosis % TUNEL Positive nuclei Advanced lesion NS *p<.5 Necrosis Figure 3. Advanced Lesions of RIP3 / ; Ldlr / Mice Have Reduced Macrophage Necrosis as Compared to RIP3 +/+ ;Ldlr / Mice (A) Statistics of apoptotic and necrotic macrophages within intimal area from early and advanced lesions at mouse aortic roots. A total of 2 macrophages within the lesional intima were counted, and quantifications of apoptotic and necrotic macrophage percentages are shown for RIP3 +/+ ;Ldlr / and RIP3 / ;Ldlr / mice after an 8-week (early lesion, three pairs, left panel) or a 16-week (advanced lesion, four pairs, right panel) Western diet. Data shown represent mean ± SEM. NS, no significant difference; *p <.5. (B) Representative micrographs showing activated caspase-3 in aortic root lesions from RIP3 +/+ ;Ldlr / (n = 1) and RIP3 / ;Ldlr / (n = 1) mice by immunohistochemistry. Bar scales: 5um. A quantitative analysis of aortic root lesional active caspase-3 positive cells for each group is shown. Data shown represent mean ± SEM. NS, no significant difference. (C) Representative micrographs showing TUNEL positive cells in aortic root lesions from RIP3 +/+ ; Ldlr / and RIP3 / ;Ldlr / mice by immunefluorescence. Bar scales, 1 mm. A quantitative analysis of aortic root lesional TUNEL positive cells for each group (n = 5) is shown. Data shown represent mean ± SEM. NS, no significant difference. See also Figures S2 and S3. inflammation in lesions. First, RIP3 +/+ and RIP3 / peritoneal macrophages were treated with oxldl and the expressions of a number of different cytokines and chemokines were measured; no differences were found between RIP3 +/+ and RIP3 / cells in oxldl-induced tumor necrosis factor (TNF), interleukin 1b, monocyte chemoattractant protein 1 (MCP1), MCP2, MCP3, macrophage inflammatory protein 1-a (MIP1a), MIP1b, or interferon gamma-induced protein 1 transcription (Figure S5A). This is consistent with a published study that showed that RIP3 does not regulate cytokine production (Newton et al., 24). We next evaluated the efficiency of phagocytic clearance of dead cells (efferocytosis) by RIP3 +/+ and RIP3 / macrophages. There was no difference in the ability of RIP3 +/+ versus RIP3 / macrophages in engulfing oxldl-induced apoptotic macrophages, regardless of whether the apoptotic cells were RIP3 +/+ or RIP3 / (Figure S5B), or in engulfing oxldl-pluszvad-induced necrotic macrophages (Figure S5B). It is important to note that necrotic macrophages are poorer substrate for phagocytes compared to apoptotic macrophages (Figure S5B), which is consistent with previous studies (Kono and Rock, 28). The inefficient clearance of necrotic macrophages could aggravate the proinflammatory effect of RIP3- dependent macrophage necrosis in advanced atherosclerosis, because cellular necrosis can release damaging intracellular molecules into the extracellular milieu and stimulate inflammatory responses. To evaluate whether inflammatory responses are different between RIP3 +/+ and RIP3 / mice at the onset of atherosclerosis development, total RNA from whole lesions of RIP3 +/+ ;Ldlr / and RIP3 / ;Ldlr / mice in advanced stages was extracted, and the results showed that overall, the mrna levels of the cytokines tested were slightly higher in RIP3 +/+ ; Ldlr / advanced lesions (Figure 5A). Statistically significant differences were observed in the mrna levels of MIP1b, TNF, and vascular cell adhesion molecule 1 (VCAM-1). VCAM-1 expression by vascular cells is a characteristic feature of atherosclerosis, reflecting the inflammatory state in the plaque (Cybulsky and Gimbrone, 1991; Libby, 22; Tupin et al., 24). Similar to the lower VCAM-1 mrna level in RIP3 / ;Ldlr / advanced lesion, there was also reduction of VCAM-1 positive areas in RIP3 / ;Ldlr / lesions (Figure 5B). Immunohistological detection of macrophages, using a Mac-2 antibody, showed greatly reduced staining in lesions at the aortic roots of RIP3 / ;Ldlr / mice compared to those of RIP3 +/+ ;Ldlr / littermate mice (Figure 5C). A significant decrease in Mac-2-positive areas was also seen in RIP3 / ;Apoe / mice compared to RIP3 +/+ ;Apoe / littermate mice (Figure S2F). These data indicate that reduced lesions in the absence of RIP3 are partially due to the decreased accumulation of macrophages in lesions, which are known to participate in a maladaptive, nonresolving inflammatory response that expands the subendothelial layer due to the 24 Cell Reports 3, 2 21, January 31, 213 ª213 The Authors

6 A C B D Figure 4. RIP3-Dependent Necrosis Is Not a Postapoptotic Cell Death (A) Peritoneal macrophages from RIP3 +/+ and RIP3 / mice were treated for 12 hr with vehicle control (DMSO), zvad (2 mm), oxldl (15 mg/ml), and oxldl (15 mg/ml) plus zvad (2 mm). Percentages of annexin V- or PI-positive macrophages are shown. Data are expressed as mean ± SEM. (B) The RIP3 +/+ and RIP3 / peritoneal macrophages were treated for 1 hr with vehicle control, zvad, oxldl, and oxldl plus zvad. Representative morphologies of alive and necrotic peritoneal macrophages are shown (upper panel). Scale bars, 1 mm. Among the 2 macrophages counted, the number of necrotic cells was recorded for each sample and is shown (lower panel). (C) Peritoneal macrophages from RIP3 +/+ and RIP3 / mice were loaded with [ 3 H]adenine for 2 hr and then treated with vehicle control, zvad, oxldl, and oxldl plus zvad. The supernatants were harvested and the cells were lysed; radioactivity was measured by scintillation counting, and the percentage of released radioactivity was calculated. Data are expressed as mean ± SEM. (D) Peritoneal macrophages from RIP3 +/+ and RIP3 / mice were loaded with oxldl and oxldl plus zvad for the time indicated. Quantification of annexin V- or PI-positive macrophage percentages at each time point is shown. Data are expressed as mean ± SEM. See also Figure S4. infiltration of macrophages and accumulation of lipid and matrix (Moore and Tabas, 211). Caspase-8 Inhibition or RIP3 Upregulation Sensitize Macrophages to oxldl-induced Necrosis Studies from our and other laboratories have demonstrated that caspase-8-dependent apoptosis and RIP3-dependent necrosis compete with each other in mediating cell death (Han et al., 211; Kaiser et al., 211; Oberst et al., 211). Increased expression of RIP3 can convert cell death from apoptosis to necrosis (Zhang et al., 29). On the other hand, a complex of caspase-8 and FLIP L, in which caspase-8 is not processed but retains protease activity, can prevent RIP3 from mediating necrosis (Oberst et al., 211). The data in Figure 4 already showed cross-inhibition between apoptosis and necrosis in oxldl-treated macrophages, because inhibition of caspases promotes RIP3-dependent necrosis. To determine which caspase(s) plays a key role in blocking oxldl-induced macrophage necrosis, we tested different caspase inhibitors. Cell necrosis was determined by Annexin V/PI double staining (Figure 6A). Another pan-caspase inhibitor similar to zvad, QVD, also enhanced oxldl-induced macrophage necrosis. Caspase-8-specific inhibitor IETD, but neither caspase-3 inhibitor DEVD nor caspase-1 inhibitor WEHD, promoted oxldlinduced macrophage necrosis. Thus, inhibition of caspase-8 plays a major role in promoting oxldl-induced macrophage necrosis. Next, whether increasing RIP3 activity by overexpression of RIP3 in macrophages can convert apoptosis to necrosis was examined. Indeed, oxldl alone triggered necrosis in macrophage cell line Raw by overexpressing RIP3 over a certain level (Figure 6B), similar to the reported conversion of apoptosis to necrosis by increased RIP3 expression in fibroblast and epithelial cell lines (Sun et al., 212; Zhang et al., 29). It is noteworthy that percentages of necrotic cells positively correlate with the RIP3 level in the macrophages. Cell Reports 3, 2 21, January 31, 213 ª213 The Authors 25

7 A B Relative mrna in lesion MCP1 MCP2 MCP3 MIP1 alpha MIP1 beta TNF IP1 IL1 beta * * * VCAM-1 VCAM-1 relative level *P<.5 Figure 5. Less Inflammation and Macrophage Infiltration in the Advanced Lesions of RIP3 / ;Ldlr / Mice (A) Relative expression levels of cytokine and chemokine mrna in lesions from 16-week fat-fed RIP3 +/+ ;Ldlr / and RIP3 / ;Ldlr / mice were measured by quantitative RT-PCR. The gene expression levels were normalized to the expression of GAPDH. Data are presented as means ± SEM. *p <.5. (B) Representative photographs of VCAM-1 expression in advanced lesions from aortic roots. Lesions developed in RIP3 +/+ ;Ldlr / or RIP3 / ;Ldlr / mice after 16 weeks on a Western diet were stained for VCAM-1 (upper; red signal is VCAM-1 positive, blue signal is DAPI) and Oil Red O (lower). Bar scale, 1 mm. A quantitative analysis of VCAM-1 expression at aortic root lesions in four pairs of RIP3 +/+ ;Ldlr / and RIP3 / ;Ldlr / mice is shown. The mean value of VCAM-1-positive areas from the four RIP3 +/+ ; Ldlr / mice was set as 1. Data are expressed as mean ± SEM. *p <.5. (C) Representative micrographs show Mac-2- positive cells in aortic root lesions from RIP3 +/+ ; Ldlr / and RIP3 / ;Ldlr / mice by immunohistochemical staining. A quantitative analysis of aortic root lesional macrophage infiltration in RIP3 +/+ ;Ldlr / (n = 5) and RIP3 / ;Ldlr / (n = 7) mice is shown. Scale bars, 5 mm. Data are expressed as mean ± SEM. *p <.5. See also Figure S5. C RIP3 Upregulation in Lesional Macrophages during Lesion Development Induces Macrophage Necrosis and Promotes Lesion Development We then speculated that RIP3-dependent necrosis of macrophages in advanced atherosclerotic lesions resulted from either increased RIP3 expression or reduced caspase-8 expression. To evaluate this hypothesis, the levels of RIP3 and active or fulllength caspase-8 in early and advanced lesions were examined. No difference was detected in active caspase-8 or full-length caspase-8 between early and advanced lesions from either RIP3 +/+ ;Ldlr / or RIP3 / ;Ldlr / mice by western blotting (Figure S5C) However, the RIP3 level is higher in advanced lesions compared to early lesions in RIP3. +/+ ;Ldlr / mice (Figure S5C). To demonstrate that the RIP3 level is increased in macrophages in advanced lesions, laser capture microdissection was carried out to isolate smooth muscle cells and macrophages % Mac-2 positive cells *P<.5 and to determine their RIP3 mrna levels (Figure S6). It was found that although RIP3 level did not change between nonlesional smooth muscle cells and lesional smooth muscle cells (Figure 7A), its level in lesional macrophages increased as the lesion developed from a small size to large size (Figure 7B). Because cell-based experiments showed that the RIP3 level should exceed a certain threshold in order to induce cellular necrosis (Figure 6B), it is very likely that the RIP3 level in lesional macrophages should also exceed a certain threshold in order to induce necrosis. The importance of RIP3 expression level in determining the mode of cell death in vivo is supported by the comparison of lesion areas in RIP3 +/+ ;Apoe /, RIP3 +/ ;Apoe /, and RIP3 +/ ;Apoe / mice. It was found that RIP3 +/ ;Apoe / mice had less lesion areas compared to RIP3 +/+ ; Apoe / mice, but more lesion areas compared to RIP3 / ;Apoe / mice (Figure 7C). Thus, RIP3- mediated macrophage necrosis in advanced lesions most likely can be attributed to the increase of RIP3 expression in macrophages during the development of atherosclerosis. DISCUSSION Both apoptotic and necrotic macrophages were detected in atherosclerotic lesions from human patients and model animals. 26 Cell Reports 3, 2 21, January 31, 213 ª213 The Authors

8 A Figure 6. Caspase-8 Inhibition or RIP3 Overexpression Exceeding a Certain 8 25 Threshold Switches oxldl-induced Apoptosis to Necrosis * 2 6 (A) RIP3 +/+ and RIP3 / peritoneal macrophages 4 * were treated for 12 hr with vehicle control, oxldl, 15 oxldl plus DEVD (caspase-3 inhibitor), oxldl plus WEHD (caspase-1 inhibitor), oxldl plus QVD 1 * * (pan caspase inhibitor), and oxldl plus IETD 2 5 (caspase-8 inhibitor), and then were analyzed by annexin V and PI staining. Percentage of annexin V and PI double-positive macrophages is shown (left panel). Data are expressed as mean ± SEM. *p <.5. Inhibition of caspase-8 by IETD and QVD is shown in the right panel. (B) Raw cells were infected with different doses of a RIP3-expressing lentivirus or an empty B vector. The cells were then treated with or without RIP3 oxldl for 48 hr. Percentage of annexin V and - RIP3 PI double-positive cells is shown. Data are expressed GAPDH as mean ± SEM. NS, no significant difference. *p <.5. RIP3 expression level was 8 ctrl oxldl determined by western blot with an anti-rip3 antibody. See also Figure S5. % of Annexin V/PI double positive % of Annexin V/PI double positive RIP3 - ctrl oxldl+wehd oxldl+devd oxldl+qvd oxldl+ietd While the role of apoptosis in the development of atherosclerosis has been well studied, the involvement of necrosis has never been experimentally evaluated. Because of the limitation in our understanding toward the mechanisms of cellular necrosis, only postapoptotic cell disruption, termed secondary or postapoptotic necrosis, was considered in the formation of necrotic core in the lesions. Because of very recent advances in defining the mechanisms of programmed necrosis, we used RIP3 knockout mice to address whether RIP3-dependent necrosis is involved in atherosclerosis. We found that RIP3- dependent macrophage death is primary necrosis and is not important in the early stages of the disease, but plays an important role in advanced atherosclerosis. RIP3-dependent necrosis accounts for half of the necrotic cells in advanced lesions, while the other half could be postapoptotic cells or primary necrosis controlled by yet unknown mechanisms. It appears that the conversion of macrophage apoptosis to RIP3-dependent necrosis occurs during the development of atherosclerosis toward the advanced stage. The underlying mechanism of this conversion most likely is the upregulation of RIP3 expression in lesional macrophages toward the later stages of the disease. Given the fact that necrotic cells are more difficult to clear than apoptotic cells, primary necrosis of macrophages in advanced lesions may not only be involved in plaque formation, but also cause more inflammatory cell infiltra- * * Active casp 8 activity (units) ctrl oxldl oxldl+wehd oxldl+devd oxldl+qvd oxldl+ietd tion, and thus have a strong proinflammatory and proatherogenic role. The necrotic morphology of RIP3- dependent macrophage death was determined by electron microscope analysis (Figure 4B). Although these necrotic macrophages are annexin V/PI double positive as determined by cellular assays (Figures 4A, 4D, and S4A), they lack apoptotic markers such as active caspase-3 or TUNEL-positive signals (Figures S4B and S4C). The annexin V-positive staining of necrotic cells could have resulted from loss of integrity of the plasma membrane, which allows annexin V to interact with the phosphatidylserine located in the inner part of the plasma membrane. This also explains why inhibition of caspases by zvad had no effect on annexin V staining in oxldl-treated cells. Reactive oxygen species (ROS) derived from mitochondria has been proven to play a crucial role in the execution of RIP3-dependent necrosis (Zhang et al., 29). We and others have revealed that RIP1 and RIP3 are required for mitochondrial ROS production in some cell lines during necrosis (Zhang et al., 29). Similarly, the ROS scavenger butylated hydroxyanisole (BHA) and the RIP1 inhibitor Nec-1 (necrostatin-1) also effectively blocked RIP3-mediated necrosis and ROS production in oxldl-pluszvad-treated RIP3 +/+ macrophages (data not shown), as did the inhibitor of glycolysis (2-DG), and inhibitors of the complexes of the electron transport chain (Amytal, TTFA, antimycin A, NaN3) (data not shown). Inhibitors of ATP synthase (oligomycin) and lactate dehydrogenase (oxamate) had no effect on oxldlplus-zvad-induced necrosis and ROS production (data not shown). Therefore, oxldl-plus-zvad-induced RIP3-mediated macrophage necrosis is dependent on RIP1 kinase activity and mitochondrial ROS production. Similarly, the enhanced Cell Reports 3, 2 21, January 31, 213 ª213 The Authors 27

9 A Relative RIP3 mrna in SMC C Non-lesional SMC Lesional SMC NS B Relative RIP3 mrna in Mac *P<.5 Small lesion Middle lesion Large lesion *P<.5 *P<.5 RIP3 GAPDH Figure 7. Increased RIP3 Expression during Lesion Development and the Correlation between In Vivo RIP3 Level and the Severity of Atherosclerotic Lesions (A) RIP3 mrna levels were compared between nonlesional SMC and lesional SMC isolated by laser capture microdissection from atherosclerotic lesions of 16-week fat-fed RIP3 +/+ ;Ldlr / mice. Data are presented as mean ± SEM. NS, no significant difference. (B) RIP3 mrna levels in lesional macrophages were compared among atherosclerotic lesions of different sizes (small, middle, and large). Data are expressed as mean ± SEM. *p <.5. (C) Eight-week-old RIP3 +/+ ;Apoe / (n = 5), RIP3 +/ ;Apoe / (n = 6), and RIP3 / ;Apoe / (n = 4) mice were fed with a Western diet for 16 weeks. Representative images of pinned Sudan-IV-stained aortas and quantification of the surface area occupied by the lesions (percent area occupied by the lesions) are shown. Horizontal lines within the data sets represent means. Each symbol represents a measurement from a single mouse. *p <.5. See also Figures S5 and S6. 25 RIP3+/- oxldl-induced cell death in the presence of QVD or IETD can also be inhibited by Nec-1 (data not shown). As anticipated, BHA, Nec-1, and inhibitors of glycolysis and the electron transport chain could not inhibit oxldl-induced apoptosis in either RIP3 +/+ or RIP3 / macrophages (data not shown). It is clear that macrophage necrosis uses the same execution pathway as the necrosis observed in other cell types, excepting extracellular stimuli. The cellular receptor/sensor(s) of oxldl or other lipids that initiate the signaling linking to the RIP3-dependent necrosis pathway is currently unclear. It has been well established that macrophage apoptosis occurs in atherosclerotic lesions, and stimuli (including oxldl, 7-KC, 25-HOC, free cholesterol, etc.) that are thought to be proatherogenic also induce macrophage apoptosis in vitro. We found that these proapoptotic stimuli could be pronecrotic under conditions of caspase inhibition or RIP3 induction. Because caspase-8-dependent apoptosis and RIP3-dependent necrosis compete with each other in mediating cell death, the relative level of the two central mediators of cell death might determine the cell death mode responding to different upstream signals. An increase of RIP3 in advanced lesional macrophages was Lesion area (%) *P<.5 RIP3+/- detected, but which atherosclerosisrelated factors can induce RIP3 expression is currently unknown. Given the complicated in vivo situation for the development of atherosclerosis, it is possible that multiple factors are involved. Although our data suggest that the increase of RIP3 expression converts macrophage apoptosis to necrosis in advanced lesions, we cannot exclude other mechanisms, such as increasing RIP3 activity by eliminating RIP3 phosphatase and expression of caspase-8 inhibitor, that can modulate the balance between RIP3 and caspase-8 and thus are involved in the regulation of macrophage necrosis in advanced atherosclerosis. Nevertheless, the balance between RIP3 and caspase-8 should be a determinant of whether the cells undergo necrosis or apoptosis. In short, the correlation between decreased macrophage primary necrosis in advanced lesions and improved disease condition in RIP3 / mice demonstrates the contribution of RIP3-dependent macrophage necrosis in the development of atherosclerosis toward advanced stage. Although postapoptotic necrosis is well accepted to play a role in atherosclerosis development, the data reported here demonstrate a previously unidentified primary necrosis that plays an essential role in the development of advanced atherosclerosis. RIP3-mediated primary necrosis of macrophages could contribute to lesion development by directly coalescing into the necrotic core and secondarily exacerbating the maladaptive inflammatory response, thus promoting advanced atherosclerotic lesions. Interventions by the RIP3 signaling pathway may be used to modulate the development of atherosclerosis. 28 Cell Reports 3, 2 21, January 31, 213 ª213 The Authors

10 EXPERIMENTAL PROCEDURES Mice and Diets Mice were housed in a specific pathogen-free environment. All experiments were conducted in compliance with the regulations of Xiamen University. RIP3 / mice from the C57BL6/J background were bred onto the Ldlr / and Apoe / C57BL6/J backgrounds to generate RIP3 +/ ;Apoe / and RIP3 +/ ;Ldlr / breeding pairs. Starting at 8 weeks of age, female progeny (RIP3 +/+ ;Ldlr / and RIP3 / ;Ldlr / or RIP3 +/+ ;Apoe / and RIP3 / ; Apoe / ) from these breeding pairs were fed a high-cholesterol Westerntype diet for either 12 weeks (Apoe / study) or 16 weeks (Ldlr / study). For studying early lesions, female RIP3 +/+ ;Ldlr / and RIP3 / ;Ldlr / mice were fed a Western-type diet for 8 weeks. The Western diet was produced by Vital River Laboratories (Beijing, China) according to the Formula from Harlan (TD.88137). Oxidized Low-Density Lipoprotein oxldl was purchased from Unionbiol (Beijing, China). LDL was isolated from human plasma (density: ; purity: 98%). Oxidized LDL was obtained by oxidizing LDL with copper sulfate. Peritoneal Macrophages Thioglycollate-elicited macrophages were obtained from 8-week-old RIP3 +/+ ; Ldlr /, RIP3 / ;Ldlr /, RIP3 +/+, and RIP3 / mice using a method described previously (Osuga et al., 2). Because it was found that having a Ldlr / background did not affect the macrophages response to the treatment of oxldl or oxldl plus zvad, RIP3 +/+ and RIP3 / macrophages were used in most of our experiments. The cells were resuspended in Dulbecco s modified Eagle medium containing 1% fetal calf serum (FCS), and cells were plated in one well of a 48-well plate and cultured in 37 C, 5% CO 2 conditions. Atherosclerotic Lesion Analysis Mice were euthanized and their hearts and aortas were isolated. The degree of atherosclerosis was assessed by determining lesion sizes on both pinnedopen aortas and serial cross-sections through the aortic root as modified from previous studies (Seimon et al., 29; Sekiya et al., 29; Thorp et al., 29). The aorta was opened longitudinally along the ventral midline from the iliac arteries to the aortic root. After branching vessels were removed, the aorta was pinned out flat on a black wax surface. The lesions were treated with 7% ethanol and then stained with Sudan IV for 15 min, destained with 8% ethanol, and then washed with PBS. Aortic images were analyzed with Adobe Photoshop software, data are reported as the percentage of the aortic surface covered by lesions. The hearts were perfused with saline containing 4% paraformaldehyde and fixed for more than 48 hr in the same solution. The basal halves of the hearts were embedded in Tissue-Tek OCT compound (or paraffin), and serial sections (6 mm thick) were captured using a Cryostat microtome (Leica, Germany) or Paraffin microtome (Leica, Germany). As described in a previous study (Seimon et al., 29), for morphometric lesion analysis, sections were stained with Harris hematoxylin and eosin (H&E). Total intimal lesion area (from internal elastic lamina to the lumen) and acellular/anuclear areas (negative for hematoxylin-positive nuclei) per cross-section were quantified by taking the average of six sections spaced 28 mm apart beginning at the base of the aortic root. The necrotic core was defined as a clear area that was H&E free. Boundary lines were drawn around these regions, and the area measurements were obtained by image analysis software. Lipids in lesion areas were stained with.5% Oil Red O (Sangon Biotech, Shanghai, China) in propylene glycol for 1 min at 6 C. Morphological analysis of collagen contents in the lesional area and of fibrous cap thickness was performed with a Masson trichrome stain kit (Maixin-Bio, Liaoning, China), whereas elastin breaks were conducted by an elastin stain kit (Maixin-Bio). Fibrous cap thickness was quantified by choosing the largest necrotic core from duplicate sections and measuring the thickness of the thinnest part of the cap. Analysis was performed by two independent experienced morphologists blinded to the genotype. In Vitro Peritoneal Macrophage Cell Death Assay Macrophage cell death was measured by annexin V-EGFP and PI staining as previously described (Feng et al., 23). Macrophages were stained with annexin V-EGFP in annexin V binding buffer on ice for 2 min in the dark. The annexin V labeling solution was then removed; and PI labeling solution was added. Cells were immediately viewed with a 23 objective using an Olympus IX-7 inverted fluorescence microscope equipped with filters appropriate for fluorescein and PI. Four fields of cells for each condition (more than 1, cells) were counted. The total number of cells was measured by counterstaining with DAPI. The data are expressed as the percentage of these cells per total cells counted. Percentages of green fluorescent protein-positive and PI-positive cells were counted separately. Statistics Absent error bars in graphs signify that SEM values were smaller than the graphic symbols. Comparison of mean values between groups was evaluated by the two-tailed Student s t test, Mann-Whitney U test, or ANOVA. A p value of less than.5 was considered significant. For further details, please refer to Extended Experimental Procedures. SUPPLEMENTAL INFORMATION Supplemental Information includes Extended Experimental Procedures and six figures and can be found with this article online at 116/j.celrep LICENSING INFORMATION This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-No Derivative Works License, which permits non-commercial use, distribution, and reproduction in any medium, provided the original author and source are credited. ACKNOWLEDGMENTS We thank Ping Chen at the School of Life Sciences, Xiamen University for her help with electron microscopy analysis. We thank K. Newton and V.M. Dixit for RIP3 / mice. This work was supported by 973 Program 29CB5222, Natural Science Foundation of China grants 38392, 39215, , , 863 program 212AA2A21, and 111 project B121. Received: May 31, 212 Revised: November 19, 212 Accepted: December 18, 212 Published: January 17, 213 REFERENCES Bao, L.P., Li, Y.K., Deng, S.X., Landry, D., and Tabas, I. (26). 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12 Supplemental Information EXTENDED EXPERIMENTAL PROCEDURES Quantitative Transmission Electron Microscopy Peritoneal macrophages after treatment in vitro and lesional intimas from mice aorta roots were prepared for transmission electron microscopy (TEM) (JEM 21 HC, JEOL USA) by the conventional method. For quantitative analysis of necrotic macrophages in vitro, a total of 2 peritoneal macrophages were counted and the number of necrotic macrophages was recorded. A necrotic macrophage was defined as a macrophage with morphology characteristics of primary necrosis (plasma membrane ruptures without nuclear condensation). For quantitative analysis of apoptotic and necrotic macrophages in vivo, atherosclerotic lesions from the aorta root were serially sectioned at 2 mm intervals (about the size of a macrophage). In vivo quantitative TEM analysis was focused on the atherosclerotic intimal area, and the morphometric quantification was done by an experienced morphologist blinded to the genotype. Intimal areas within those sections were scanned for macrophage until 2 macrophages were counted for each mouse (1525 sections). Criteria for discriminating cell types in atherosclerotic lesions were adapted from previous studies (Bauriedel et al., 1998; Crisby et al., 1997; Tranfield et al., 21; Yamada et al., 1988). Briefly, contractile SMCs were identified with cytoplasm rich in myofilaments, the presence of dense bodies and circumferential basement membranes. Synthetic SMCs characterized with large endoplasmic reticula and Golgi complexes make up only a minority of the total SMCs. Lymphocytes (which are very rare in atherosclerotic plaques) were small, rounded and well- reserved cells with little cytoplasm and few organelles. Macrophages were characterized by no myofilaments or dense bodies, and the presence of pseudopods. As for the morphology of lesional macrophages, apoptotic macrophages were identified based on the presence of nuclear condensation or nuclear fragments surrounded by membrane (apoptotic bodies); macrophages with ruptured cellular membrane and no apparent nuclear condensation are defined as primary necrosis. Macrophages with intact plasma membrane and without nuclear change are defined as alive. The number of apoptotic and necrotic macrophages in the lesions were recorded. Antibodies, Immunohistochemistry, Immunofluorescence Anti- Mac-2 antibody (Santa Cruz Biotechnology, California, USA; 1:2 dilution), anti- activated caspase-3 antibody (Abcam; 1:5 dilution), and anti- active caspase-8 antibody (Cell Signaling Technology, Danvers, Massachusetts, USA; 1:2 dilution) were used for immunohistochemistry. Anti- VCAM-1 antibody (Biolegend, California, USA; 1:2 dilution) was used for immunofluorescence. Anti-RIP3 polyclonal antibodies raised in rabbits using E. coli-expressed GST-RIP3 ( amino acids), anti caspase-8 antibody (Cell Signaling Technology, Danvers, Massachusetts, USA; 1:1 dilution), and anti-active caspase-8 antibody (Cell Signaling Technology, Danvers, Massachusetts, USA; 1:5 dilution) were used for Western blotting. For immunohistochemistry, antigen retrieval was achieved by boiling in citrate buffer (ph 6.), followed by hydrogen peroxide/pbs blocking of endogenous peroxidase. Blocking was performed with immunoglobulin from the species of the secondary antibody. Primary antibodies, including the Mac-2 antibody (Santa Cruz Biotechnology, California, USA; 1:2 dilution), active caspase-3 antibody (Abcam; 1:2 dilution), and active caspase-8 antibody (Cell Signaling Technology, Danvers, Massachusetts, USA; 1:2 dilution) were used. Negative controls included omission of the primary antibody and use of non-immune IgG or secondary antibody only; in all cases, negative controls showed insignificant staining. Secondary antibodies were from Dako ChemMateTM EnVision System. The horseradish peroxidase substrate was diaminobenzidine. To verify the genotype and treatment, quantitative histology involved standard procedures, and results were confirmed by independent pathologists blinded to genotype or treatment group. Images were viewed and captured by use of a Nikon Labophot 2 microscope equipped with a Sony CCD-Iris/RGB color video camera attached to a computerized imaging system and analyzed by ImagePro Plus 3. (ECLIPSE8i/9i; Nikon, Tokyo, Japan). The expressions of macrophage markers, active caspase3, or active caspase8 were calculated as a proportion of positive area to total tissue area for all measurements of the section. For immunofluorescence, frozen sections from 8 C refrigerator were incubated at room temperature for 3 min, merged in PBS for 5 min, then fixed in 4%PFA for 15 min. Next, those sections were washed with cold PBS (5 min, twice), merged in.3 M glycine for 15 min, and washed again with PBS (5 min, twice). Permeablization was done in.2% PBST for 1 min, then washed in PBS (5 min, twice). Blocking was carried out in 1% goat serum in.1% PBST tween (37 C, 1 hr). The primary antibody was incubated at 4 C for 14 hr, and the secondary antibody (for VCAM-1, Alexa Fluorâ 555 Goat Anti-Rat IgG (H + L) from Invitrogen, California was used) was incubated at room temperature for 1 hr. Immunofluorescence of peritoneal macrophages was carried out as follows: fixed in 4% PFA for 2 min;,permeabilized in.2% PBST for 1 min, then washed in PBS (5 min, twice);,blocked in BSA at room temperature for an hour, incubated with primary antibody at room temperature for an hour, then washed with PBS for five times. Then they were incubated with secondary antibody at room temperature for an hour, counterstained with DAPI, washed in PBS for five times and then observed under microscopy. All images were captured and processed using identical settings in the Zeiss LSM 78 Laser Scanning Confocal Microscope at Xiamen University. TUNEL assay Apoptotic cells in lesions were detected by TUNEL using an ApoAlertâ DNA Fragmentation Assay Kit. Briefly, after de-paraffinizing, tissue sections were pretreated with 2ug/mL proteinase K for 15 min. The specimen was rinsed twice with PBS or water for 5 min each time, then incubated in equilibration buffer for at least 1 s at room temperature. Excess liquid was taped off. After carefully Cell Reports 3, 2 21, January 31, 213 ª213 The Authors S1

13 blotting or aspirating around the section, it was then incubated in the mixture of reaction buffer and TdT enzyme for 37 C 1 hr. The nuclei were stained with DAPI. The cells were viewed under the Laser Scanning Confocal Microscope (Karl Zeiss, Germany) and photographed for analysis. The apoptotic results were calculated as a proportion of TUNEL positive cell number to total lesion area cells number for all measurements of the section. All images were captured and processed using identical settings in the Zeiss LSM 78 Laser Scanning Confocal Microscope at Xiamen University. The TUNEL assay of peritoneal macrophages was carried out with the ApoAlert DNA Fragmentation Assay Kit (Clontech Laboratories, California, USA) according to the manufacturer s instruction. Efferocytosis Assays Efferocytosis was assessed as previously described (Li et al., 26; Thorp et al., 28), but modified a little. Peritoneal macrophages rendered apoptotic by loading with oxldl for 3 days or rendered necrotic by loading with oxldl plus zvad were harvested and marked with Annexin V-EGFP. And then the apoptotic/necrotic macrophages were added to monolayers of unloaded macrophages ( efferocytes ) marked by DAPI at a ratio of 3:1. After 3 min of co-culture, the non-ingested apoptotic/necrotic cells were removed and the ingested apoptotic/necrotic cells were quantified as described (Li et al., 26). Plasma Cholesterol and Triglyceride Measurements After an overnight fast, 1 ml of blood was drawn from anesthetized mice. The concentrations of total cholesterol, LDL-Cholesterol, HDL-Cholesterol and triglycerides in plasma were determined using enzymatic colorimetric assays (Zhongsheng Beikong Bio-technology and Science Inc., Beijing, China) according to the manufacturer s instructions. Bone Marrow Transplantation Bone marrow was harvested from donor mouse femurs and tibias using 27G syringe Needles and flushed with PBS with penicillin (1 IU/ml), streptomycin (1 g/ml) into tubes. The cell pellet was spun down at 12 rpm, supernatant was aspirated and the pellet was hemolyzed with hemolytic buffer for 2min. The cells were then spun down again and supernatant was aspirated; then they were resuspended, counted, and diluted to 1 7 cells/ml in PBS for use. Recipient mice were exposed to lethal X-ray irradiation (7.5Gy), and then transplanted with 2x1 6 cells each mouse by tail vein injection. Membrane Integrity Membrane integrity was assessed using the [ 3 H]adenine release method described by Reid and Mitchinson (Reid and Mitchinson, 1993). Briefly, macrophages were loaded with [2,8-3H]adenine (1 mci/ml, PerkinElmer, Boston, Massachusetts) in culture medium with 5% FBS for 2 hr and washed twice with PBS for 5 min at 37 C. Cells were then incubated for 48 hr. Loss of membrane integrity was measured as the percentage of radioactivity released into the supernatant. Intracellular radioactivity was measured after cell lysis with 1% (by volume) Triton X-1. Radioactivity was determined by scintillation counting. Quantitative Real-Time PCR All the RNA was prepared from peritoneal macrophages incubated with oxldl for 24h using TRIzol reagent (Invitrogen, California). Lower dose of oxldl (5 mg/ml) was used in the experiments for analyzing cytokine or chemokine expression in order to avoid possible cell death induced by the high dose oxldl. Four micrograms of total RNA were reverse-transcribed using the oligo dt. Quantitative real-time PCR was performed using SYBR Green Dye and a PCR instrument (Bio-RAD CFX96Ô Real-time system, Bio-rad, California). The relative abundance of each transcript was calculated from a standard curve of cycle thresholds for serial dilutions of a cdna sample and normalized to GAPDH. Primer sequences are available upon request (Burke-Gaffney et al., 22). RNA and Protein Extraction from Atherosclerotic Lesions for Quantitative PCR and Western Blotting Atherosclerotic lesions were isolated from mouse aortas under anatomical lens and divided into early lesions and advanced lesions by an experienced morphologist. For RNA extraction, lesions were lysed in TRIzol (Invitrogen, California), facilitated by homogenization, and then were subject to RNA extraction according to the manufacturer s instructions. The protein extraction method was adapted from a previous study (Wisniewski et al., 21). Lesions were lysed in 2% SDS,.1 M DTT in.1 M Tris/HCl, ph 7.6. Lysis was facilitated by homogenization using an IKA Ultra Turbax blender at maximum speed for 1 min. After 5 min of incubation in boiling water, the suspensions were sonicated using the Branson SONIFIER 25 (Heinemann, Germany) for 3 s (output control 3.5; duty cycle 2%). Then, the mixture was incubated at 1 C for 5 min. The crude extract was clarified by centrifugation at 16 g at 3 C for 1 min. The supernatant was mixed with loading buffer for Western blotting. Overexpression of RIP3 Raw was transfected with plasmid pbabe-puro-large T and screened with 5ug/mL puromycin for 1 days, then the screened cells were infected with lentivirus expressing either pbobi-vector or pbobi-rip3. 4 days after infection, cells were treated with 15ug/mL oxldl, and cell death was detected with PI and Annexin V double staining after 48 hr. S2 Cell Reports 3, 2 21, January 31, 213 ª213 The Authors

14 Measurement of Intracellular Reactive Oxygen Species and Active Caspase-8 Activity Formation of intracellular ROS was assessed with the redox-sensitive dyes 6-carboxy-2,7 -dichlorodihydrofluorescein (CDC-H 2 F, Sigma-Aldrich, St. Louis, Missouri, USA) as previously described (Asmis and Begley, 23). After stimulation, peritoneal macrophages were incubated for 3min at 37 C with 5mM CDC-H 2 F diacetate di(acethoxymethyl) ester. Fluorescence was measured by FCS. Active caspase-8 activity was detected using a Caspase-Glo 8 assay kit (Promega, Madison, Wisconsin, USA) according to the manufacturer s instructions. Rapid Immunohistochemistry, Laser Capture Micro-dissection, Trace RNA Extraction, and One-Step Quantitative PCR The proximal aorta was embedded in the OCT compound (SAKURA).7um thick sections were cut onto the flat side of the mmi Membrane Slide (Molecular Machines & Industries). Frozen sections were fixed in 7% ethanol for 15 s followed by cold acetone for 5 min. The slides were rinsed with RNase-free water for 3 s. Slides were incubated with rabbit anti-mouse MAC-2 IgG (Santa Cruz Biotechnology, California, USA; 1:1 dilution) for 2 min, washed in RNase-free PBS, and incubated with biotinylated goatanti-rabbit IgG secondary antibody (ZSGB-bio, Beijing China; 1:5 dilution) for 5 min. The slides were washed in PBS, then incubated with alkaline phosphatase enzyme complex (ZSGB-bio, Beijing China; 1:5 dilution) for 5 min. The staining was detected with the Vector Red Alkaline Phosphatase substrate Kit 1 (Vector Labs, Burlingame, California, USA). Nuclei were stained with Harris hematoxylin (Sigma-Aldrich, St. Louis, Missouri, USA). Smooth muscle cells and macrophages were isolated from immune-stained frozen sections by laser micro-dissection with MMI cellcut plus (Molecular Machines & Industries). Non-lesional smooth cells and lesional smooth muscle cells were isolated as well. Lesional macrophages from small, middle, and large-size lesions were also isolated separately. All the RNA was extracted with PicoPure RNA Isolation Kit (Invitrogen, California) according to the manufacturer s instructions. Quantification of RIP3 mrna levels was carried out with iscript One-Step RT-PCR Kit With SYBRâ Green from Bio-Rad according to the manufacturer s instructions. SUPPLEMENTAL REFERENCES Asmis, R., and Begley, J.G. (23). Oxidized LDL promotes peroxide-mediated mitochondrial dysfunction and cell death in human macrophages: a caspase-3- independent pathway. Circ. Res. 92, e2 e29. Burke-Gaffney, A., Brooks, A.V., and Bogle, R.G. (22). Regulation of chemokine expression in atherosclerosis. Vascul. Pharmacol. 38, Li, Y., Gerbod-Giannone, M.C., Seitz, H., Cui, D., Thorp, E., Tall, A.R., Matsushima, G.K., and Tabas, I. (26). Cholesterol-induced apoptotic macrophages elicit an inflammatory response in phagocytes, which is partially attenuated by the Mer receptor. J. Biol. Chem. 281, Reid, V.C., and Mitchinson, M.J. (1993). Toxicity of oxidised low density lipoprotein towards mouse peritoneal macrophages in vitro. Atherosclerosis 98, Thorp, E., Cui, D., Schrijvers, D.M., Kuriakose, G., and Tabas, I. (28). Mertk receptor mutation reduces efferocytosis efficiency and promotes apoptotic cell accumulation and plaque necrosis in atherosclerotic lesions of apoe-/- mice. Arterioscler. Thromb. Vasc. Biol. 28, Tranfield, E.M., van Eeden, S.F., Yatera, K., Hogg, J.C., and Walker, D.C. (21). Ultrastructural changes in atherosclerotic plaques following the instillation of airborne particulate matter into the lungs of rabbits. Can. J. Cardiol. 26, e258 e269. Wisniewski, J.R., Nagaraj, N., Zougman, A., Gnad, F., and Mann, M. (21). Brain phosphoproteome obtained by a FASP-based method reveals plasma membrane protein topology. J. Proteome Res. 9, Yamada, T., Press, M., Vesselinovitch, D., and Wissler, R.W. (1988). Quantitative ultrastructural analysis of coronary atherosclerotic involvement in two macaque species. Exp. Mol. Pathol. 48, Cell Reports 3, 2 21, January 31, 213 ª213 The Authors S3

15 A RIP3 -/- Elastin breaks number NS B *P<.5 5 RIP3 -/- Collagen cotents(%) C RIP3 -/- Fibrous cap thickness(µm) *P<.5 D Body weight (g) Body weight (g) Lipid level (mg/dl) before diet after diet TC LDLC HDLC TG Figure S1. Elastin Break Number, Collagen Content, Fibrous Cap Thickness, Body Weight, and Cholesterol Levels of Female RIP3 +/+ ;Ldlr / and RIP3 / ;Ldlr / Mice, Related to Figure 1 (A) Representative pictures showing elastin break number of lesions from female RIP3 +/+ ;Ldlr / (n = 6) and RIP3 / ;Ldlr / (n = 6) mice; elastin (purple) break number statistics are also shown. Data are expressed as mean ± SEM. NS, no significant difference. (B) Representative pictures showing the collagen (blue) content of female RIP3 +/+ ;Ldlr / (n = 6) and RIP3 / ;Ldlr / (n = 6) mice; collagen content statistics are also shown. Data are expressed as mean ± SEM. *p <.5. (C) Bracketed regions shows fibrous cap thickness of lesions from female RIP3 +/+ ;Ldlr / (n = 6) and RIP3 / ;Ldlr / (n = 6) mice; fibrous cap thickness statistics are also shown. Data are expressed as mean ± SEM. *p <.5. (D) Body weight of female RIP3 +/+ ;Ldlr / (n = 1) and RIP3 / ;Ldlr / (n = 1) mice before and after being fed a 16 week Western diet. The levels of total cholesterol (TC), low density lipoprotein cholesterol (LDLC), high density lipoprotein cholesterol (HDLC), and triglyceride (TG) of female RIP3 +/+ ;Ldlr / (n = 1) and RIP3 / ;Ldlr / (n = 1) mice after being fed a 16 week Western diet. Data are expressed as mean ± SEM. None of the differences between the two groups of mice in these parameters were statistically significant. Scale Bars: 1 mm. S4 Cell Reports 3, 2 21, January 31, 213 ª213 The Authors

16 A 3 *P<.5 B 8 *P<.5 Lesion area (%) 2 1 Total lesion area (μm ) C 1 *P<.5 D 3 1 Total necrotic area (μm ) Body weight (g) 2 1 Lipid level (mg/dl) after diet TC LDLC HDLC TG E % activated caspase3 positive cells NS F % mac-2 positive cells 8 *P< Figure S2. Reduced Lesion and Necrotic Area in RIP3 / ;Apoe / Mice Compared to RIP3 +/+ ;Apoe / Mice after Being Fed a Western-type Diet for 12 Weeks, Related to Figures 1, 3, and 5 (A) Representative images of pinned Sudan-IV stained aortas of female RIP3 +/+ ;Apoe / (n = 9) and RIP3 / ;Apoe / (n = 6) mice and quantification of the surface area occupied by the lesions (% area occupied by the lesions) are shown. Horizontal lines within the data sets represent means. Each symbol represents a measurement from a single mouse. *p <.5. (B) Quantitative analysis of atherosclerotic lesion size in hematoxylin and eosin-stained aortic root sections are from 12 week Western diet-fed female RIP3 +/+ ;Apoe / (n = 6) and RIP3 / ;Apoe / (n = 6) mice. Data are expressed as mean ± SEM. *p <.5. (C) Quantification of anuclear, afibrotic, and eosin-negative necrotic areas of 12 week Western diet-fed female RIP3 +/+ ;Apoe / (n = 6) and RIP3 / ;Apoe / (n = 6) mice. Data are expressed as mean ± SEM. *p <.5. (D) Body weight and levels of total cholesterol (TC), low density lipoprotein cholesterol (LDLC), high density lipoprotein cholesterol (HDLC), and triglyceride (TG) of 12 week Western diet-fed female RIP3 +/+ ;Apoe / (n = 9) and RIP3 / ;Apoe / (n = 6) mice. Data are expressed as mean ± SEM. None of the differences between the two groups of mice in these parameters were statistically significant. (E) Quantitative analysis of aortic root lesional active caspase-3 positive cells from each group is shown. Data are expressed as mean ± SEM. NS, no significant difference. (F) Quantitative analysis of aortic root lesional macrophage infiltration is shown for each group. Data are expressed as mean ± SEM. *p <.5. Cell Reports 3, 2 21, January 31, 213 ª213 The Authors S5

17 Figure S3. Cellular Morphologies within Lesional Intimal Area, Related to Figure 3 (A) Non-lesional aorta intima, which is very thin, comprises of endothelial cells (ECs) lining the surface of lumen (L); the media, separated from the intima by the basement membrane (BM), comprises of multiple sheets of smooth muscle cells (SMC). The middle SMC is in a longitudinal section with an elongated nucleus, the two SMCs in the upper right corner are in the cross section. (B) Lesional intima is infiltrated with SMC and macrophage (Mac) between ECs and basement membrane. Quantitative transmission electron microscopy analysis focused on the lesional intimal area. A foam cell (FC) of macrophage origin is beneath an EC. (C) Three adjacent macrophages containing many vacuoles in the cytoplasm and numerous pseudopods on the cell membranes. The top two macrophages are alive without any sign of apoptosis or necrosis, while the third macrophage is characterized with apoptotic features (cell and nuclear shrinkage, and chromatin condensation). There are long elongated free cholesterol crystals to the right of the SMC. (D) Beneath the EC, a macrophage shows signs of necrosis with a ruptured plasma membrane and no nuclear condensation. A SMC in the cross section lies to the left of the necrotic macrophage. Scale Bar: 5 mm. S6 Cell Reports 3, 2 21, January 31, 213 ª213 The Authors

18 A ctrl oxldl + zvad oxldl RIP3 +/+ RIP3 -/- B ctrl oxldl + zvad oxldl RIP3 +/+ RIP3 -/- C ctrl oxldl + zvad oxldl RIP3 +/+ RIP3 -/- Figure S4. The Absence of Apoptosis Markers in Necrotic Macrophages Treated with oxldl plus zvad, Related to Figure 4 (A) Annexin V/PI double staining of peritoneal macrophages. RIP3 +/+ and RIP3 / macrophages were treated with oxldl for 72 hr or oxldl plus zvad for 16 hr, and then stained with Annexin V and PI. Green: annexin V positive; Red: PI positive. Scale Bar: 1 mm. (B) Active caspase-3 staining of peritoneal macrophages. The cells described in (A) were stained with an antibody for active caspase-3 (red). Blue: DAPI. Scale Bar: 2 mm. (C) The cells described in (A) were subjected to TUNEL assay. Arrows denote the TUNEL positive cells (green). Scale Bar: 2 mm. oxldl plus zvad induced Annexin V/PI double positive cells showed no sign of caspase-3 activation or DNA fragmentation (TUNEL positive). Cell Reports 3, 2 21, January 31, 213 ª213 The Authors S7

19 A Folds up-regulated C MCP1 MCP2 MCP3 MIP1 alpha MIP1 beta TNF IP1 IL1 beta apoptotic MФ apoptotic MФ necrotic MФ () () () Substrates B % efferocytosis E1 A1 E2 A2 E3 A3 E1 A1 E2 A2 E3 A3 RIP1 RIP3 Pro-caspase 8 p43 GAPDH Early lesion Early lesion Advanced lesion Advanced lesion *P<.5 *P<.5 Fold over early lesion (RIP3) Relative RIP3 mrna Figure S5. Cytokine Production and Efferocytosis in Peritoneal Macrophages from RIP3 +/+ and RIP3 / Mice Are the Same; Protein Levels of RIP1, Pro-Caspase-8, Cleaved Caspase-8 Are the Same in Both Early and Advanced Lesions; RIP3 Level Is Higher in Advanced Lesion, Related to Figures 5, 6, and 7 (A) Relative expression levels of chemokine mrna in peritoneal macrophages from RIP3 +/+ and RIP3 / mice treated with or without oxldl (5ug/mL, 8 hr) were measured by QRT-PCR. Chemokine gene expression was normalized to the expression of GAPDH. The folds of the upregulation of mrna level were calculated by comparing samples treated with or without oxldl. Data are presented as means ± SEM. None of the differences shown were statistically significant. (B) Efferocytosis assays were conducted using various combinations of peritoneal macrophages from RIP3 +/+ and RIP3 / mice, where the macrophages served as the source of the apoptotic cells, necrotic cells, or the efferocytes. Apoptotic macrophages were obtained by treating RIP3 +/+ and RIP3 / peritoneal macrophages with oxldl for 3 days, and necrotic macrophages were obtained by treating RIP3 +/+ peritoneal macrophages with oxldl plus zvad for 12 hr. Data are expressed as mean ± SEM. (C) Protein levels of RIP1, RIP3, pro-caspase-8, and cleaved caspase 8 (p43 fragment) in early or advanced lesions from RIP3 +/+ ;Ldlr / and RIP3 / ;Ldlr / mice. Lesions from each individual mouse were separated into early or advanced lesions. Each lane is a mixed sample from three individual mice. E1 and A1 (E2 and A2, E3 and A3) are early and advanced lesions, respectively. Protein extracts from the lesions were analyzed by Western blot. Densitometric quantification is shown for RIP3 level in early and advanced lesions from RIP3 +/+ ;Ldlr / mice. RIP3 mrna levels in both early and advanced lesions were also determined by QRT-PCR. Data are presented as mean ± SEM. *p <.5. S8 Cell Reports 3, 2 21, January 31, 213 ª213 The Authors

20 Figure S6. Laser Capture Micro-dissection of Smooth Muscle Cells and Macrophages, Related to Figure 7 (A) Representative pictures showing isolation of macrophages from lesions of different sizes (small, middle, and large). Macrophages from the small-sized lesion (panels a and b); macrophages from the middle-sized lesion (panels c and d); macrophages from the large-sized lesion (panels e and f). (B) Representative pictures showing isolation of smooth muscle cells from non-lesional (panels a and b) and lesional areas (panels c and d). Lesion sections were first immuno-stained with rabbit anti-mouse MAC-2 (red) and then with Harris hematoxylin for nuclei. Black arrows indicate the isolated areas. Scale Bar: 1 mm. Cell Reports 3, 2 21, January 31, 213 ª213 The Authors S9