Youmin Zhang

Macrophage LRP-1 Controls Plaque Cellularity by Regulating Efferocytosis and Akt Activation

Objective—The balance between apoptosis susceptibility and efferocytosis of macrophages is central to plaque remodeling and inflammation. LRP-1 and its ligand, apolipoprotein E, have been implicated in efferocytosis and apoptosis in some cell types. We investigated the involvement of the macrophage LRP-1/apolipoprotein E axis in controlling plaque apoptosis and efferocytosis. Method and Results—LRP-1−/− macrophages displayed nearly 2-fold more TUNEL positivity compared to wild-type cells in the presence of DMEM alone or with either lipopolysaccharide or oxidized low-density lipoprotein. The survival kinase, phosphorylated Akt, was barely detectable in LRP-1−/− cells, causing decreased phosphorylated Bad and increased cleaved caspase-3. Regardless of the apoptotic stimulation and degree of cell death, LRP-1−/− macrophages displayed enhanced inflammation with increased IL-1&bgr;, IL-6, and tumor necrosis factor-&agr; expression. Efferocytosis of apoptotic macrophages was reduced by 60% in LRP-1−/− vs wild-type macrophages despite increased apolipoprotein E expression by both LRP-1−/− phagocytes and wild-type apoptotic cells. Compared to wild-type macrophage lesions, LRP-1−/− lesions had 5.7-fold more necrotic core with more dead cells not associated with macrophages. Conclusion—Macrophage LRP-1 deficiency increases cell death and inflammation by impairing phosphorylated Akt activation and efferocytosis. Increased apolipoprotein E expression in LRP-1−/− macrophages suggests that the LRP-1/apolipoprotein E axis regulates the balance between apoptosis and efferocytosis, thereby preventing necrotic core formation.

Local effects of human PCSK9 on the atherosclerotic lesion

Proprotein convertase subtilisin/kexin type 9 (PCSK9) promotes atherosclerosis by increasing low‐density lipoprotein (LDL) cholesterol levels through degradation of hepatic LDL receptor (LDLR). Studies have described the systemic effects of PCSK9 on atherosclerosis, but whether PCSK9 has local and direct effects on the plaque is unknown. To study the local effect of human PCSK9 (hPCSK9) on atherosclerotic lesion composition, independently of changes in serum cholesterol levels, we generated chimeric mice expressing hPCSK9 exclusively from macrophages, using marrow from hPCSK9 transgenic (hPCSK9tg) mice transplanted into apoE−/− and LDLR−/− mice, which were then placed on a high‐fat diet (HFD) for 8 weeks. We further characterized the effect of hPCSK9 expression on the inflammatory responses in the spleen and by mouse peritoneal macrophages (MPM) in vitro. We found that MPMs from transgenic mice express both murine (m) Pcsk9 and hPCSK9 and that the latter reduces macrophage LDLR and LRP1 surface levels. We detected hPCSK9 in the serum of mice transplanted with hPCSK9tg marrow, but did not influence lipid levels or atherosclerotic lesion size. However, marrow‐derived PCSK9 progressively accumulated in lesions of apoE−/− recipient mice, while increasing the infiltration of Ly6Chi inflammatory monocytes by 32% compared with controls. Expression of hPCSK9 also increased CD11b‐ and Ly6Chi‐positive cell numbers in spleens of apoE−/− mice. In vitro, expression of hPCSK9 in LPS‐stimulated macrophages increased mRNA levels of the pro‐inflammatory markers Tnf and Il1b (40% and 45%, respectively) and suppressed those of the anti‐inflammatory markers Il10 and Arg1 (30% and 44%, respectively). All PCSK9 effects were LDLR‐dependent, as PCSK9 protein was not detected in lesions of LDLR−/− recipient mice and did not affect macrophage or splenocyte inflammation. In conclusion, PCSK9 directly increases atherosclerotic lesion inflammation in an LDLR‐dependent but cholesterol‐independent mechanism, suggesting that therapeutic PCSK9 inhibition may have vascular benefits secondary to LDL reduction. Copyright

Low-Density Lipoprotein Receptor–Related Protein 1 Prevents Early Atherosclerosis by Limiting Lesional Apoptosis and Inflammatory Ly-6Chigh Monocytosis Evidence That the Effects Are Not Apolipoprotein E Dependent

Background— We previously demonstrated that macrophage low-density lipoprotein receptor (LDLR)–related protein 1 (LRP1) deficiency increases atherosclerosis despite antiatherogenic changes including decreased uptake of remnants and increased secretion of apolipoprotein E (apoE). Thus, our objective was to determine whether the atheroprotective effects of LRP1 require interaction with apoE, one of its ligands with multiple beneficial effects. Methods and Results— We examined atherosclerosis development in mice with specific deletion of macrophage LRP1 (apoE−/− M&PHgr;LRP1−/−) and in LDLR−/− mice reconstituted with apoE−/− M&PHgr;LRP1−/− bone marrow. The combined absence of apoE and LRP1 promoted atherogenesis more than did macrophage apoE deletion alone in both apoE-producing LDLR−/− mice (+88%) and apoE−/− mice (+163%). The lesions of both mouse models with apoE−/− LRP1−/− macrophages had increased macrophage content. In vitro, apoE and LRP1 additively inhibit macrophage apoptosis. Furthermore, there was excessive accumulation of apoptotic cells in lesions of both LDLR−/− mice (+110%) and apoE−/− M&PHgr;LRP1−/− mice (+252%). The apoptotic cell accumulation was partially due to decreased efferocytosis as the ratio of free to cell-associated apoptotic nuclei was 3.5-fold higher in lesions of apoE−/− M&PHgr;LRP1−/− versus apoE−/− mice. Lesion necrosis was also increased (6 fold) in apoE−/− M&PHgr;LRP1−/− versus apoE−/− mice. Compared with apoE−/− mice, the spleens of apoE−/− M&PHgr;LRP1−/− mice contained 1.6- and 2.4-fold more total and Ly6-Chigh monocytes. Finally, there were 3.6- and 2.4-fold increases in Ly6-Chigh and CC-chemokine receptor 2–positive cells in lesions of apoE−/− M&PHgr;LRP1−/− versus apoE−/− mice, suggesting that accumulation of apoptotic cells enhances lesion development and macrophage content by promoting the recruitment of inflammatory monocytes. Conclusion— Low-density lipoprotein receptor protein 1 exerts antiatherogenic effects via pathways independent of apoE involving macrophage apoptosis and monocyte recruitment.

Macrophage Apolipoprotein E Reduces Atherosclerosis and Prevents Premature Death in Apolipoprotein E and Scavenger Receptor–Class BI Double-Knockout Mice

Objective—Mice null for both apolipoprotein (apo)E and scavenger receptor (SR)-BI (DKO) develop severe hypercholesterolemia, occlusive coronary atherosclerosis, myocardial infarction, and premature death. The current study examines the ability of macrophage apoE to improve the dyslipidemia, reduce atherosclerosis, and rescue the lethal phenotype of DKO mice. Methods and Results—Initially, bone marrow transplantation (BMT) was unsuccessful, because the DKO mice died from a rapidly fatal anemia 3 to 5 days after lethal irradiation. Therefore, probucol was used to rescue the DKO mice during BMT and was discontinued 2-weeks after BMT, allowing successful reconstitution with donor marrow. Twelve male apoE−/−SR-BI−/− mice fed 0.5% probucol in a chow diet were lethally irradiated and transplanted with either wild-type (WT) or DKO bone marrow. Two-weeks after BMT, apoE was detected in serum in WT→DKO mice, and mean serum cholesterol levels were reduced by 70% versus DKO→DKO mice. Lipoprotein profiles and HDL subpopulations in WT→DKO mice were similar to apoE+/+SR-BI−/−→DKO mice and resembled those of SR-BI−/− mice. In WT→DKO mice, aortic atherosclerosis was reduced by 88% to 90% versus DKO→DKO mice. Furthermore, the DKO→DKO mice died ≈8 weeks after BMT, whereas WT→DKO mice exhibited a life span >40 weeks after BMT. Conclusions—Macrophage apoE is able to rescue the lethal phenotype of apoE−/−SR-BI−/− mice by improving the dyslipidemia and dramatically reducing atherosclerotic lesion development.

Macrophage deficiency of Akt2 reduces atherosclerosis in Ldlr null mice

Macrophages play crucial roles in the formation of atherosclerotic lesions. Akt, a serine/threonine protein kinase B, is vital for cell proliferation, migration, and survival. Macrophages express three Akt isoforms, Akt1, Akt2, and Akt3, but the roles of Akt1 and Akt2 in atherosclerosis in vivo remain unclear. To dissect the impact of macrophage Akt1 and Akt2 on early atherosclerosis, we generated mice with hematopoietic deficiency of Akt1 or Akt2. After 8 weeks on Western diet, Ldlr−/− mice reconstituted with Akt1−/− fetal liver cells (Akt1−/−→Ldlr−/−) had similar atherosclerotic lesion areas compared with control mice transplanted with WT cells (WT→Ldlr−/−). In contrast, Akt2−/−→Ldlr−/− mice had dramatically reduced atherosclerotic lesions compared with WT→Ldlr−/− mice of both genders. Similarly, in the setting of advanced atherosclerotic lesions, Akt2−/−→Ldlr−/− mice had smaller aortic lesions compared with WT→Ldlr−/− and Akt1−/−→Ldlr−/− mice. Importantly, Akt2−/−→Ldlr−/− mice had reduced numbers of proinflammatory blood monocytes expressing Ly-6Chi and chemokine C-C motif receptor 2. Peritoneal macrophages isolated from Akt2−/− mice were skewed toward an M2 phenotype and showed decreased expression of proinflammatory genes and reduced cell migration. Our data demonstrate that loss of Akt2 suppresses the ability of macrophages to undergo M1 polarization reducing both early and advanced atherosclerosis.

Macrophage Mal1 Deficiency Suppresses Atherosclerosis in Low-Density Lipoprotein Receptor–Null Mice by Activating Peroxisome Proliferator-Activated Receptor-γ–Regulated Genes

Objective—The adipocyte/macrophage fatty acid-binding proteins aP2 (FABP4) and Mal1 (FABP5) are intracellular lipid chaperones that modulate systemic glucose metabolism, insulin sensitivity, and atherosclerosis. Combined deficiency of aP2 and Mal1 has been shown to reduce the development of atherosclerosis, but the independent role of macrophage Mal1 expression in atherogenesis remains unclear. Methods and Results—We transplanted wild-type (WT), Mal1−/−, or aP2−/− bone marrow into low-density lipoprotein receptor–null (LDLR−/−) mice and fed them a Western diet for 8 weeks. Mal1−/−→LDLR−/− mice had significantly reduced (36%) atherosclerosis in the proximal aorta compared with control WT→LDLR−/− mice. Interestingly, peritoneal macrophages isolated from Mal1-deficient mice displayed increased peroxisome proliferator-activated receptor-&ggr; (PPAR&ggr;) activity and upregulation of a PPAR&ggr;-related cholesterol trafficking gene, CD36. Mal1−/− macrophages showed suppression of inflammatory genes, such as COX2 and interleukin 6. Mal1−/−→LDLR−/− mice had significantly decreased macrophage numbers in the aortic atherosclerotic lesions compared with WT→LDLR−/− mice, suggesting that monocyte recruitment may be impaired. Indeed, blood monocytes isolated from Mal1−/−→LDLR−/− mice on a high-fat diet had decreased CC chemokine receptor 2 gene and protein expression levels compared with WT monocytes. Conclusion—Taken together, our results demonstrate that Mal1 plays a proatherogenic role by suppressing PPAR&ggr; activity, which increases expression of CC chemokine receptor 2 by monocytes, promoting their recruitment to atherosclerotic lesions.

Macrophage SR-BI mediates efferocytosis via Src/PI3K/Rac1 signaling and reduces atherosclerotic lesion necrosis

Macrophage apoptosis and efferocytosis are key determinants of atherosclerotic plaque inflammation and necrosis. Bone marrow transplantation studies in ApoE- and LDLR-deficient mice revealed that hematopoietic scavenger receptor class B type I (SR-BI) deficiency results in severely defective efferocytosis in mouse atherosclerotic lesions, resulting in a 17-fold higher ratio of free to macrophage-associated dead cells in lesions containing SR-BI−/− cells, 5-fold more necrosis, 65.2% less lesional collagen content, nearly 7-fold higher dead cell accumulation, and 2-fold larger lesion area. Hematopoietic SR-BI deletion elicited a maladaptive inflammatory response [higher interleukin (IL)-1β, IL-6, and TNF-α lower IL-10 and transforming growth factor β]. Efferocytosis of apoptotic thymocytes was reduced by 64% in SR-BI−/− versus WT macrophages, both in vitro and in vivo. In response to apoptotic cells, macrophage SR-BI bound with phosphatidylserine and induced Src phosphorylation and cell membrane recruitment, which led to downstream activation of phosphoinositide 3-kinase (PI3K) and Ras-related C3 botulinum toxin substrate 1 (Rac1) for engulfment and clearance of apoptotic cells, as inhibition of Src decreased PI3K, Rac1-GTP, and efferocytosis in WT cells. Pharmacological inhibition of Rac1 reduced macrophage efferocytosis in a SR-BI-dependent fashion, and activation of Rac1 corrected the defective efferocytosis in SR-BI−/− macrophages. Thus, deficiency of macrophage SR-BI promotes defective efferocytosis signaling via the Src/PI3K/Rac1 pathway, resulting in increased plaque size, necrosis, and inflammation.

Lentiviral Transduction of ApoAI Into Hematopoietic Progenitor Cells and Macrophages Applications to Cell Therapy of Atherosclerosis

Objective—We used genetically engineered mouse hematopoietic progenitor cells (HPCs) to investigate the therapeutic effects of human apoAI on atherosclerosis in apoE−/− mice. Methods and Results—Lentiviral constructs expressing either human apoAI (LV-apoAI) or green fluorescent protein (LV-GFP) cDNA under a macrophage specific promoter (CD68) were generated and used for ex vivo transduction of mouse HPCs and macrophages. The transduction efficiency was >25% for HPCs and >70% for macrophages. ApoAI was found in the macrophage culture media, mostly associated with the HDL fraction. Interestingly, a significant increase in mRNA and protein levels for ATP binding cassette A1 (ABCA1) and ABCG1 were found in apoAI-expressing macrophages after acLDL loading. Expression of apoAI significantly increased cholesterol efflux in wild-type and apoE−/− macrophages. HPCs transduced with LV-apoAI ex vivo and then transplanted into apoE−/− mice caused a 50% reduction in atherosclerotic lesion area compared to GFP controls, without influencing plasma HDL-C levels. Conclusions—Lentiviral transduction of apoAI into HPCs reduces atherosclerosis in apoE−/− mice. Expression of apoAI in macrophages improves cholesterol trafficking in wild-type apoE-producing macrophages and causes upregulation of ABCA1 and ABCG1. These novel observations set the stage for a cell therapy approach to atherosclerosis regression, exploiting the cooperation between apoE and apoAI to maximize cholesterol exit from the plaque.

Macrophage IKKα Deficiency Suppresses Akt Phosphorylation, Reduces Cell Survival, and Decreases Early Atherosclerosis

Objective— The I&kgr;B kinase (IKK) is an enzyme complex that initiates the nuclear factor &kgr;B transcription factor cascade, which is important in regulating multiple cellular responses. IKK&agr; is directly associated with 2 major prosurvival pathways, PI3K/Akt and nuclear factor &kgr;B, but its role in cell survival is not clear. Macrophages play critical roles in the pathogenesis of atherosclerosis, yet the impact of IKK&agr; signaling on macrophage survival and atherogenesis remains unclear. Approach and Results— Here, we demonstrate that genetic IKK&agr; deficiency, as well as pharmacological inhibition of IKK, in mouse macrophages significantly reduces Akt S473 phosphorylation, which is accompanied by suppression of mTOR complex 2 signaling. Moreover, IKK&agr; null macrophages treated with lipotoxic palmitic acid exhibited early exhaustion of Akt signaling compared with wild-type cells. This was accompanied by a dramatic decrease in the resistance of IKK&agr;−/− monocytes and macrophages to different proapoptotic stimuli compared with wild-type cells. In vivo, IKK&agr; deficiency increased macrophage apoptosis in atherosclerotic lesions and decreased early atherosclerosis in both female and male low-density lipoprotein receptor (LDLR)−/− mice reconstituted with IKK&agr;−/− hematopoietic cells and fed with the Western diet for 8 weeks compared with control LDLR−/− mice transplanted with wild-type cells. Conclusions— Hematopoietic IKK&agr; deficiency in mouse suppresses Akt signaling, compromising monocyte/macrophage survival and this decreases early atherosclerosis.

Macrophage IκB Kinase α Deficiency Suppresses Akt Phosphorylation, Reduces Cell Survival, and Decreases Early Atherosclerosis

Objective— The I&kgr;B kinase (IKK) is an enzyme complex that initiates the nuclear factor &kgr;B transcription factor cascade, which is important in regulating multiple cellular responses. IKK&agr; is directly associated with 2 major prosurvival pathways, PI3K/Akt and nuclear factor &kgr;B, but its role in cell survival is not clear. Macrophages play critical roles in the pathogenesis of atherosclerosis, yet the impact of IKK&agr; signaling on macrophage survival and atherogenesis remains unclear. Approach and Results— Here, we demonstrate that genetic IKK&agr; deficiency, as well as pharmacological inhibition of IKK, in mouse macrophages significantly reduces Akt S473 phosphorylation, which is accompanied by suppression of mTOR complex 2 signaling. Moreover, IKK&agr; null macrophages treated with lipotoxic palmitic acid exhibited early exhaustion of Akt signaling compared with wild-type cells. This was accompanied by a dramatic decrease in the resistance of IKK&agr;−/− monocytes and macrophages to different proapoptotic stimuli compared with wild-type cells. In vivo, IKK&agr; deficiency increased macrophage apoptosis in atherosclerotic lesions and decreased early atherosclerosis in both female and male low-density lipoprotein receptor (LDLR)−/− mice reconstituted with IKK&agr;−/− hematopoietic cells and fed with the Western diet for 8 weeks compared with control LDLR−/− mice transplanted with wild-type cells. Conclusions— Hematopoietic IKK&agr; deficiency in mouse suppresses Akt signaling, compromising monocyte/macrophage survival and this decreases early atherosclerosis.