Objective-Atherosclerosis is an inflammatory disease characterized by innate and adaptive immune responses. We investigated the role of B cells and antibodies in the development of atherosclerosis in low density lipoprotein (LDL) receptor-deficient (LDLR Ϫ/Ϫ ) mice. Methods and Results-Using wild-type and B cell-deficient mice as bone marrow donors, we were able to generate LDLR Ϫ/Ϫ mice that possessed Ͻ1.0% of their normal B cell population. B cell-deficient LDLR Ϫ/Ϫ mice on a Western diet showed marked decreases in total serum antibody and anti-oxidized LDL antibody. B cell deficiency was associated with a 30% to 40% increase in the lesion area in the proximal and distal aortas. Real-time reverse transcription-polymerase chain reaction and enzyme-linked immunospot analyses showed a decrease in proatherogenic (interferon-␥) and antiatherogenic (interleukin-10 and transforming growth factor-␤) cytokine mRNA and a decrease in interleukin-4 -and interferon-␥-producing cells. Additionally, we observed a decrease in splenocyte proliferation to oxidized LDL in the B cell-deficient LDLR Ϫ/Ϫ mice, suggesting that B lymphocytes may play a role in the presentation of lipid antigen. Conclusions

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Abstract-Macrophage low-density lipoprotein receptor-related protein (LRP) mediates internalization of remnant lipoproteins, and it is generally thought that blocking lipoprotein internalization will reduce foam cell formation and atherogenesis. Therefore, our study examined the function of macrophage LRP in atherogenesis. We generated transgenic mice that specifically lack macrophage LRP through Cre/lox recombination. Transplantation of macrophage LRP Ϫ/Ϫ bone marrow into lethally irradiated female LDLR Ϫ/Ϫ recipient mice resulted in a 40% increase in atherosclerosis. The difference in atherosclerosis was not caused by altered serum lipoprotein levels. Furthermore, deletion of macrophage LRP decreased uptake of 125 I-very-low-density lipoprotein compared with wild-type cells in vitro. The increase in atherosclerosis was accompanied by increases in monocyte chemoattractant protein type-1, tumor necrosis factor-␣, and proximal aorta macrophage cellularity. We also found that deletion of macrophage LRP increases matrix metalloproteinase-9. This increase in matrix metalloproteinase-9 was associated with a higher frequency of breaks in the elastic lamina. Contrary to what was found with other lipoprotein receptors, deletion of LRP increases atherogenesis in hypercholesterolemic mice. Our data support the hypothesis that macrophage LRP modulates atherogenesis through regulation of inflammatory responses. (Circ Res. 2007;100:670-677.) Key Words: low-density lipoprotein receptor-related protein Ⅲ atherosclerosis Ⅲ lipoproteins Ⅲ metalloproteinase Ⅲ macrophage F irst defined as a complex for removal of ␣ 2 -macroglobulin, 1,2 and then later identified as the lowdensity lipoprotein receptor (LDLR)-related protein (LRP), 3 LRP is a 600-kDa membrane receptor linked to numerous cellular functions and intracellular signaling events. 4 It has multiple extracellular ligands, including apolipoprotein E (apoE), lipoprotein lipase, plasma proteases (urokinase-type and tissue plasminogen activators), fibrinolytic factors (IXa and VIIIa), thrombospondin 1 and 2, and chaperone proteins receptor associated protein and heat shock protein-96 (reviewed elsewhere 5 ). The cytoplasmic tail of LRP binds to multiple intracellular adapter and scaffold proteins including disabled-1 and FE65. 6,7 LRP is present in numerous cell types, including macrophages and hepatocytes, and its systemic expression is essential for embryonic development. 8 A fundamental role for hepatic LRP in the clearance of plasma remnants has been demonstrated, as conditional hepatic LRP deletion results in increased plasma triglyceride and chylomicron levels, particularly in the absence of the LDLR. 9 Furthermore, decreased expression of hepatic LRP causes delayed chylomicron remnant clearance, supporting a protective effect of hepatic LRP on atherogenesis via reduced plasma lipoprotein burden. 10 Besides the effect on lipoprotein remnants, hepatic LRP may provide additional vascular protection by mediating the clearance of other proinflammatory ligands including matrix...

Proprotein Convertase Subtilisin/Kexin type 9 (PCSK9) promotes atherosclerosis by increasing low-density lipoprotein (LDL) cholesterol levels through degradation of hepatic LDL receptors (LDLR). Studies have described the systemic effects of PCSK9 on atherosclerosis, but whether PCSK9 has local and direct effects on the plaque in 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 for 8 wk. 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 MPM from transgenic mice express both murine (m) Pcsk9 and hPCSK9 and that the latter reduces macrophage LDLR and LRP1 surface levels. hPCSK9 was detected in 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.

The immune system is complex, with multiple layers of regulation that serve to prevent the production of self-antigens. One layer of regulation involves regulatory T cells (Tregs) that play an essential role in maintaining peripheral self-tolerance. Patients with autoimmune diseases such as systemic lupus erythematosus and rheumatoid arthritis have decreased levels of HDL, suggesting that apoA-I concentrations may be important in preventing autoimmunity and the loss of self-tolerance. In published studies, hypercholesterolemic mice lacking HDL apoA-I or LDLr ؊/؊ , apoA-I ؊/؊ (DKO), exhibit characteristics of autoimmunity in response to an atherogenic diet. This phenotype is characterized by enlarged cholesterol-enriched lymph nodes (LNs), as well as increased T cell activation, proliferation, and the production of autoantibodies in plasma. In this study, we investigated whether treatment of mice with lipid-free apoA-I could attenuate the autoimmune phenotype. To do this, DKO mice were first fed an atherogenic diet containing 0.1% cholesterol, 10% fat for 6 weeks, after which treatment with apoA-I was begun. Subcutaneous injections of 500 g of lipid-free apoA-I was administered every 48 h during the treatment phase. These and control mice were maintained for an additional 6 weeks on the diet. At the end of the 12-week study, DKO mice showed decreased numbers of LN immune cells, whereas Tregs were proportionately increased. Accompanying this increase in Tregs was a decrease in the percentage of effector/effector memory T cells. Furthermore, lipid accumulation in LN and skin was reduced. These results suggest that treatment with apoA-I reduces inflammation in DKO mice by augmenting the effectiveness of the LN Treg response.

Objective-The purpose of this study was to determine the effects of an atherogenic diet on immune function in LDLr Ϫ/Ϫ , ApoA-I Ϫ/Ϫ mice. Methods and Results-When LDLrϪ/Ϫ , ApoA-I Ϫ/Ϫ (DKO), and LDLr Ϫ/Ϫ (SKO) mice were fed an atherogenic diet, DKO had larger peripheral lymph nodes (LNs) and spleens compared to SKO mice. LNs were enriched in cholesterol and contain expanded populations of T, B, dendritic cells, and macrophages. Expansion of all classes of LN cells was accompanied by a Ϸ1.5-fold increase in T cell proliferation and activation. Plasma antibodies to dsDNA, ␤2-glycoprotein I, and oxidized LDL were increased in DKO, similar to levels in diet-fed Fas lpr/lpr mice, suggesting the development of an autoimmune phenotype. Both LN enlargement and cellular cholesterol expansion were "prevented" when diet-fed DKO mice were treated with helper dependent adenovirus expressing apoA-I. Independent of the amount of dietary cholesterol, DKO mice consistently showed lower plasma cholesterol than SKO mice, yet greater aortic cholesterol deposition and inflammation. Conclusions-ApoA-I prevented cholesterol-associated lymphocyte activation and proliferation in peripheral LN of diet-fed DKO mice. A Ϸ1.5-fold increase in T cell activation and proliferation was associated with a Ϸ3-fold increase in concentrations of circulating autoantibodies and Ϸ2-fold increase in the severity of atherosclerosis suggesting a common link between plasma apoA-I, inflammation, and atherosclerosis.

Background-Atherosclerosis is a disease marked by lipid accumulation and inflammation. Recently, atherosclerosis has gained recognition as an autoimmune-type syndrome characterized by increased activation of the innate and acquired immune systems. Natural killer T (NKT) cells have characteristics of both conventional T cells and NK cells and recognize glycolipid antigens presented in association with CD1d molecules on antigen-presenting cells. The capacity of NKT cells to respond to lipid antigens and modulate innate and acquired immunity suggests that they may play a role in atherogenesis. Methods and Results-We examined the role of NKT cells in atherogenesis and how the atherosclerotic environment affects the NKT cell population itself. The data show that CD1d-deficiency in male apolipoprotein E-deficient (apoE 0 ) mice results in reduction in atherosclerosis, and treatment of apoE 0 mice with ␣-galactosylceramide, a potent and specific NKT cell activator, results in a 2-fold increase in atherosclerosis. Interestingly, we demonstrate that ␣-galactosylceramide-induced interferon-␥ responses and numbers of NKT cells in apoE 0 mice show age-dependent qualitative and quantitative differences as compared with age-matched wild-type mice. Conclusions-Collectively, these findings reveal that hyperlipidemia and atherosclerosis have significant effects on NKT cell responses and that these cells are proatherogenic. ϩ or CD4 Ϫ CD8 Ϫ and express varying levels of CD161 (NK1.1 in mice). NKT cells are present in both humans and mice, are found in lymphoid organs and tissues, have a restricted T cell receptor (TCR) expression (V␣14-J␣18/V␤8 in mice and V␣24-J␣18/V␤11 in humans), and recognize glycolipid antigens presented in the context of the major histocompatibility complex (MHC) class I-like molecule CD1d. 1,2 Although a physiological ligand for NKT cells is still not known, these cells respond strongly to the marine sponge-derived glycolipid ␣-galactosylceramide (␣-GalCer), 3 which specifically binds to CD1d and selectively activates invariant NKT cells. Once activated, NKT cells rapidly produce large amounts of cytokines, including interleukin (IL)-4 and IL-10, which are associated with an antiinflammatory T helper 2 (Th2) response, and interferon (IFN)-␥ and tumor necrosis factor-␣, which are associated with a proinflammatory Th1 response. Recent studies have shown that activation of NKT cells by in vivo administration of ␣-GalCer, or its synthetic homolog KRN7000, has antimetastatic activities and suppresses inflammation in chronic autoimmune diseases such as type 1 diabetes and experimental autoimmune encephalomyelitis in mice. 3 Because atherosclerosis is a lipid-associated disease and has many characteristics in common with other autoimmune disorders, we hypothesized that NKT cells regulate immunity and progression of lesion growth in the artery wall. This hypothesis is consistent with the recent findings that CD1d is expressed in human atherosclerotic lesions and that numbers of CD4 ϩ NK1.1 ϩ cells producing IL-4 a...

BackgroundElevated cholesterol and triglycerides in blood lead to atherosclerosis and fatty liver, contributing to rising cardiovascular and hepatobiliary morbidity and mortality worldwide.Methods and ResultsA cell‐penetrating nuclear transport modifier (NTM) reduced hyperlipidemia, atherosclerosis, and fatty liver in low‐density lipoprotein receptor‐deficient mice fed a Western diet. NTM treatment led to lower cholesterol and triglyceride levels in blood compared with control animals (36% and 53%, respectively; P<0.005) and liver (41% and 34%, respectively; P<0.05) after 8 weeks. Atherosclerosis was reduced by 63% (P<0.0005), and liver function improved compared with saline‐treated controls. In addition, fasting blood glucose levels were reduced from 209 to 138 mg/dL (P<0.005), and body weight gain was ameliorated (P<0.005) in NTM‐treated mice, although food intake remained the same as that in control animals. The NTM used in this study, cSN50.1 peptide, is known to modulate nuclear transport of stress‐responsive transcription factors such as nuclear factor kappa B, the master regulator of inflammation. This NTM has now been demonstrated to also modulate nuclear transport of sterol regulatory element‐binding protein (SREBP) transcription factors, the master regulators of cholesterol, triglyceride, and fatty acid synthesis. NTM‐modulated translocation of SREBPs to the nucleus was associated with attenuated transactivation of their cognate genes that contribute to hyperlipidemia.ConclusionsTwo‐pronged control of inflammation and dyslipidemia by modulating nuclear transport of their critical regulators offers a new approach to comprehensive amelioration of hyperlipidemia, atherosclerosis, fatty liver, and their potential complications.