Loss of Macrophage Low-Density Lipoprotein Receptor-Related Protein 1 Confers Resistance to the Antiatherogenic Effects of Tumor Necrosis Factor-? Inhibition.
ABSTRACT: Antiatherosclerotic effects of tumor necrosis factor-? (TNF-?) blockade in patients with systemic inflammatory states are not conclusively demonstrated, which suggests that effects depend on the cause of inflammation. Macrophage LRP1 (low-density lipoprotein receptor-related protein 1) and apoE contribute to inflammation through different pathways. We studied the antiatherosclerosis effects of TNF-? blockade in hyperlipidemic mice lacking either LRP1 (M?LRP1(-/-)) or apoE from macrophages.Lethally irradiated low-density lipoprotein receptor (LDLR)(-/-) mice were reconstituted with bone marrow from either wild-type, M?LRP1(-/-), apoE(-/-) or apoE(-/-)/M?LRP1(-/-)(DKO) mice, and then treated with the TNF-? inhibitor adalimumab while fed a Western-type diet. Adalimumab reduced plasma TNF-? concentration, suppressed blood ly6C(hi) monocyte levels and their migration into the lesion, and reduced lesion cellularity and inflammation in both wild-type?LDLR(-/-) and apoE(-/-)?LDLR(-/-) mice. Overall, adalimumab reduced lesion burden by 52% to 57% in these mice. Adalimumab reduced TNF-? and blood ly6C(hi) monocyte levels in M?LRP1(-/-)?LDLR(-/-) and DKO?LDLR(-/-) mice, but it did not suppress ly6C(hi) monocyte migration into the lesion or atherosclerosis progression.Our results show that TNF-? blockade exerts antiatherosclerotic effects that are dependent on the presence of macrophage LRP1.
Project description: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 Ly6C(hi) inflammatory monocytes by 32% compared with controls. Expression of hPCSK9 also increased CD11b- and Ly6C(hi) -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.
Project description: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.We examined atherosclerosis development in mice with specific deletion of macrophage LRP1 (apoE(-/-) M?LRP1(-/-)) and in LDLR(-/-) mice reconstituted with apoE(-/-) M?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?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?LRP1(-/-) versus apoE(-/-) mice. Lesion necrosis was also increased (6 fold) in apoE(-/-) M?LRP1(-/-) versus apoE(-/-) mice. Compared with apoE(-/-) mice, the spleens of apoE(-/-) M?LRP1(-/-) mice contained 1.6- and 2.4-fold more total and Ly6-C(high) monocytes. Finally, there were 3.6- and 2.4-fold increases in Ly6-C(high) and CC-chemokine receptor 2-positive cells in lesions of apoE(-/-) M?LRP1(-/-) versus apoE(-/-) mice, suggesting that accumulation of apoptotic cells enhances lesion development and macrophage content by promoting the recruitment of inflammatory monocytes.Low-density lipoprotein receptor protein 1 exerts antiatherogenic effects via pathways independent of apoE involving macrophage apoptosis and monocyte recruitment.
Project description:1-Methylnicotinamide (MNA), the major endogenous metabolite of nicotinic acid (NicA), may partially contribute to the vasoprotective properties of NicA. Here we compared the antiatherosclerotic effects of MNA and NicA in apolipoprotein E (ApoE)/low-density lipoprotein receptor (LDLR)-deficient mice. ApoE/LDLR(-/-) mice were treated with MNA or NicA (100 mg/kg). Plaque size, macrophages, and cholesterol content in the brachiocephalic artery, endothelial function in the aorta, systemic inflammation, platelet activation, as well as the concentration of MNA and its metabolites in plasma and urine were measured. MNA and NicA reduced atherosclerotic plaque area, plaque inflammation, and cholesterol content in the brachiocephalic artery. The antiatherosclerotic actions of MNA and NicA were associated with improved endothelial function, as evidenced by a higher concentration of 6-keto-prostaglandin F1 ? and nitrite/nitrate in the aortic ring effluent, inhibition of platelets (blunted thromboxane B2 generation), and inhibition of systemic inflammation (lower plasma concentration of serum amyloid P, haptoglobin). NicA treatment resulted in an approximately 2-fold higher concentration of MNA and its metabolites in urine and a 4-fold higher nicotinamide/MNA ratio in plasma, compared with MNA treatment. In summary; MNA displays pronounced antiatherosclerotic action in ApoE/LDLR(-/-) mice, an effect associated with an improvement in prostacyclin- and nitric oxide-dependent endothelial function, inhibition of platelet activation, inhibition of inflammatory burden in plaques, and diminished systemic inflammation. Despite substantially higher MNA availability after NicA treatment, compared with an equivalent dose of MNA, the antiatherosclerotic effect of NicA was not stronger. We suggest that detrimental effects of NicA or its metabolites other than MNA may limit beneficial effects of NicA-derived MNA.
Project description:We investigated atheroprotective properties of apolipoprotein (apo) E beyond its ability to lower plasma cholesterol. We hypothesized that apoE reduces atherosclerosis by decreasing lipid accumulation in circulating monocytes and the inflammatory state of monocytes and the vascular endothelium.We developed mice with spontaneous hyperlipidemia with and without plasma apoE. Hypomorphic apoE mice deficient in low-density lipoprotein receptor (Apoe(h/h)Ldlr(-/-)) were compared to Apoe(-/-)Ldlr(-/-) mice. Despite 4-fold more plasma apoE than WT mice, Apoe(h/h)Ldlr(-/-) mice displayed similar plasma cholesterol as Apoe(-/-) Ldlr(-/-) mice but developed 4-fold less atherosclerotic lesions by 5 months of age. The aortic arch of Apoe(h/h)Ldlr(-/-) mice showed decreased endothelial expression of ICAM-1, PECAM-1, and JAM-A. In addition, Apoe(h/h)Ldlr(-/-) mice had less circulating leukocytes and proinflammatory Ly6C(high) monocytes. These monocytes had decreased neutral lipid content and reduced surface expression of ICAM-1, VLA-4, and L-Selectin. Apoe(h/h)Ldlr(-/-) mice displayed increased levels of apoA1-rich HDL that were potent in promoting cellular cholesterol efflux.Our findings suggest that apoE reduces atherosclerosis in the setting of hyperlipidemia by increasing plasma apoA1-HDL that likely contribute to reduce intracellular lipid accumulation and thereby the activation of circulating leukocytes and the vascular endothelium.
Project description:Hepatic inflammation is associated with the development of insulin resistance, which can perpetuate the disease state and may increase the risk of metabolic syndrome and diabetes. Despite recent advances, mechanisms linking hepatic inflammation and insulin resistance are still unclear. The low-density lipoprotein receptor-related protein 1 (LRP1) is a large endocytic and signaling receptor that is highly expressed in macrophages, adipocytes, hepatocytes, and vascular smooth muscle cells. To investigate the potential role of macrophage LRP1 in hepatic inflammation and insulin resistance, we conducted experiments using macrophage-specific LRP1-deficient mice (macLRP1-/- ) generated on a low-density lipoprotein receptor knockout (LDLR-/- ) background and fed a Western diet. LDLR-/-; macLRP1-/- mice gained less body weight and had improved glucose tolerance compared to LDLR-/- mice. Livers from LDLR-/-; macLRP1-/- mice displayed lower levels of gene expression for several inflammatory cytokines, including Ccl3, Ccl4, Ccl8, Ccr1, Ccr2, Cxcl9, and Tnf, and reduced phosphorylation of GSK3? and p38 MAPK proteins. Furthermore, LRP1-deficient peritoneal macrophages displayed altered cholesterol metabolism. Finally, circulating levels of sFRP-5, a potent anti-inflammatory adipokine that functions as a decoy receptor for Wnt5a, were elevated in LDLR-/-; macLRP1-/- mice. Surface plasmon resonance experiments revealed that sFRP-5 is a novel high affinity ligand for LRP1, revealing that LRP1 regulates levels of this inhibitor of Wnt5a-mediated signaling. Collectively, our results suggest that LRP1 expression in macrophages promotes hepatic inflammation and the development of glucose intolerance and insulin resistance by modulating Wnt signaling.
Project description:Dipeptidyl-peptidase 4 (DPP-4) inhibitors are increasingly used to accomplish glycemic targets in patients with type II diabetes mellitus. Because DPP-4 is expressed in inflammatory cells, we hypothesized that its inhibition will exert favorable effects in atherosclerosis.Male LDLR(-/-) mice (6 weeks) were fed a high-fat diet or normal chow diet for 4 weeks and then randomized to vehicle or alogliptin, a high-affinity DPP-4 inhibitor (40 mg · kg(-1) · d(-1)), for 12 weeks. Metabolic parameters, blood pressure, vascular function, atherosclerosis burden, and indexes of inflammation were obtained in target tissues, including the vasculature, adipose, and bone marrow, with assessment of global and cell-specific inflammatory pathways. In vitro and in vivo assays of DPP-4 inhibition (DPP-4i) on monocyte activation/migration were conducted in both human and murine cells and in a short-term ApoE(-/-) mouse model. DPP-4i improved markers of insulin resistance and reduced blood pressure. DPP-4i reduced visceral adipose tissue macrophage content (adipose tissue macrophages; CD11b(+), CD11c(+), Ly6C(hi)) concomitant with upregulation of CD163. DPP-4 was highly expressed in bone marrow-derived CD11b(+) cells, with DPP-4i downregulating proinflammatory genes in these cells. DPP-4i decreased aortic plaque with a striking reduction in plaque macrophages. DPP-4i prevented monocyte migration and actin polymerization in in vitro assays via Rac-dependent mechanisms and prevented in vivo migration of labeled monocytes to the aorta in response to exogenous tumor necrosis factor-? and DPP-4.DPP-4i exerts antiatherosclerotic effects and reduces inflammation via inhibition of monocyte activation/chemotaxis. These findings have important implications for the use of this class of drugs in atherosclerosis.
Project description:LDL cholesterol (LDL-C) contributes to coronary heart disease. Proprotein convertase subtilisin/kexin type 9 (PCSK9) increases LDL-C by inhibiting LDL-C clearance. The therapeutic potential for PCSK9 inhibitors is highlighted by the fact that PCSK9 loss-of-function carriers exhibit 15-30% lower circulating LDL-C and a disproportionately lower risk (47-88%) of experiencing a cardiovascular event. Here, we utilized pcsk9(-/-) mice and an anti-PCSK9 antibody to study the role of the LDL receptor (LDLR) and ApoE in PCSK9-mediated regulation of plasma cholesterol and atherosclerotic lesion development. We found that circulating cholesterol and atherosclerotic lesions were minimally modified in pcsk9(-/-) mice on either an LDLR- or ApoE-deficient background. Acute administration of an anti-PCSK9 antibody did not reduce circulating cholesterol in an ApoE-deficient background, but did reduce circulating cholesterol (-45%) and TGs (-36%) in APOE*3Leiden.cholesteryl ester transfer protein (CETP) mice, which contain mouse ApoE, human mutant APOE3*Leiden, and a functional LDLR. Chronic anti-PCSK9 antibody treatment in APOE*3Leiden.CETP mice resulted in a significant reduction in atherosclerotic lesion area (-91%) and reduced lesion complexity. Taken together, these results indicate that both LDLR and ApoE are required for PCSK9 inhibitor-mediated reductions in atherosclerosis, as both are needed to increase hepatic LDLR expression.
Project description:After an ischemic stroke, mononuclear phagocytic cells such as microglia, macrophages, and monocytes migrate to the lesion site and coordinate an immune response. Monocytes, which are recruited from the bloodstream after ischemic brain injury, can be categorized into two subsets in mice: inflammatory and patrolling monocytes. Although inflammatory monocytes (Ly6C(hi)) seem to have a protective role in stroke progression, the impact of patrolling monocytes (Ly6C(low)) is unknown. To address the role of Ly6C(low) monocytes in stroke, we generated bone marrow chimeric mice in which their hematopoietic system was replaced by Nr4a1(-/-) cells, allowing the complete and permanent ablation of Ly6C(low) monocytes without affecting the Ly6C(hi) subset. We then subjected adult mice to cerebral hypoxia-ischemia using the Levine/Vannucci model. Functional outcomes after stroke such as body weight change, neurologic score, motor functions and spatial learning were not affected. Moreover, depletion in Ly6C(low) monocytes did not change significantly the total infarct size, cell loss, atrophy, the number, or the activation state of microglia/macrophages at the lesion site. These data suggest that Ly6C(low) patrolling monocytes are redundant in the progression and recovery of ischemic stroke.
Project description:The role of farnesoid X receptor (FXR) in the development of atherosclerosis has been unclear. Here, LDL receptor (LDLR(-/-)) or apolipoprotein E (apoE(-/-)) female or male mice were fed a Western diet and treated with a potent synthetic FXR agonist, WAY-362450. Activation of FXR blocked diet-induced hypertriglyceridemia and elevations of non-HDL cholesterol and produced a near complete inhibition of aortic lesion formation. WAY-362450 also induced small heterodimer partner (SHP) expression and repressed cholesterol 7alpha-hydroxylase (CYP7A1) and sterol 12 alpha-hydroxylase (CYP8B1) expression. To determine if SHP was essential for these protective activities, LDLR(-/-)SHP(-/-) and apoE(-/-)SHP(-/-) mice were similarly treated with WAY-362450. Surprisingly, a notable sex difference was observed in these mice. In male LDLR(-/-)SHP(-/-) or apoE(-/-)SHP(-/-) mice, WAY-362450 still repressed CYP7A1 and CYP8B1 expression by 10-fold and still strongly reduced non-HDL cholesterol levels and aortic lesion area. In contrast, in the female LDLR(-/-)SHP(-/-) or apoE(-/-)SHP(-/-) mice, WAY-362450 only slightly repressed CYP7A1 and CYP8B1 expression and did not reduce non-HDL cholesterol or aortic lesion size. WAY-362450 inhibition of hypertriglyceridemia remained intact in LDLR(-/-) or apoE(-/-) mice lacking SHP of both sexes. These results suggest that activation of FXR protects against atherosclerosis in the mouse, and this protective effect correlates with repression of bile acid synthetic genes, with mechanistic differences between male and female mice.
Project description:Numerous risk factors are responsible for the development of atherosclerosis, for which an increased serum level of low-density lipoprotein cholesterol (LDL-C) is a driving force. By binding to the low-density lipoprotein cholesterol receptor (LDLR) and inducing LDLR degradation, proprotein convertase subtilisin/kexin type 9 (PCSK9) plays a key role in cholesterol homeostasis regulation. The inducement of PCSK9 expression is also an important reason for statin intolerance. The Di'ao Xinxuekang (DXXK) capsule extracted from Dioscorea nipponica Makino is a well-known traditional Chinese herbal medicinal product used in atherosclerotic cardiovascular disease. Although DXXK has been widely used in atherosclerotic cardiovascular treatment for nearly 30 years, studies on the potential mechanisms of the lipid-lowering effect are very limited. The purpose of the present study was to demonstrate the possible involvement of the PCSK9/LDLR signaling pathway in the lipid-lowering and antiatherosclerotic effect of DXXK in high-fat diet-fed ApoE-/- mice. The results showed that DXXK treatment alleviated hyperlipidemia, fat accumulation, and atherosclerosis formation in ApoE-/- mice. Furthermore, changes in the expression of PCSK9 mRNA in liver tissue and the circulating PCSK9 level in ApoE-/- mice were both reversed after DXXK treatment, and upregulation of LDLR in the liver was also detected in the protein level in DXXK-treated mice. Our study is the first to show that DXXK could alleviate lipid disorder and ameliorate atherosclerosis with downregulation of the PCSK9 in high-fat diet-fed ApoE-/- mice, suggesting that DXXK may be a potential novel therapeutic treatment and may support statin action in the treatment of atherosclerosis.