??T cells are prevalent in the proximal aorta and drive nascent atherosclerotic lesion progression and neutrophilia in hypercholesterolemic mice.
ABSTRACT: Unique innate immunity-linked ??T cells have been seen in early human artery lesions, but their role in lesion development has received little attention. Here we investigated whether ??T cells modulate atherogenesis in apolipoprotein E-deficient (ApoE KO) mice. We found that ??T cell numbers were markedly increased in the proximal aorta of ApoE-deficient vs. wild-type mice during early atherogenesis, particularly in the aortic root and arch, where they comprised most of the T cells and lesion progression is most rapid. ??T cells infiltrated intimal lesions in ApoE KO mice, but only the adventitia in wild-type mice, and were more prevalent than CD4+ T cells in early nascent lesions, as evaluated by en face confocal microscopy. These aortic ??T cells produced IL-17, but not IFN-?, analyzed by ex vivo FACS. Furthermore, aortic arch lipid accumulation correlated strongly with abundance of IL-17-expressing splenic ??T cells in individual ApoE KO mice. To investigate the role of these ??T cells in early atherogenesis, we analyzed ApoE/??T double knockout (DKO) compared to ApoE KO mice. We observed reduced early intimal lipid accumulation at sites of nascent lesion formation, both in chow-fed (by 40%) and Western diet-fed (by 44%) ApoE/??T DKO mice. In addition, circulating neutrophils were drastically reduced in these DKO mice on Western diet, while expansion of inflammatory monocytes and splenic Th1 or Th17 lymphocytes was not affected. These data reveal, for the first time, a pathogenic role of ??T cells in early atherogenesis in ApoE KO mice, by mechanisms likely to involve their IL-17 production and induction of neutrophilia. Targeting ??T cells thus might offer therapeutic benefit in atherosclerosis or other inflammatory vascular diseases.
Project description:Atherosclerosis encompasses a conspicuously maladaptive inflammatory response that might involve innate immunity. Here, we compared the role of Toll-like receptor 4 (TLR4) with that of TLR2 in intimal foam cell accumulation and inflammation in apolipoprotein E (ApoE) knockout (KO) mice in vivo and determined potential mechanisms of upstream activation and downstream action.We measured lipid accumulation and gene expression in the lesion-prone lesser curvature of the aortic arch. TLR4 deficiency reduced intimal lipid by ?75% in ApoE KO mice, despite unaltered total serum cholesterol and triglyceride levels, whereas TLR2 deficiency reduced it by ?45%. TLR4 deficiency prevented the increased interleukin-1? (IL-1?) and monocyte chemoattractant protein-1 mRNA levels seen within lesional tissue, and it also lowered serum IL-1? levels. Smooth muscle cells (SMC) were present within the intima of the lesser curvature of the aortic arch at this early lesion stage, and they enveloped and permeated nascent lesions, which consisted of focal clusters of foam cells. Cholesterol enrichment of SMC in vitro stimulated acyl-coenzyme A:cholesterol acyltransferase-1 mRNA expression, cytoplasmic cholesterol ester accumulation, and monocyte chemoattractant protein-1 mRNA and protein expression in a TLR4-dependent manner.TLR4 contributes to early-stage intimal foam cell accumulation at lesion-prone aortic sites in ApoE KO mice, as does TLR2 to a lesser extent. Intimal SMC surround and penetrate early lesions, where TLR4 signaling within them may influence lesion progression.
Project description:Apoptosis signal-regulating kinase 1-interacting protein-1 (AIP1) is a signaling adaptor molecule implicated in stress and apoptotic signaling induced by proinflammatory mediators. However, its function in atherosclerosis has not been established. In the present study, we use AIP1-null (AIP1(-/-)) mice to examine its effect on atherosclerotic lesions in an apolipoprotein E-null (ApoE(-/-)) mouse model of atherosclerosis.ApoE(-/-) control mice developed atherosclerosis in the aortic roots and descending aortas on Western-type diet for 10 weeks, whereas the atherosclerotic lesions are significantly augmented in ApoE(-/-)AIP1(-/-) double knockout (DKO) mice. DKO mice show increases in plasma inflammatory cytokines with no significant alterations in body weight, total cholesterol levels, or lipoprotein profiles. Aortas in DKO mice show increased inflammation and endothelial cell (EC) dysfunction with nuclear factor-?B activity, correlating with increased accumulation of macrophages in the lesion area. Importantly, macrophages from DKO donors are not sufficient to augment inflammatory responses and atherogenesis when transferred to ApoE-KO recipients. Mechanistic studies suggest that AIP1 is highly expressed in aortic EC, but not in macrophages, and AIP1 deletion in EC significantly enhance oxidized low-density lipoprotein-induced nuclear factor-?B signaling, gene expression of inflammatory molecules, and monocyte adhesion, suggesting that vascular EC are responsible for the increased inflammatory responses observed in DKO mice.Our data demonstrate that loss of AIP1 in aortic EC primarily contributes to the exacerbated lesion expansion in the ApoE(-/-)AIP1(-/-) mice, revealing an important role of AIP1 in limiting inflammation, EC dysfunction, and atherosclerosis.
Project description:AIMS:The goal of this study was to determine whether the A1 adenosine receptor (AR) plays a role in atherosclerosis development and to explore its potential mechanisms. METHODS AND RESULTS:Double knockout (DKO) mice, deficient in the genes encoding A1 AR and apolipoprotein E (apoE), demonstrated reduced atherosclerotic lesions in aortic arch (en face), aortic root, and innominate arteries when compared with apoE-deficient mice (APOE-KO) of the same age. Treating APOE-KO with an A1 AR antagonist (DPCPX) also led to a concentration-dependent reduction in lesions. The total plasma cholesterol and triglyceride levels were not different between DKO and APOE-KO; however, higher triglyceride was observed in DKO fed a high-fat diet. DKO also had higher body weights than APOE-KO. Plasma cytokine concentrations (IL-5, IL-6, and IL-13) were significantly lower in DKO. Proliferating cell nuclear antigen expression was also significantly reduced in the aorta from DKO. Despite smaller lesions in DKO, the composition of the innominate artery lesion and cholesterol loading and efflux from bone marrow-derived macrophages of DKO were not different from APOE-KO. CONCLUSION:The A1 AR may play a role in the development of atherosclerosis, possibly due to its pro-inflammatory and mitogenic properties.
Project description:Atherosclerotic plaque rupture is a major cause of myocardial infarction and ischemic stroke. The adhesive strength of the bond between a plaque and the vascular wall, measured as local energy release rate, G, is used for quantitative plaque stability estimation. We tested the hypothesis that adhesive strength varies with plaque composition. Matrix metalloproteinase-12 (MMP12) deficiency was previously reported to alter lesion composition. To estimate G values, peeling experiments are performed on aortic plaques from apolipoprotein E knockout (apoE KO) and apoE MMP12 double knockout (DKO) male mice after 8 months on high-fat diet. For plaques in apoE KO and apoE MMP12 DKO mice, experimental values for G differ significantly (p<0.002) between genotypes, averaging 19.2J/m(2) and 12.1J/m(2), respectively. Histology confirms that plaques delaminate along their interface with the underlying internal elastic lamina (IEL) in both genotypes. Quantitative image analysis of stained tissue sections demonstrates a significant positive correlation (p<0.05) between local collagen content of lesions and G values in both genotypes, indicating that adhesive strength of plaques depends on local collagen content. Surprisingly, macrophage content of aortic plaques is neither significantly correlated with G values nor significantly different between genotypes. The IEL underlying plaques in apoE KO mice is significantly more fragmented (number of breaks and length of breaks) than in apoE MMP12 DKO mice, suggesting that elastin fragmentation also influences adhesion strength of plaques. Overall, our results suggest that plaques adhere more strongly to the underlying IEL in apoE KO mice than in apoE MMP12 DKO mice.
Project description:Hypercholesterolemia is associated with decreased vascular nitric oxide bioavailability and deletion of endothelial nitric oxide synthase (eNOS) markedly accelerates atherosclerosis development in apolipoprotein E knockout (apoE ko) mice. The current study tests whether atheroprotection provided by a lipid lowering therapy with Ezetimibe depends on eNOS.ApoE ko and apoE/eNOS double ko (dko) mice received a high fat diet with or without 0.05% Ezetimibe. Ezetimibe significantly reduced plasma cholesterol concentrations and atherogenic lipoproteins in both genotypes to a similar extent. Moreover, the drug reduced vascular inflammation, as it significantly reduced vascular cell adhesion molecule-1 (VCAM-1) expression and vascular CD14 expression, a marker for mononuclear cell infiltration, in both genotypes. Neither NOS protein expression nor vascular reactivity of aortic rings was changed in apoE ko mice following Ezetimibe treatment. Significant lesion reduction was seen in Ezetimibe-treated male and female apoE ko and apoE/eNOS dko animals (p<or=0.05). Interestingly, the drug-mediated additional atheroprotection in male apoE ko, compared to male eNOS dko mice, suggesting that lipid lowering does provide additional eNOS-dependent atheroprotection in this experimental group.Lipid lowering with Ezetimibe potently reduces atherosclerosis and vascular inflammation independent of eNOS. Moreover, Ezetimibe did not exert any effects on eNOS protein expression or enzyme activity. However, additional atheroprotection by Ezetimibe was observed in eNOS competent apoE ko mice, suggesting that some of the drug's anti-atherosclerotic effects are mediated by the eNOS pathway.
Project description:Macrophages in the arterial intima sustain chronic inflammation during atherogenesis. Under hypercholesterolemic conditions murine Ly6C(high) monocytes surge in the blood and spleen, infiltrate nascent atherosclerotic plaques, and differentiate into macrophages that proliferate locally as disease progresses. Spleen tyrosine kinase (SYK) may participate in downstream signaling of various receptors that mediate these processes. We tested the effect of the SYK inhibitor fostamatinib on hypercholesterolemia-associated myelopoiesis and plaque formation in Apoe(-/-) mice during early and established atherosclerosis. Mice consuming a high cholesterol diet supplemented with fostamatinib for 8 weeks developed less atherosclerosis. Histologic and flow cytometric analysis of aortic tissue showed that fostamatinib reduced the content of Ly6C(high) monocytes and macrophages. SYK inhibition limited Ly6C(high) monocytosis through interference with GM-CSF/IL-3 stimulated myelopoiesis, attenuated cell adhesion to the intimal surface, and blocked M-CSF stimulated monocyte to macrophage differentiation. In Apoe(-/-) mice with established atherosclerosis, however, fostamatinib treatment did not limit macrophage accumulation or lesion progression despite a significant reduction in blood monocyte counts, as lesional macrophages continued to proliferate. Thus, inhibition of hypercholesterolemia-associated monocytosis, monocyte infiltration, and differentiation by SYK antagonism attenuates early atherogenesis but not established disease when local macrophage proliferation dominates lesion progression.
Project description:RATIONALE:Atherosclerosis is, in part, caused by immune and inflammatory cell infiltration into the vascular wall, leading to enhanced inflammation and lipid accumulation in the aortic endothelium. Understanding the molecular mechanisms underlying this disease is critical for the development of new therapies. Our recent studies demonstrate that epsins, a family of ubiquitin-binding endocytic adaptors, are critical regulators of atherogenicity. Given the fundamental contribution lesion macrophages make to fuel atherosclerosis, whether and how myeloid-specific epsins promote atherogenesis is an open and significant question. OBJECTIVE:We will determine the role of myeloid-specific epsins in regulating lesion macrophage function during atherosclerosis. METHODS AND RESULTS:We engineered myeloid cell-specific epsins double knockout mice (LysM-DKO) on an ApoE-/- background. On Western diet, these mice exhibited marked decrease in atherosclerotic lesion formation, diminished immune and inflammatory cell content in aortas, and reduced necrotic core content but increased smooth muscle cell content in aortic root sections. Epsins deficiency hindered foam cell formation and suppressed proinflammatory macrophage phenotype but increased efferocytosis and anti-inflammatory macrophage phenotype in primary macrophages. Mechanistically, we show that epsin loss specifically increased total and surface levels of LRP-1 (LDLR [low-density lipoprotein receptor]-related protein 1), an efferocytosis receptor with antiatherosclerotic properties. We further show that epsin and LRP-1 interact via epsin's ubiquitin-interacting motif domain. ox-LDL (oxidized LDL) treatment increased LRP-1 ubiquitination, subsequent binding to epsin, and its internalization from the cell surface, suggesting that epsins promote the ubiquitin-dependent internalization and downregulation of LRP-1. Crossing ApoE-/-/LysM-DKO mice onto an LRP-1 heterozygous background restored, in part, atherosclerosis, suggesting that epsin-mediated LRP-1 downregulation in macrophages plays a pivotal role in propelling atherogenesis. CONCLUSIONS:Myeloid epsins promote atherogenesis by facilitating proinflammatory macrophage recruitment and inhibiting efferocytosis in part by downregulating LRP-1, implicating that targeting epsins in macrophages may serve as a novel therapeutic strategy to treat atherosclerosis.
Project description:The key initial step in atherogenesis is the subendothelial retention of apolipoprotein B-containing lipoproteins. Acid sphingomyelinase (acid SMase), an enzyme present extracellularly within the arterial wall, strongly enhances lipoprotein retention in model systems in vitro, and retained lipoproteins in human plaques are enriched in ceramide, a product of SMase. We now sought to test a direct causative role for acid SMase in lipoprotein retention and atherogenesis in vivo.We studied atherogenesis and lipoprotein retention in Asm(-/-) versus Asm(+/+) mice on the Apoe(-/-) and Ldlr(-/-) backgrounds. Asm(-/-);Apoe(-/-) mice had a approximately 40% to 50% decrease in early foam cell aortic root lesion area compared with Asm(+/+);Apoe(-/-) mice (P<0.05) despite no difference in plasma cholesterol or lipoproteins. To assay lipoprotein retention in vivo, the two groups of mice were injected with fluorescently labeled Apoe(-/-) lipoproteins. Early foam cell lesions of Asm(-/-);Apoe(-/-) mice showed a striking 87% reduction in lipoprotein trapping (P<0.0001) compared with Asm(+/+);Apoe(-/-) lesions. Similar results were obtained with Ldlr(-/-) mice, including an 81% reduction in lipoprotein retention within Asm(-/-);Ldlr(-/-) lesions compared with Asm(+/+);Ldlr(-/-) lesions (P<0.0005).These findings support a causal role for acid SMase in lipoprotein retention and lesion progression and provides further support for the response-to-retention model of atherogenesis.
Project description:Ecto-nucleotide pyrophosphatase/phosphodiesterase 1 (NPP1) generates inorganic pyrophosphate (PP(i)), a physiologic inhibitor of hydroxyapatite deposition. In a previous study, we found NPP1 expression to be inversely correlated with the degree of atherosclerotic plaque calcification. Moreover, function-impairing mutations of ENPP1, the gene encoding for NPP1, are associated with severe, artery tunica media calcification and myointimal hyperplasia with infantile onset in human beings. NPP1 and PP(i) have the potential to modulate atherogenesis by regulating arterial smooth muscle cell (SMC) differentiation and function, including increase of pro-atherogenic osteopontin (OPN) expression. Hence, this study tested the hypothesis that NPP1 deficiency modulates both atherogenesis and atherosclerotic intimal plaque calcification. Npp1/ApoE double deficient mice were generated by crossing mice bearing the ttw allele of Enpp1 (that encodes a truncation mutation) with ApoE null mice and fed with high-fat/high-cholesterol atherogenic diet. Atherosclerotic lesion area and calcification were examined at 13, 18, 23 and 28 weeks of age. The aortic SMCs isolated from both ttw/ttw ApoE(-/-) and ttw/+ ApoE(-/-) mice demonstrated decreased Opn expression. The 28-week-old ttw/ttw ApoE(-/-) and ttw/+ ApoE(-/-) had significantly smaller atherosclerotic lesions compared with wild-type congenic ApoE(-/-) mice. Only ttw/ttw but not ttw/+ mice developed artery media calcification. Furthermore in ttw/+ mice, there was a tendency towards increased plaque calcification compared to ApoE(-/-) mice without Npp1 deficiency. We conclude that Npp1 promotes atherosclerosis, potentially mediated by Opn expression in ApoE knockout mice.
Project description:We hypothesized that NADPH oxidase 4 (Nox4) is involved in the formation of neointimal atherosclerotic plaques through the migration of smooth muscle cells (SMCs) in response to flagellin. Here, we demonstrate that TLR5-mediated Nox4 activation regulates the migration of SMCs, leading to neointimal plaque formation in atherosclerosis. To investigate the molecular mechanism by which the TLR5-Nox4 cascade mediates SMC migration, we analyzed the signaling cascade in primary vascular SMCs (VSMCs) from wild-type (WT) or Nox4 KO mice. Stimulation of VSMCs from Nox4 KO mice with flagellin failed to induce H2O2 production and Rac activation compared with stimulation of VSMCs from WT mice. Moreover, the migration of Nox4-deficient VSMCs was attenuated in response to flagellin in transwell migration and wound healing assays. Finally, we performed partial carotid artery ligation in ApoE KO and Nox4ApoE DKO mice fed a high-fat diet (HFD) with or without recombinant FliC (rFliC) injection. Injection of rFliC into ApoE KO mice fed a HFD resulted in significantly increased SMC migration into the intimal layer, whereas SMC accumulation was not detected in Nox4ApoE DKO mice. We conclude that activation of the TLR5-Nox4 cascade plays an important role in the formation of neointimal atherosclerotic plaques.