Objective-To investigate whether high-density lipoproteins (HDLs) suppress chemokine (CCL2, CCL5, and CX 3 CL1) and chemokine receptor (CCR2 and CX 3 CR1) expression, a mechanism for the atheroprotective properties of HDLs. Methods and Results-Apolipoprotein (apo) EϪ/Ϫ mice were fed a high-fat diet for 12 weeks. Before being euthanized, the mice received 5 consecutive daily injections of lipid-free apoA-I, 40 mg/kg, or saline (control). The injection of apoA-I reduced CCR2 and CX 3 CR1 expression in plaques compared with controls (PϽ0.05). ApoA-I-injected mice had lower plasma CCL2 and CCL5 levels. Hepatic CCL2, CCL5, and CX 3 CL1 levels were also reduced (PϽ0.05). In vitro studies found that reconstituted HDL (rHDL) reduced monocyte CCR2 and CX 3 CR1 expression and inhibited their migration toward CCL2 and CX 3 CL1 (PϽ0.05). Preincubation with rHDL reduced CCL2, CCL5, and CX 3 CL1 expression in monocytes and human coronary artery endothelial cells. The stimulation of CX 3 CR1 with peroxisome proliferator-activated receptor ␥ agonist CAY10410 was suppressed by preincubation with rHDL but did not affect the peroxisome proliferator-activated receptor ␥ antagonist (GW9664)-mediated increase in CCR2. In monocytes and human coronary artery endothelial cells, rHDL reduced the expression of the nuclear p65 subunit, IB kinase activity, and the phosphorylation of IB␣ (PϽ0.05). Key Words: high-density lipoproteins Ⅲ inflammation Ⅲ chemokines Ⅲ chemokine receptors Ⅲ atherosclerosis H igh-density lipoproteins (HDLs) are atheroprotective. 1 Human and animal intervention studies have found that infusion of reconstituted HDL (rHDL) or overexpression of apolipoprotein (apo) A-I reduces atherosclerotic plaque size 2 and macrophage and lipid content. [3][4][5][6] The mechanisms for this have been predominantly attributed to reverse cholesterol transport. However, HDLs also have potent anti-inflammatory properties. 7 For example, HDLs reduce endothelial expression of adhesion molecules, such as vascular cell adhesion molecule-1 and intercellular adhesion molecule-1. This project seeks to investigate if HDLs also reduce the expression of chemokines and their receptors, which are key mediators of inflammatory processes and atherosclerotic lesion development. Conclusion-Lipid-freeIn the early stages of atherosclerosis, chemokines and chemokine receptors are critical for the recruitment of circulating monocytes to the endothelium and assist in their migration into the artery wall. 8 The monocytes differentiate into macrophages and express more chemokines, thereby exacerbating the disease process. 8 The importance of the chemokine receptors (CCR2 and CX 3 CR1) and the chemokines (CCL2 [monocyte chemotactic protein-1], CCL5 [regulated upon activation, normal T-cell expressed, and secreted (RANTES)], and CX 3 CL1 [fractalkine]) in atherosclerosis has been demonstrated in human studies and animal knockout models. For example, CCR2Ϫ/Ϫ and CX 3 CR1 Ϫ/Ϫ mice, crossed with apoE Ϫ/Ϫ mice, develop smaller atherosclerotic plaques 9 -11 ; and ...

Key Points• ADAM10 but not ADAM17 on leukocytes is essential for chemokine-induced signaling, adhesion, cytoskeletal rearrangement, and migration.• Leukocyte-expressed ADAM10 promotes leukocyte recruitment and edema formation in a murine model of acute pulmonary inflammation.Inflammation is a key process in various diseases, characterized by leukocyte recruitment to the inflammatory site. This study investigates the role of a disintegrin and a metalloproteinase (ADAM) 10 and ADAM17 for leukocyte migration in vitro and in a murine model of acute pulmonary inflammation. Inhibition experiments or RNA knockdown indicated that monocytic THP-1 cells and primary human neutrophils require ADAM10 but not ADAM17 for efficient chemokine-induced cell migration. Signaling and adhesion events that are linked to cell migration such as p38 and r GTPase-family activation, F-actin polymerization, adhesion to fibronectin, and upregulation of a 5 integrin were also dependent on ADAM10 but not ADAM17. This was confirmed with leukocytes isolated from mice lacking either ADAM10 or ADAM17 in all hematopoietic cells (vav 1 guanine nucleotide exchange factor [Vav]-Adam10 2/2 or Vav-Adam17 2/2 mice). In lipopolysaccharide-induced acute pulmonary inflammation, alveolar recruitment of neutrophils and monocytes was transiently increased in Vav-Adam17 2/2 but steadily reduced in Vav-Adam10 2/2 mice. This deficit in alveolar leukocyte recruitment was also observed in LysM-Adam10 2/2 mice lacking ADAM10in myeloid cells and correlated with protection against edema formation. Thus, with regard to leukocyte migration, leukocyteexpressed ADAM10 but not ADAM17 displays proinflammatory activities and may therefore serve as a target to limit inflammatory cell recruitment. (Blood. 2014;123(26):4077-4088)

ObjectiveTo investigate therapeutic effects of annexin A1 (anxA1) on atherogenesis in LDLR-/- mice.MethodsHuman recombinant annexin A1 (hr-anxA1) was produced by a prokaryotic expression system, purified and analysed on phosphatidylserine (PS) binding and formyl peptide receptor (FPR) activation. Biodistribution of 99mTechnetium-hr-anxA1 was determined in C57Bl/6J mice. 12 Weeks old LDLR-/- mice were fed a Western Type Diet (WTD) during 6 weeks (Group I) or 12 weeks (Group P). Mice received hr-anxA1 (1 mg/kg) or vehicle by intraperitoneal injection 3 times per week for a period of 6 weeks starting at start of WTD (Group I) or 6 weeks after start of WTD (Group P). Total aortic plaque burden and phenotype were analyzed using immunohistochemistry.ResultsHr-anxA1 bound PS in Ca2+-dependent manner and activated FPR2/ALX. It inhibited rolling and adherence of neutrophils but not monocytes on activated endothelial cells. Half lives of circulating 99mTc-hr-anxA1 were <10 minutes and approximately 6 hours for intravenously (IV) and intraperitoneally (IP) administered hr-anxA1, respectively. Pharmacological treatment with hr-anxA1 had no significant effect on initiation of plaque formation (-33%; P = 0.21)(Group I) but significantly attenuated progression of existing plaques of aortic arch and subclavian artery (plaque size -50%, P = 0.005; necrotic core size -76% P = 0.015, hr-anxA1 vs vehicle) (Group P).ConclusionHr-anxA1 may offer pharmacological means to treat chronic atherogenesis by reducing FPR-2 dependent neutrophil rolling and adhesion to activated endothelial cells and by reducing total plaque inflammation.

The microbiota has been linked to the development of atherosclerosis, but the functional impact of these resident bacteria on the lesion size and cellular composition of atherosclerotic plaques in the aorta has never been experimentally addressed with the germ-free low-density lipoprotein receptor-deficient (Ldlr −/-) mouse atherosclerosis model. Here, we report that 16 weeks of high-fat diet (HFD) feeding of hypercholesterolemic Ldlr −/mice at germ-free (GF) housing conditions did not impact relative aortic root plaque size, macrophage content, and necrotic core area. Likewise, we did not find changes in the relative aortic arch lesion size. However, late atherosclerotic GF Ldlr −/mice had altered inflammatory plasma protein markers and reduced smooth muscle cell content in their atherosclerotic root plaques relative to CONV-R Ldlr −/mice. Neither absolute nor relative aortic root or aortic arch plaque size correlated with age. Our analyses on GF Ldlr −/mice did not reveal a significant contribution of the microbiota in late aortic atherosclerosis.