Atherosclerosis is an immunoinflammatory disease elicited by accumulation of lipids in the artery wall and leads to myocardial infarction and stroke. Here, we show that naturally arising CD4(+)CD25(+) regulatory T cells, which actively maintain immunological tolerance to self and nonself antigens, are powerful inhibitors of atherosclerosis in several mouse models. These results provide new insights into the immunopathogenesis of atherosclerosis and could lead to new therapeutic approaches that involve immune modulation using regulatory T cells.

Abstract-The potential role of anti-inflammatory cytokines in the modulation of the atherosclerotic process remains unknown. Interleukin (IL)-10 has potent deactivating properties in macrophages and T cells and modulates many cellular processes that may interfere with the development and stability of the atherosclerotic plaque. IL-10 is expressed in human atherosclerosis and is associated with decreased signs of inflammation. In the present study, we show that IL-10 -deficient C57BL/6J mice fed an atherogenic diet and raised under specific pathogen-free conditions exhibit a significant 3-fold increase in lipid accumulation compared with wild-type mice. Interestingly, the susceptibility of IL-10 -deficient mice to atherosclerosis was exceedingly high (30-fold increase) when the mice were housed under conventional conditions. Atherosclerotic lesions of IL-10 -deficient mice showed increased T-cell infiltration, abundant interferon-␥ expression, and decreased collagen content. In vivo, transfer of murine IL-10 achieved 60% reduction in lesion size. These results underscore the critical roles of IL-10 in both atherosclerotic lesion formation and stability. Moreover, IL-10 appears to be crucial as a protective factor against the effect of environmental pathogens on atherosclerosis. The full text of this article is available at http://www.circresaha.org. (Circ Res. 1999;85:e17-e24.)

Background-Monocytes are critical mediators of atherogenesis. Deletion of individual chemokines or chemokine receptors leads to significant but only partial inhibition of lesion development, whereas deficiency in other signals such as CXCL16 or CCR1 accelerates atherosclerosis. Evidence that particular chemokine pathways may cooperate to promote monocyte accumulation into inflamed tissues, particularly atherosclerotic arteries, is still lacking. Methods and Results-Here, we show that chemokine-mediated signals critically determine the frequency of monocytes in the blood and bone marrow under both noninflammatory and atherosclerotic conditions. Particularly, CCL2-, CX3CR1-, and CCR5-dependent signals differentially alter CD11b ϩ Ly6G Ϫ 7/4 hi (also known as Ly6C hi ) and CD11b ϩ Ly6G Ϫ 7/4 lo (Ly6C lo ) monocytosis. Combined inhibition of CCL2, CX3CR1, and CCR5 in hypercholesterolemic, atherosclerosis-susceptible apolipoprotein E-deficient mice leads to abrogation of bone marrow monocytosis and to additive reduction in circulating monocytes despite persistent hypercholesterolemia. These effects are associated with a marked and additive 90% reduction in atherosclerosis. Interestingly, lesion size highly correlates with the number of circulating monocytes, particularly the CD11b ϩ Ly6G Ϫ 7/4 lo subset. Conclusions-CCL2, CX3CR1, and CCR5 play independent and additive roles in atherogenesis. Signals mediated through these pathways critically determine the frequency of circulating monocyte subsets and thereby account for almost all macrophage accumulation into atherosclerotic arteries. (Circulation. 2008;117:1649-1657.)

B cell depletion significantly reduces the burden of several immune-mediated diseases. However, B cell activation has been until now associated with a protection against atherosclerosis, suggesting that B cell–depleting therapies would enhance cardiovascular risk. We unexpectedly show that mature B cell depletion using a CD20-specific monoclonal antibody induces a significant reduction of atherosclerosis in various mouse models of the disease. This treatment preserves the production of natural and potentially protective anti–oxidized low-density lipoprotein (oxLDL) IgM autoantibodies over IgG type anti-oxLDL antibodies, and markedly reduces pathogenic T cell activation. B cell depletion diminished T cell–derived IFN-γ secretion and enhanced production of IL-17; neutralization of the latter abrogated CD20 antibody–mediated atheroprotection. These results challenge the current paradigm that B cell activation plays an overall protective role in atherogenesis and identify new antiatherogenic strategies based on B cell modulation.

Abstract-Atherosclerosis is a disease of the arterial wall that seems to be tightly modulated by the local inflammatory balance. Whereas a large body of evidence supports a role for proinflammatory mediators in disease progression, the understanding of the role of the antiinflammatory component in the modulation of plaque progression is only at its beginning. TGF-␤1, -␤2, and -␤3 are cytokines/growth factors with broad activities on cells and tissues in the cardiovascular system and have been proposed to play a role in the pathogenesis of atherosclerosis. However, no study has examined the direct role of TGF-␤ in the development and composition of advanced atherosclerotic lesions. In the present study, we show that inhibition of TGF-␤ signaling using a neutralizing anti-TGF-␤1, -␤2, and -␤3 antibody accelerates the development of atherosclerotic lesions in apoE-deficient mice. Moreover, inhibition of TGF-␤ signaling favors the development of lesions with increased inflammatory component and decreased collagen content. These results identify a major protective role for TGF-␤ in atherosclerosis.

Background-Fractalkine (CX3CL1), a CX3C chemokine, is expressed in the vessel wall and mediates the firm adhesion and chemotaxis of leukocytes expressing its receptor, CX3CR1. A polymorphism in the CX3CR1 gene is associated with low CX3CR1 expression and reduced risk of acute coronary disease in humans. Methods and Results-We generated CX3CR1-deficient mice (CX3CR1 Ϫ/Ϫ ) by targeted gene disruption and crossed them with the proatherogenic apolipoprotein E-deficient mice (apoE Ϫ/Ϫ ). Here we show that the extent of lipid-stained lesions in the thoracic aorta was reduced by 59% in CX3CR1/apoE double knockout mice compared with their CX3CR1 ϩ/ϩ / apoE Ϫ/Ϫ littermates. The development of atherosclerosis in the aortic sinus was also markedly altered in the double knockout mice, with 50% reduction in macrophage accumulation. Although lesions of CX3CR1 Ϫ/Ϫ mice were smaller in size, they retained a substantial accumulation of smooth muscle cells and collagen, features consistent with a stable plaque phenotype. Finally, CX3CR1 ϩ/Ϫ /apoE Ϫ/Ϫ mice showed the same reduction in atherosclerosis as the CX3CR1 Ϫ/Ϫ / apoE Ϫ/Ϫ mice. Conclusions-The CX3CR1-CX3CL1 pathway seems to play a direct and critical role in monocyte recruitment and atherosclerotic lesion development in a mouse model of human atherosclerosis.

Flow-induced changes in vessel caliber tend to restore baseline wall shear stress (WSS) and have been reported to be endothelium-dependent. To investigate the role of endothelium-derived nitric oxide (NO) in the adaptive increase in artery diameter in response to a chronic increase in blood flow, an arteriovenous fistula was constructed between the left common carotid artery (CCA) and the external jugular vein in 22 New Zealand White rabbits, and NO synthesis was inhibited in 14 animals by long-term administration of NG-nitro-L-arginine-methyl ester (L-NAME) in drinking water given for 4 weeks. The remaining 8 animals served as controls. Mean arterial blood pressure was not significantly altered by L-NAME treatment (91 +/- 2 in control versus 98 +/- 3 mm Hg in L-NAME-treated rabbits). Blood flow significantly increased in the left CCA in both groups but was lower in L-NAME-treated than control animals (106.1 +/- 10.7 versus 196.2 +/- 32.3 mL/min, P < .003). The diameter of the flow-loaded left CCA also increased significantly in both groups compared with the right CCA (2.15 +/- 0.12 and 2.54 +/- 0.1 mm, respectively, P < .02), but the increase was less in the L-NAME-treated than the control group (3.24 +/- 0.09 and 4.64 +/- 0.17 mm, respectively, P < .0001). The diameter of the anastomosed veins was also increased but to a much lesser degree in L-NAME-treated animals than in controls (4.14 +/- 0.29 versus 7.94 +/- 0.51 mm, P < .0001). As a result of artery enlargement, WSS was normalized in the flow-loaded left CCA of the control group (8.87 +/- 0.77 dynes/cm2) regardless of blood flow values. In L-NAME-treated animals, however, WSS was only partially regulated, the mean value being significantly increased (18.7 +/- 2.2 dynes/cm2, P < .006). Moreover, a highly significant positive correlation between WSS and blood flow was obtained in L-NAME-treated animals (r = .84, P < .0001). We also found remodeling of the artery wall, with a larger increase in the medial cross-sectional area associated with an increased number of smooth muscle cells, in the control group compared with the L-NAME-treated group (0.75 +/- 0.09 versus 0.49 +/- 0.04 mm2 and 4504 +/- 722 versus 2717 +/- 282 cells/mm2, P < .03). We conclude that NO plays a role in the increase of vessel caliber in response to chronic increase in blood flow. As yet unidentified additional metabolic processes appear to be necessary for a complete regulatory response.

Rationale for Study: MicroRNAs (miRNAs) are small noncoding RNAs that regulate protein expression at post-transcriptional level. We hypothesized that a specific pool of endothelial miRNAs could be selectively regulated by flow conditions and inflammatory signals, and as such be involved in the development of atherosclerosis. Objective: To identify miRNAs, called atheromiRs, which are selectively regulated by shear stress and oxidized low-density lipoproteins (oxLDL), and to determine their role in atherogenesis. Methods and Results: Large-scale miRNA profiling in HUVECs identified miR-92a as an atheromiR candidate, whose expression is preferentially upregulated by the combination of low shear stress (SS) and atherogenic oxLDL. Ex vivo analysis of atheroprone and atheroprotected areas of mouse arteries and human atherosclerotic plaques demonstrated the preferential expression of miR-92a in atheroprone low SS regions. In Ldlr −/− mice, miR-92a expression was markedly enhanced by hypercholesterolemia, in particular in atheroprone areas of the aorta. Assessment of endothelial inflammation in gain- and loss-of-function experiments targeting miR-92a expression revealed that miR-92a regulated endothelial cell activation by oxLDL, more specifically under low SS conditions, which was associated with modulation of Kruppel-like factor 2 (KLF2), Kruppel-like factor 4 (KLF4), and suppressor of cytokine signaling 5. miR-92a expression was regulated by signal transducer and activator of transcription 3 in SS- and oxLDL-dependent manner. Furthermore, specific in vivo blockade of miR-92a expression in Ldlr −/− mice reduced endothelial inflammation and altered the development of atherosclerosis, decreasing plaque size and promoting a more stable lesion phenotype. Conclusions: Upregulation of miR-92a by oxLDL in atheroprone areas promotes endothelial activation and the development of atherosclerotic lesions. Therefore, miR-92a antagomir seems as a new atheroprotective therapeutic strategy.