Olga Ovchinnikova

Enhanced T-cell expression of RANK ligand in acute coronary syndrome: possible role in plaque destabilization.

Objective—Based on its role in inflammation and matrix degradation, we hypothesized a role for osteoprotegerin (OPG), RANK, and RANK ligand (RANKL) in coronary artery disease. Methods and Results—We examined the expression of various members of the OPG/RANKL/RANK axis in patients with stable and unstable angina and in the atherosclerotic lesions of apolipoprotein E–deficient (apoE−/−) mice. Our findings were: (1) Serum levels of OPG were raised in patients with unstable angina (n=40), but not in those with stable angina (n=40), comparing controls (n=20); (2) mRNA levels of RANKL were increased in T-cells in unstable angina patients accompanied by increased expression of RANK in monocytes; (3) strong immunostaining of OPG/RANKL/RANK was seen within thrombus material obtained at the site of plaque rupture during acute myocardial infarction; (4) OPG/RANKL/RANK was expressed in the atherosclerotic plaques of apoE−/− mice, with RANKL located specifically to the plaques; and (5) RANKL enhanced the release of monocyte chemoattractant peptide-1 in mononuclear cells from unstable angina patients, and promoted matrix metalloproteinase (MMP) activity in vascular smooth muscle cells. Conclusions—We show enhanced expression of the OPG/RANKL/RANK system both in clinical and experimental atherosclerosis, with enhanced T-cell expression of RANKL as an important feature of unstable disease.

5-Lipoxygenase–Activating Protein A Potential Link Between Innate and Adaptive Immunity in Atherosclerosis and Adipose Tissue Inflammation

Transforming growth factor-&bgr; (TGF-&bgr;) is a major antiinflammatory mediator in atherosclerosis. Transgenic ApoE−/− mice with a dominant-negative TGF&bgr; type II receptor (dnTGF&bgr;RII) on CD4+ and CD8+ T cells display aggravated atherosclerosis. The aim of the present study was to elucidate the mechanisms involved in this enhanced inflammatory response. Gene array analyses identified the 5-lipoxygenase–activating protein (FLAP) among the most upregulated genes in both the aorta and adipose tissue of dnTGF&bgr;RII transgenic ApoE−/− mice compared with their ApoE−/− littermates, a finding that was confirmed by real-time quantitative RT-PCR. Aortas from the former mice in addition produced increased amounts of the lipoxygenase product leukotriene B4 after ex vivo stimulation. FLAP protein expression in both the aorta and adipose tissue was detected in macrophages, but not in T cells. Four weeks of treatment with the FLAP inhibitor MK-886 (10 mg/kg in 1% tylose delivered by osmotic pumps) significantly reduced atherosclerotic lesion size and T-cell content. Finally, FLAP mRNA levels were upregulated approximately 8-fold in adipose tissue derived from obese ob/ob mice. In conclusion, the results of the present study suggest a key role for mediators of the 5-lipoxygenase pathway in inflammatory reactions of atherosclerosis and metabolic disease.

Enhanced Expression of the Homeostatic Chemokines CCL19 and CCL21 in Clinical and Experimental Atherosclerosis. Possible Pathogenic Role in Plaque Destabilization

Objective—Based on their role in T-cell homing into nonlymphoid tissue, we examined the role of the homeostatic chemokines CCL19 and CCL21 and their common receptor CCR7 in coronary artery disease (CAD). Methods and Results—We performed studies in patients with stable (n=40) and unstable (n=40) angina and healthy controls (n=20), in vitro studies in T-cells and macrophages, and studies in apolipoprotein-E–deficient (ApoE−/−) mice and human atherosclerotic carotid plaques. We found increased levels of CCL19 and CCL21 within the atherosclerotic lesions of the ApoE−/− mice, in human atherosclerotic carotid plaques, and in plasma of CAD patients. Whereas strong CCR7 expression was seen in T-cells from murine and human atherosclerotic plaques, circulating T-cells from angina patients showed decreased CCR7 expression. CCL19 and CCL21 promoted an inflammatory phenotype in T-cells and macrophages and increased matrix metalloproteinase (MMP) and tissue factor levels in the latter cell type. Although aggressive statin therapy increased CCR7 and decreased CCL19/CCL21 levels in peripheral blood from CAD patients, conventional therapy did not. Conclusions—The abnormal regulation of CCL19 and CCL21 and their common receptor in atherosclerosis could contribute to disease progression by recruiting T-cells and macrophages to the atherosclerotic lesions and by promoting inflammatory responses in these cells.

Transforming Growth Factor–β Signaling in T Cells Promotes Stabilization of Atherosclerotic Plaques Through an Interleukin-17–Dependent Pathway

Enhanced TGF-β signaling may promote plaque stability and prevent clinical manifestations of atherosclerosis. IL-17 Helps Plaques Lie Dormant Like a dormant volcano, stable atherosclerotic plaques can lull you into a false sense of security. The accumulation of lipids and inflammatory mediators results in arterial hardening and lack of flexibility, but individuals with these plaques may be asymptomatic for decades. However, when an unstable plaque ruptures, thrombi forming on the exposed tissue can block blood flow, resulting in heart attack or stroke. Gisterå et al. now report that transforming growth factor–β (TGF-β) promotes plaque stabilization through the effects of interleukin-17 (IL-17). The authors looked at T cells with enhanced expression of TGF-β in a mouse model of atherosclerosis. They found that these animals had larger atherosclerotic lesions, but these lesions were more stable. Inhibiting IL-17 through neutralizing antibodies decreased the stability of these plaques, whereas IL-17 expression correlated to expression of components of the fibrous cap in human atherosclerotic plaques. These data suggest that patients treated with IL-17 receptor blockers should be closely monitored for cardiovascular events and provide IL-17 as a therapeutic option to prevent plaque eruption. Adaptive immunity has a major impact on atherosclerosis, with pro- and anti-atherosclerotic effects exerted by different subpopulations of T cells. Transforming growth factor–β (TGF-β) may promote development either of anti-atherosclerotic regulatory T cells or of T helper 17 (TH17) cells, depending on factors in the local milieu. We have addressed the effect on atherosclerosis of enhanced TGF-β signaling in T cells. Bone marrow from mice with a T cell–specific deletion of Smad7, a potent inhibitor of TGF-β signaling, was transplanted into hypercholesterolemic Ldlr−/− mice. Smad7-deficient mice had significantly larger atherosclerotic lesions that contained large collagen-rich caps, consistent with a more stable phenotype. The inflammatory cytokine interleukin-6 (IL-6) was expressed in the atherosclerotic aorta, and increased mRNA for IL-17A and the TH17-specific transcription factor RORγt were detected in draining lymph nodes. Treating Smad7-deficient chimeras with neutralizing IL-17A antibodies reversed stable cap formation. IL-17A stimulated collagen production by human vascular smooth muscle cells, and RORγt mRNA correlated positively with collagen type I and α-smooth muscle actin mRNA in a biobank of human atherosclerotic plaques. These data link IL-17A to induction of a stable plaque phenotype, could lead to new plaque-stabilizing therapies, and should prompt an evaluation of cardiovascular events in patients treated with IL-17 receptor blockade.

The tryptophan metabolite 3-hydroxyanthranilic acid lowers plasma lipids and decreases atherosclerosis in hypercholesterolaemic mice

AIMS Cardiovascular disease is the most common cause of death in the world and atherosclerosis, an inflammatory process in the vessel wall, accounts for the majority of these deaths. The tryptophan metabolite 3-hydroxyanthranilic acid (3-HAA) has been shown to inhibit inflammation in different experimental autoimmune disease models. However, the effect of 3-HAA in atherosclerosis has never been explored. METHODS AND RESULTS In this study, we used the atherosclerosis prone Ldlr-/- mice, and cell culture experiments to evaluate the role of 3-HAA in atherosclerosis. Eight weeks treatment with 3-HAA significantly reduced the lesion size in the aorta, and modulated local and systemic inflammatory responses. 3-hydroxyanthranilic acid inhibited the uptake of oxLDL by macrophages, an initiating event in the formation of foam cells, a major cellular component of atherosclerotic lesions. Surprisingly, 3-HAA significantly affected plasma cholesterol and triglyceride levels in Ldlr-/- mice, likely due to modulation of signalling through peroxisome proliferator-activated receptors. CONCLUSION 3-Hydroxyanthranilic acid inhibits atherosclerosis by regulating lipid metabolism and inflammation, two major components of this disease.

T-Cell Activation Leads to Reduced Collagen Maturation in Atherosclerotic Plaques of Apoe−/− Mice

Rupture of the collagenous, fibrous cap of an atherosclerotic plaque commonly causes thrombosis. Activated immune cells can secrete mediators that jeopardize the integrity of the fibrous cap. This study aimed to determine the relationship between T-cell-mediated inflammation and collagen turnover in a mouse model of experimental atherosclerosis. Both Apoe(-/-) x CD4dnTbetaRII mice with defective transforming growth factor-beta receptors in T cells (and hence released from tonic suppression of T-cell activation) and lesion size-matched Apoe(-/-) mice were used. Picrosirius red staining showed a lower content of thick mature collagen fibers in lesions of Apoe(-/-) x CD4dnTbetaRII mice, although both groups had similar levels of procollagen type I or III mRNA and total collagen content in lesions. Analysis of both gene expression and protein content showed a significant decrease of lysyl oxidase, the extracellular enzyme needed for collagen cross-linking, in aortas of Apoe(-/-)--CD4dnTbetaRII mice. T-cell-driven inflammation provoked a selective and limited increase in the expression of proteinases that catabolize the extracellular matrix. Atheromata of Apoe(-/-)--CD4dnTbetaRII mice had increased levels of matrix metalloproteinase-13 and cathepsin S mRNAs and of the active form of cathepsin S protein but no increase was detected in collagen fragmentation. Our results suggest that exaggerated T-cell-driven inflammation limits collagen maturation in the atherosclerotic plaque while having little effect on collagen degradation.

Short-term delivery of anti-PlGF antibody delays progression of atherosclerotic plaques to vulnerable lesions

AIMS Placental growth factor (PlGF), a homologue of vascular endothelial growth factor, is a pleiotropic cytokine with a pro-inflammatory activity. Previous gene-inactivation studies revealed that the loss of PlGF delays atherosclerotic lesion development and inhibits macrophage infiltration, but the activity of an anti-PlGF antibody (alphaPlGF mAb) has not been evaluated yet. METHODS AND RESULTS We characterized the potential of short-term delivery of alphaPlGF mAb in inhibiting lesion development in ApoE-deficient mice (apoE(-/-)) and in CD4:TGFbetaRII(DN) x apoE(-/-) mice, a more severe atherosclerosis model. Short-term treatment of alphaPlGF mAb reduces early atherosclerotic plaque size and inflammatory cell infiltration in the lesion. CONCLUSION These pharmacological alphaPlGF mAb results confirm previous genetic evidence that inhibition of PlGF slows down early atherosclerotic lesion development. Furthermore, the phenocopy of genetic and pharmacological loss-of-function strategies underscores that alphaPlGF acts by selectively neutralizing PlGF.

Inhibition of indoleamine 2,3-dioxygenase promotes vascular inflammation and increases atherosclerosis in Apoe-/- mice.

AIMS Atherosclerosis is a chronic inflammatory disease that is initiated by the retention and accumulation of low-density lipoprotein in the artery, leading to maladaptive response of cells from the immune system and vessel wall. Strong evidence implicates indoleamine 2,3-dioxygenase (IDO), the first and rate-limiting enzyme of the kynurenine pathway of tryptophan (Trp) degradation, with immune regulation and anti-inflammatory mechanisms in different diseases. However, the role of IDO and the endogenous degradation of Trp have never been directly examined in atherosclerosis development. We used the IDO inhibitor 1-methyl-Trp (1-MT) to determine the role of IDO-mediated Trp metabolism in vascular inflammation and atherosclerosis. METHODS AND RESULTS Apoe(-/-) mice were treated with 1-MT in drinking water for 8 weeks. Systemic IDO inhibition led to a significant increase in atherosclerotic lesions that were ∼58 and 54% larger in the aortic arch and root, respectively. 1-MT treatment enhanced vascular inflammation, up-regulated VCAM-1 and CCL2, and increased CD68 macrophage accumulation into the plaque. Notably, the rise in VCAM-1 expression was not limited to the plaque but also found in smooth muscle cells (SMCs) of the tunica media. Furthermore, we found that IDO-dependent Trp metabolism by SMCs regulates VCAM-1 expression, and that 1-MT-induced acceleration of atherosclerosis and vascular inflammation can be reversed by exogenous administration of the Trp metabolite 3-hydroxyanthranilic acid (3-HAA). CONCLUSION IDO-mediated Trp metabolism regulates vascular inflammation and plaque formation in hypercholesterolaemic Apoe(-/-) mice. Our data establish that this pathway plays a major role in the pathological process of atherogenesis.

Abolished synthesis of cholic acid reduces atherosclerotic development in apolipoprotein E knockout mice.

To investigate the effects of abolished cholic acid (CA) synthesis in the ApoE knockout model [apolipoprotein E (apoE) KO],a double-knockout (DKO) mouse model was created by crossbreeding Cyp8b1 knockout mice (Cyp8b1 KO), unable to synthesize the primary bile acid CA, with apoE KO mice. After 5 months of cholesterol feeding, the development of atherosclerotic plaques in the proximal aorta was 50% less in the DKO mice compared with the apoE KO mice. This effect was associated with reduced intestinal cholesterol absorption, decreased levels of apoB-containing lipoproteins in the plasma, enhanced bile acid synthesis, reduced hepatic cholesteryl esters, and decreased hepatic activity of ACAT2. The upregulation of Cyp7a1 in DKO mice seemed primarily caused by reduced expression of the intestinal peptide FGF15. Treatment of DKO mice with the farnesoid X receptor (FXR) agonist GW4064 did not alter the intestinal cholesterol absorption, suggesting that the action of CA in this process is confined mainly to formation of intraluminal micelles and less to its ability to activate the nuclear receptor FXR. Inhibition of CA synthesis may offer a therapeutic strategy for the treatment of hyperlipidemic conditions that lead to atherosclerosis.

Sterile inflammation in the spleen during atherosclerosis provides oxidation-specific epitopes that induce a protective B-cell response

Significance In this study we investigate the origin of the protective B-cell response in the spleen in atherosclerosis. We find an ongoing B-cell activation with production of antibodies against oxidation-specific epitopes. In addition, this response can be accelerated using apoptotic cells alone that reduce lesion development and serum cholesterol in a B-cell–dependent manner. This study pinpoints the spleen as an important organ for atherosclerosis-associated immunity and provides novel pathways to use for treatment. The B-cell response in atherosclerosis is directed toward oxidation-specific epitopes such as phosphorylcholine (PC) that arise during disease-driven oxidation of self-antigens. PC-bearing antigens have been used to induce atheroprotective antibodies against modified low-density lipoproteins (oxLDL), leading to plaque reduction. Previous studies have found that B-cell transfer from aged atherosclerotic mice confers protection to young mice, but the mechanism is unknown. Here, we dissected the atheroprotective response in the spleen and found an ongoing germinal center reaction, accumulation of antibody-forming cells, and inflammasome activation in apolipoprotein E-deficient mice (Apoe−/−). Specific B-cell clone expansion involved the heavy chain variable region (Vh) 5 and Vh7 B-cell receptor families that harbor anti-PC reactivity. oxLDL also accumulated in the spleen. To investigate whether protection could be induced by self-antigens alone, we injected apoptotic cells that carry the same oxidation-specific epitopes as oxLDL. This treatment reduced serum cholesterol and inhibited the development of atherosclerosis in a B-cell–dependent manner. Thus, we conclude that the spleen harbors a protective B-cell response that is initiated in atherosclerosis through sterile inflammation. These data highlight the importance of the spleen in atherosclerosis-associated immunity.