Endothelial Cell Indoleamine 2, 3-Dioxygenase 1 Alters Cardiac Function After Myocardial Infarction Through Kynurenine.

BACKGROUND: Ischemic cardiovascular diseases, particularly acute myocardial infarction (MI), is one of the leading causes of mortality worldwide. Indoleamine 2, 3-dioxygenase 1 (IDO) catalyzes 1 rate-limiting step of L-tryptophan metabolism, and emerges as an important regulator of many pathological conditions. We hypothesized that IDO could play a key role to locally regulate cardiac homeostasis after MI. METHODS: Cardiac repair was analyzed in mice harboring specific endothelial or smooth muscle cells or cardiomyocyte or myeloid cell deficiency of IDO and challenged with acute myocardial infarction. RESULTS: We show that kynurenine generation through IDO is markedly induced after MI in mice. Total genetic deletion or pharmacological inhibition of IDO limits cardiac injury and cardiac dysfunction after MI. Distinct loss of function of IDO in smooth muscle cells, inflammatory cells, or cardiomyocytes does not affect cardiac function and remodeling in infarcted mice. In sharp contrast, mice harboring endothelial cell-specific deletion of IDO show an improvement of cardiac function as well as cardiomyocyte contractility and reduction in adverse ventricular remodeling. In vivo kynurenine supplementation in IDO-deficient mice abrogates the protective effects of IDO deletion. Kynurenine precipitates cardiomyocyte apoptosis through reactive oxygen species production in an aryl hydrocarbon receptor-dependent mechanism. CONCLUSIONS: These data suggest that IDO could constitute a new therapeutic target during acute MI.

Thymic stromal lymphopoietin is a key cytokine for the immunomodulation of atherogenesis with Freund's adjuvant.

Adaptive immune responses regulate the development of atherosclerosis, with a detrimental effect of type 1 but a protective role of type 2 immune responses. Immunization of Apolipoprotein E-deficient (ApoE-/- ) mice with Freund's adjuvant inhibits the development of atherosclerosis. However, the underlying mechanisms are not fully understood. Thymic stromal lymphopoietin (TSLP) is an IL7-like cytokine with essential impact on type 2 immune responses (Th2). Thymic stromal lymphopoietin is strongly expressed in epithelial cells of the skin, but also in various immune cells following appropriate stimulation. In this study, we investigated whether TSLP may be crucial for the anti-atherogenic effect of Freund's adjuvant. Subcutaneous injection of complete Freund's adjuvant (CFA) rapidly led to the expression of TSLP and IL1ß at the site of injection. In male mice, CFA-induced TSLP occurred in immigrated monocytes-and not epithelial cells-and was dependent on NLRP3 inflammasome activation and IL1ß-signalling. In females, CFA-induced TSLP was independent of IL1ß and upon ovariectomy. CFA/OVA led to a more pronounced imbalance of the T cell response in TSLPR-/- mice, with increased INFγ/IL4 ratio compared with wild-type controls. To test whether TSLP contributes to the anti-atherogenic effects of Freund's adjuvant, we treated ApoE-/- and ApoE-/- /TSLPR-/- mice with either CFA/IFA or PBS. ApoE-/- mice showed less atherogenesis upon CFA/IFA compared with PBS injections. ApoE-/- /TSLPR-/- mice had no attenuation of atherogenesis upon CFA/IFA treatment. Freund's adjuvant executes significant immune-modulating effects via TSLP induction. TSLP-TSLPR signalling is critical for CFA/IFA-mediated attenuation of atherosclerosis.

A defect in endothelial autophagy occurs in patients with non-alcoholic steatohepatitis and promotes inflammation and fibrosis.

BACKGROUND & AIMS: Previous studies demonstrated that autophagy is protective in hepatocytes and macrophages, but detrimental in hepatic stellate cells in chronic liver diseases. The role of autophagy in liver sinusoidal endothelial cells (LSECs) in non-alcoholic steatohepatitis (NASH) is unknown. Our aim was to analyze the potential implication of autophagy in LSECs in NASH and liver fibrosis. METHODS: We analyzed autophagy in LSECs from patients using transmission electron microscopy. We determined the consequences of a deficiency in autophagy: (a) on LSEC phenotype, using primary LSECs and an LSEC line; (b) on early stages of NASH and on advanced stages of liver fibrosis, using transgenic mice deficient in autophagy specifically in endothelial cells and fed a high-fat diet or chronically treated with carbon tetrachloride, respectively. RESULTS: Patients with NASH had half as many LSECs containing autophagic vacuoles as patients without liver histological abnormalities, or with simple steatosis. LSECs from mice deficient in endothelial autophagy displayed an upregulation of genes implicated in inflammatory pathways. In the LSEC line, deficiency in autophagy enhanced inflammation (Ccl2, Ccl5, Il6 and VCAM-1 expression), features of endothelial-to-mesenchymal transition (α-Sma, Tgfb1, Col1a2 expression) and apoptosis (cleaved caspase-3). In mice fed a high-fat diet, deficiency in endothelial autophagy induced liver expression of inflammatory markers (Ccl2, Ccl5, Cd68, Vcam-1), liver cell apoptosis (cleaved caspase-3) and perisinusoidal fibrosis. Mice deficient in endothelial autophagy treated with carbon tetrachloride also developed more perisinusoidal fibrosis. CONCLUSIONS: A defect in autophagy in LSECs occurs in patients with NASH. Deficiency in endothelial autophagy promotes the development of liver inflammation, features of endothelial-to-mesenchymal transition, apoptosis and liver fibrosis in the early stages of NASH, but also favors more advanced stages of liver fibrosis. LAY SUMMARY: Autophagy is a physiological process controlling endothelial homeostasis in vascular beds outside the liver. This study demonstrates that autophagy is defective in the liver endothelial cells of patients with non-alcoholic steatohepatitis. This defect promotes liver inflammation and fibrosis at early stages of non-alcoholic steatohepatitis, but also at advanced stages of chronic liver disease.

Genetic Depletion or Hyperresponsiveness of Natural Killer Cells Do Not Affect Atherosclerosis Development.

RATIONALE: Chronic inflammation is central in the development of atherosclerosis. Both innate and adaptive immunities are involved. Although several studies have evaluated the functions of natural killer (NK) cells in experimental animal models of atherosclerosis, it is not yet clear whether NK cells behave as protective or proatherogenic effectors. One of the main caveats of previous studies was the lack of specificity in targeting loss or gain of function of NK cells. OBJECTIVES: We used 2 selective genetic approaches to investigate the role of NK cells in atherosclerosis: (1) Ncr1iCre/+R26lsl-DTA/+ mice in which NK cells were depleted and (2) Noé mice in which NK cells are hyperresponsive. METHODS AND RESULTS: No difference in atherosclerotic lesion size was found in Ldlr-/- (low-density lipoprotein receptor null) mice transplanted with bone marrow (BM) cells from Ncr1iCreR26Rlsl-DTA , Noé, or wild-type mice. Also, no difference was observed in plaque composition in terms of collagen content, macrophage infiltration, or the immune profile, although Noé chimera had more IFN (interferon)-γ-producing NK cells, compared with wild-type mice. Then, we investigated the NK-cell selectivity of anti-asialoganglioside M1 antiserum, which was previously used to conclude the proatherogenicity of NK cells. Anti-asialoganglioside M1 treatment decreased atherosclerosis in both Ldlr-/- mice transplanted with Ncr1iCreR26Rlsl-DTA or wild-type bone marrow, indicating that its antiatherogenic effects are unrelated to NK-cell depletion, but to CD8+ T and NKT cells. Finally, to determine whether NK cells could contribute to the disease in conditions of pathological NK-cell overactivation, we treated irradiated Ldlr-/- mice reconstituted with either wild-type or Ncr1iCreR26Rlsl-DTA bone marrow with the viral mimic polyinosinic:polycytidylic acid and found a significant reduction of plaque size in NK-cell-deficient chimeric mice. CONCLUSIONS: Our findings, using state-of-the-art mouse models, demonstrate that NK cells have no direct effect on the natural development of hypercholesterolemia-induced atherosclerosis, but may play a role when an additional systemic NK-cell overactivation occurs.

Anticytokine Immune Therapy and Atherothrombotic Cardiovascular Risk.

Accumulating observations in humans and animals indicate that inflammation plays a key role in atherosclerosis development and subsequent complications. Moreover, the use of loss- or gain-of-function genetically modified, atherosclerosis-prone mice has provided strong experimental evidence for a causal role of innate and adaptive immunity in atherosclerosis and has revealed the pathogenic activity of proinflammatory cytokines, including TNF (tumor necrosis factor)-α, IL (interleukin)-1ß, IL-6, and IL-18, and the atheroprotective effect of anti-inflammatory cytokines, including IL-10 and TGF-ß. For the past 15 years, treatments using monoclonal antibodies specifically targeting cytokines, commonly referred as biological therapies, have transformed the treatment of chronic inflammatory diseases, such as rheumatoid arthritis or psoriasis, both conditions associated with increased cardiovascular risk. Analyzing the impact of anticytokine therapies on the cardiovascular outcomes of patients with chronic inflammatory diseases provides insight into the clinical relevance of experimental data on the role of inflammation in atherothrombotic cardiovascular diseases. CANTOS (Canakinumab Antiinflammatory Thrombosis Outcome Study) provided the first evidence that targeting inflammation in humans with atherosclerosis could improve clinical outcomes. Treatment with the anti-IL-1ß antibody canakinumab significantly reduced recurrent cardiovascular events in individuals with stable coronary artery disease well-treated with standard-of-care measures. Other clinical studies support the protective effects of treatment with anti-TNF-α and anti-IL-6 receptor monoclonal antibodies on cardiovascular risk. Blockade of the IL-23/IL-17 axis, however, warrants caution as a cardiovascular intervention. Targeting this pathway has improved psoriasis but may augment cardiovascular risk in certain patients. Thus, careful consideration of the cardiovascular risk profile may influence the choice of the most appropriate treatment for patients with chronic inflammatory diseases.Visual Overview: An online visual overview is available for this article.

MicroRNA-21 Deficiency Alters the Survival of Ly-6Clo Monocytes in ApoE-/- Mice and Reduces Early-Stage Atherosclerosis-Brief Report.

Objective- To determine the role of microRNA-21 (miR-21) on the homeostasis of monocyte subsets and on atherosclerosis development in ApoE-/- (apolipoprotein E) mice. Approach and Results- In ApoE-/- mice, miR-21 expression was increased in circulating Ly-6Clo nonclassical monocytes in comparison to Ly-6Chi monocytes. The absence of miR-21 significantly altered the survival and number of circulating Ly-6Clo nonclassical monocytes in ApoE-/- mice. In the early stages of atherosclerosis, the absence of miR-21 limited lesion development both in the aortic sinus (by almost 30%) and in the aorta (by almost 50%). This was associated with less monocyte availability in circulation and increased apoptosis of local macrophages in plaques. At later stages of atherosclerosis, lesion size in the aortic root was similar in ApoE-/- and ApoE-/- miR-21-/- mice, but plaques showed a less stable phenotype (larger necrotic cores) in the latter. The loss of protection in advanced stages was most likely because of excessive inflammatory apoptosis related to an impairment of local efficient efferocytosis. Conclusions- Gene deletion of miR-21 in ApoE-/- mice alters Ly-6Clo nonclassical monocytes homeostasis and contribute to limit early-stage atherosclerosis.

Autophagy is required for endothelial cell alignment and atheroprotection under physiological blood flow.

It has been known for some time that atherosclerotic lesions preferentially develop in areas exposed to low SS and are characterized by a proinflammatory, apoptotic, and senescent endothelial phenotype. Conversely, areas exposed to high SS are protected from plaque development, but the mechanisms have remained elusive. Autophagy is a protective mechanism that allows recycling of defective organelles and proteins to maintain cellular homeostasis. We aimed to understand the role of endothelial autophagy in the atheroprotective effect of high SS. Atheroprotective high SS stimulated endothelial autophagic flux in human and murine arteries. On the contrary, endothelial cells exposed to atheroprone low SS were characterized by inefficient autophagy as a result of mammalian target of rapamycin (mTOR) activation, AMPKα inhibition, and blockade of the autophagic flux. In hypercholesterolemic mice, deficiency in endothelial autophagy increased plaque burden only in the atheroresistant areas exposed to high SS; plaque size was unchanged in atheroprone areas, in which endothelial autophagy flux is already blocked. In cultured cells and in transgenic mice, deficiency in endothelial autophagy was characterized by defects in endothelial alignment with flow direction, a hallmark of endothelial cell health. This effect was associated with an increase in endothelial apoptosis and senescence in high-SS regions. Deficiency in endothelial autophagy also increased TNF-α-induced inflammation under high-SS conditions and decreased expression of the antiinflammatory factor KLF-2. Altogether, these results show that adequate endothelial autophagic flux under high SS limits atherosclerotic plaque formation by preventing endothelial apoptosis, senescence, and inflammation.

The Dendritic Cell Receptor DNGR-1 Promotes the Development of Atherosclerosis in Mice.

RATIONALE: Necrotic core formation during the development of atherosclerosis is associated with a chronic inflammatory response and promotes accelerated plaque development and instability. However, the molecular links between necrosis and the development of atherosclerosis are not completely understood. Clec9a (C-type lectin receptor) or DNGR-1 (dendritic cell NK lectin group receptor-1) is preferentially expressed by the CD8α+ subset of dendritic cells (CD8α+ DCs) and is involved in sensing necrotic cells. We hypothesized that sensing of necrotic cells by DNGR-1 plays a determinant role in the inflammatory response of atherosclerosis. OBJECTIVE: We sought to address the impact of total, bone marrow-restricted, or CD8α+ DC-restricted deletion of DNGR-1 on atherosclerosis development. METHODS AND RESULTS: We show that total absence of DNGR-1 in Apoe (apolipoprotein e)-deficient mice (Apoe-/-) and bone marrow-restricted deletion of DNGR-1 in Ldlr (low-density lipoprotein receptor)-deficient mice (Ldlr-/-) significantly reduce inflammatory cell content within arterial plaques and limit atherosclerosis development in a context of moderate hypercholesterolemia. This is associated with a significant increase of the expression of interleukin-10 (IL-10). The atheroprotective effect of DNGR-1 deletion is completely abrogated in the absence of bone marrow-derived IL-10. Furthermore, a specific deletion of DNGR-1 in CD8α+ DCs significantly increases IL-10 expression, reduces macrophage and T-cell contents within the lesions, and limits the development of atherosclerosis. CONCLUSIONS: Our results unravel a new role of DNGR-1 in regulating vascular inflammation and atherosclerosis and potentially identify a new target for disease modulation.

Selective EGFR (Epidermal Growth Factor Receptor) Deletion in Myeloid Cells Limits Atherosclerosis-Brief Report.

OBJECTIVE: To determine the consequences of specific inhibition of EGFR (epidermal growth factor receptor) in myeloid cells in atherosclerosis development. APPROACH AND RESULTS: Atherosclerotic lesion size was significantly reduced in irradiated Ldlr-/- mice reconstituted with LysMCre+Egfrlox/lox bone marrow, compared with chimeric Ldlr-/- mice reconstituted with LysMCre-Egfrlox/lox bone marrow, after 4 (-43%; P<0.05), 7 (-34%; P<0.05), and 12 weeks (-54%; P<0.001) of high-fat diet. Reduction of lesion size was associated with marked reduction in macrophage accumulation and necrotic core size. Specific deletion of Egfr in myeloid cells reduced TNF-α (tumor necrosis factor-α) and IL (interleukin)-6 production by stimulated macrophages but had no effect on IL-10 and IL-12p70 secretion. Finally, we found that myeloid deletion of Egfr limited cytoskeletal rearrangements and also lipid uptake by macrophages through a downregulation of the scavenger receptor CD36 (cluster of differentiation 36). CONCLUSIONS: Gene deletion of Egfr in myeloid cells limits IL-6 and TNF-α production, lipid uptake, and consecutively reduces atherosclerosis development.

Circulating cell membrane microparticles transfer heme to endothelial cells and trigger vasoocclusions in sickle cell disease.

Intravascular hemolysis describes the relocalization of heme and hemoglobin (Hb) from erythrocytes to plasma. We investigated the concept that erythrocyte membrane microparticles (MPs) concentrate cell-free heme in human hemolytic diseases, and that heme-laden MPs have a physiopathological impact. Up to one-third of cell-free heme in plasma from 47 patients with sickle cell disease (SCD) was sequestered in circulating MPs. Erythrocyte vesiculation in vitro produced MPs loaded with heme. In silico analysis predicted that externalized phosphatidylserine (PS) in MPs may associate with and help retain heme at the cell surface. Immunohistology identified Hb-laden MPs adherent to capillary endothelium in kidney biopsies from hyperalbuminuric SCD patients. In addition, heme-laden erythrocyte MPs adhered and transferred heme to cultured endothelial cells, inducing oxidative stress and apoptosis. In transgenic SAD mice, infusion of heme-laden MPs triggered rapid vasoocclusions in kidneys and compromised microvascular dilation ex vivo. These vascular effects were largely blocked by heme-scavenging hemopexin and by the PS antagonist annexin-a5, in vitro and in vivo. Adversely remodeled MPs carrying heme may thus be a source of oxidant stress for the endothelium, linking hemolysis to vascular injury. This pathway might provide new targets for the therapeutic preservation of vascular function in SCD.

Mechanosensitive PPAP2B Regulates Endothelial Responses to Atherorelevant Hemodynamic Forces.

RATIONALE: PhosPhatidic Acid Phosphatase type 2B (PPAP2B), an integral membrane protein known as lipid phosphate phosphatase (LPP3) that inactivates lysophosphatidic acid, was implicated in coronary artery disease (CAD) by genome-wide association studies. However, it is unclear whether genome-wide association studies-identified coronary artery disease genes, including PPAP2B, participate in mechanotransduction mechanisms by which vascular endothelia respond to local atherorelevant hemodynamics that contribute to the regional nature of atherosclerosis. OBJECTIVE: To establish the critical role of PPAP2B in endothelial responses to hemodynamics. METHODS AND RESULTS: Reduced PPAP2B was detected in vivo in mouse and swine aortic arch (AA) endothelia exposed to chronic disturbed flow, and in mouse carotid artery endothelia subjected to surgically induced acute disturbed flow. In humans, PPAP2B was reduced in the downstream part of carotid plaques where low shear stress prevails. In culture, reduced PPAP2B was measured in human aortic endothelial cells under atherosusceptible waveform mimicking flow in human carotid sinus. Flow-sensitive microRNA-92a and transcription factor KLF2 were identified as upstream inhibitor and activator of endothelial PPAP2B, respectively. PPAP2B suppression abrogated atheroprotection of unidirectional flow; inhibition of lysophosphatidic acid receptor 1 restored the flow-dependent, anti-inflammatory phenotype in PPAP2B-deficient cells. PPAP2B inhibition resulted in myosin light-chain phosphorylation and intercellular gaps, which were abolished by lysophosphatidic acid receptor 1/2 inhibition. Expression quantitative trait locus mapping demonstrated PPAP2B coronary artery disease risk allele is not linked to PPAP2B expression in various human tissues but significantly associated with reduced PPAP2B in human aortic endothelial cells. CONCLUSIONS: Atherorelevant flows dynamically modulate endothelial PPAP2B expression through miR-92a and KLF2. Mechanosensitive PPAP2B plays a critical role in promoting anti-inflammatory phenotype and maintaining vascular integrity of endothelial monolayer under atheroprotective flow.

Role of Microvascular Tone and Extracellular Matrix Contraction in the Regulation of Interstitial Fluid: Implications for Aortic Dissection.

The pathophysiology of aortic dissection is poorly understood, and its risk is resistant to medical treatment. Most studies have focused on a proposed pathogenic role of transforming growth factor-ß in Marfan disease and related thoracic aortic aneurysms and aortic dissections. However, clinical testing of this concept using angiotensin II type 1 receptor antagonists to block transforming growth factor-ß signaling fell short of promise. Genetic mutations that predispose to thoracic aortic aneurysms and aortic dissections affect components of the extracellular matrix and proteins involved in cellular force generation. Thus, a role for dysfunctional mechanosensing in abnormal aortic wall remodeling is emerging. However, how abnormal mechanosensing leads to aortic dissection remains a mystery. Here, we review current knowledge about the regulation of interstitial fluid dynamics and myogenic tone and propose that alteration in contractile force reduces vascular tone in the microcirculation (here, aortic vasa vasorum) and leads to elevations of blood flow, transmural pressure, and fluid flux into the surrounding aortic media. Furthermore, reduced contractile force in medial smooth muscle cells coupled with alteration of structural components of the extracellular matrix limits extracellular matrix contraction, further promoting the formation of intramural edema, a critical step in the initiation of aortic dissection. The concept is supported by several pathophysiological and clinical observations. A direct implication of this concept is that drugs that lower blood pressure and limit interstitial fluid accumulation while preserving or increasing microvascular tone would limit the risk of dissection. In contrast, drugs that substantially lower microvascular tone would be ineffective or may accelerate the disease and precipitate aortic dissection.

Liver microRNA-21 is overexpressed in non-alcoholic steatohepatitis and contributes to the disease in experimental models by inhibiting PPARα expression.

OBJECTIVE: Previous studies suggested that microRNA-21 may be upregulated in the liver in non-alcoholic steatohepatitis (NASH), but its role in the development of this disease remains unknown. This study aimed to determine the role of microRNA-21 in NASH. DESIGN: We inhibited or suppressed microRNA-21 in different mouse models of NASH: (a) low-density lipoprotein receptor-deficient (Ldlr-/-) mice fed a high-fat diet and treated with antagomir-21 or antagomir control; (b) microRNA-21-deficient and wild-type mice fed a methionine-choline-deficient (MCD) diet; (c) peroxisome proliferation-activator receptor α (PPARα)-deficient mice fed an MCD diet and treated with antagomir-21 or antagomir control. We assessed features of NASH and determined liver microRNA-21 levels and cell localisation. MicroRNA-21 levels were also quantified in the liver of patients with NASH, bland steatosis or normal liver and localisation was determined. RESULTS: Inhibiting or suppressing liver microRNA-21 expression reduced liver cell injury, inflammation and fibrogenesis without affecting liver lipid accumulation in Ldlr-/- fed a high-fat diet and in wild-type mice fed an MCD diet. Liver microRNA-21 was overexpressed, primarily in biliary and inflammatory cells, in mouse models as well as in patients with NASH, but not in patients with bland steatosis. PPARα, a known microRNA-21 target, implicated in NASH, was decreased in the liver of mice with NASH and restored following microRNA-21 inhibition or suppression. The effect of antagomir-21 was lost in PPARα-deficient mice. CONCLUSIONS: MicroRNA-21 inhibition or suppression decreases liver injury, inflammation and fibrosis, by restoring PPARα expression. Antagomir-21 might be a future therapeutic strategy for NASH.

Microvesicles as cell-cell messengers in cardiovascular diseases.

Cell-cell communication has proven to be even more complex than previously thought since the discovery that extracellular vesicles serve as containers of biological information on various pathophysiological settings. Extracellular vesicles are classified into exosomes, microvesicles/microparticles, or apoptotic bodies, originating from different subcellular compartments. The cellular machinery controlling their formation and composition, as well as the mechanisms regulating their extracellular release, remain unfortunately much unknown. Extracellular vesicles have been found in plasma, urine, saliva, and inflammatory tissues. Their biomarker potential has raised significant interest in the cardiovascular field because the vesicle composition and microRNA content are specific signatures of cellular activation and injury. More than simply cell dust, extracellular vesicles are capable of transferring biological information to neighboring cells and play an active role in inflammatory diseases, including atherosclerosis and angiogenesis. The molecular interactions regulating these effects involve specific receptor activation, proteolytic enzymes, reactive oxygen species, or delivery of genetic information to target cells. Unraveling their mechanisms of action will likely open new therapeutic avenues.

Adaptive (T and B cells) immunity and control by dendritic cells in atherosclerosis.

Chronic inflammation in response to lipoprotein accumulation in the arterial wall is central in the development of atherosclerosis. Both innate and adaptive immunity are involved in this process. Adaptive immune responses develop against an array of potential antigens presented to effector T lymphocytes by antigen-presenting cells, especially dendritic cells. Functional analysis of the role of different T-cell subsets identified the Th1 responses as proatherogenic, whereas regulatory T-cell responses exert antiatherogenic activities. The effect of Th2 and Th17 responses is still debated. Atherosclerosis is also associated with B-cell activation. Recent evidence established that conventional B-2 cells promote atherosclerosis. In contrast, innate B-1 B cells offer protection through secretion of natural IgM antibodies. This review discusses the recent development in our understanding of the role of T- and B-cell subsets in atherosclerosis and addresses the role of dendritic cell subpopulations in the control of adaptive immunity.

Inhibition of microRNA-92a prevents endothelial dysfunction and atherosclerosis in mice.

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.

IL-17 and Th17 cells in atherosclerosis: subtle and contextual roles.

Atherosclerosis is a chronic inflammatory arterial disease driven by both innate and adaptive immune responses to modified lipoproteins and components of the injured vascular wall. Specific T lymphocyte responses driven by T helper-1 or T regulatory cells play distinct and opposing roles in atherosclerosis. More recently, T helper-17 cells, which produce the prototype cytokine interleukin-17, have been characterized and shown to be critical in mucosal host defense against microbial and fungal pathogens. Sustained production of interleukin-17 in an inflammatory context has been linked to the pathology of several autoimmune and inflammatory diseases. However, regulatory and protective roles have also been reported in selective disease settings. Studies in atherosclerosis led to conflicting results on the roles of interleukin-17 and T helper-17 cells in disease development and plaque stability. The present review provides a summary of the available evidence and putative mechanisms linking this pathway to atherosclerosis, as well as a perspective on the risks and benefits of interleukin-17-targeted cytokine therapy in patients at high cardiovascular risk.

Angiotensin II mobilizes spleen monocytes to promote the development of abdominal aortic aneurysm in Apoe-/- mice.

OBJECTIVE: Abdominal aortic aneurysm (AAA) is widespread among elderly people and results in progressive expansion and rupture of the aorta with high mortality. Macrophages, which are the main population observed within the site of aneurysm, are thought to derive from circulating monocytes although no direct evidence has been provided to date. In this study, we were particularly interested in understanding the trafficking behavior of monocyte subsets in AAA and their role in disease pathogenesis. APPROACH AND RESULTS: Using bone marrow transplantation in Apoe(-/-) mice, we showed that circulating monocytes give rise to abdominal aortic macrophages in hypercholesterolemic mice submitted to angiotensin II (AngII). Detailed monitoring of monocyte compartmentalization revealed that lymphocyte antigen 6C(high) and lymphocyte antigen 6C(low) monocytes transiently increase in blood early after AngII infusion and differentially infiltrate the abdominal aorta. The splenic reservoir accounted for the mobilization of the 2 monocyte subsets after 3 days of AngII infusion. Spleen removal or lymphocyte deficiency in Apoe(-/-) Rag2(-/-) mice similarly impaired early monocyte increase in blood in response to AngII and protected against AAA development, independently of blood pressure. Reconstitution of Apoe(-/-) Rag2(-/-) mice with total splenocytes but not with B-cell-depleted splenocytes restored monocyte mobilization in response to AngII and enhanced susceptibility to AAA. CONCLUSIONS: Taken together, the data show that lymphocyte antigen 6C(high) and lymphocyte antigen 6C(low) monocytes are mobilized from the spleen in response to AngII. Intriguingly, the process is dependent on the presence of B cells and significantly contributes to the development of AAA and the occurrence of aortic rupture.

The intravenous injection of oxidized LDL- or Apolipoprotein B100--Coupled splenocytes promotes Th1 polarization in wildtype and Apolipoprotein E--Deficient mice.

BACKGROUND: Th1 responses in atherosclerosis are mainly associated with the aggravation of atherosclerotic plaques, whereas Th2 responses lead to a less pronounced disease in mouse models. The fixation of antigens on cells by means of ethylene carbodiimide (ECDI), and subsequent injection of these antigen-coupled splenocytes (Ag-SP) to induce tolerance against the attached antigens, has been successfully used to treat murine type 1 diabetes or encephalomyelitis in. We analyzed this approach in a mouse model for atherosclerosis. METHODS AND RESULTS: OTII-transgenic mice that were treated with a single dose of 5 × 10(7) OVA-coupled splenocytes (OVA-SP), had decreased splenocyte proliferation, and lower IFNγ production in vitro upon antigen recall. However, in vivo CD4 cell activation was increased. To try lipoprotein-derived, "atherosclerosis-associated" antigens, we first tested human oxidized LDL. In wild type mice, an increase of IFNγ production upon in vitro recall was detected in the oxLDL-SP group. In Apolipoprotein E - deficient (ApoE-/-) mice that received oxLDL-SP every 5 weeks for 20 weeks, we did not find any difference of atherosclerotic plaque burden, but again increased IFNγ production. To overcome xenogenous limitations, we then examined the effects of mouse Apolipoprotein B100 peptides P3 and P6. ApoB100-SP treatment again promoted a more IFNγ pronounced response upon in vitro recall. Flow cytometry analysis of cytokine secreting spleen cells revealed CD4 positive T cells to be mainly the source for IFNγ. In ApoE-/- mice that were administered ApoB100-SP during 20 weeks, the atherosclerotic plaque burden in aortic roots as well as total aorta was unchanged compared to PBS treated controls. Splenocyte proliferation upon antigen recall was not significantly altered in ApoB100-SP treated ApoE-/- mice. CONCLUSION: Although we did not observe a relevant anti-atherosclerotic benefit, the treatment with antigen-coupled splenocytes in its present form already impacts the immune responses and deserves further exploration.