Chemical Hybridization of Glucagon and Thyroid Hormone Optimizes Therapeutic Impact for Metabolic Disease.

Glucagon and thyroid hormone (T3) exhibit therapeutic potential for metabolic disease but also exhibit undesired effects. We achieved synergistic effects of these two hormones and mitigation of their adverse effects by engineering chemical conjugates enabling delivery of both activities within one precisely targeted molecule. Coordinated glucagon and T3 actions synergize to correct hyperlipidemia, steatohepatitis, atherosclerosis, glucose intolerance, and obesity in metabolically compromised mice. We demonstrate that each hormonal constituent mutually enriches cellular processes in hepatocytes and adipocytes via enhanced hepatic cholesterol metabolism and white fat browning. Synchronized signaling driven by glucagon and T3 reciprocally minimizes the inherent harmful effects of each hormone. Liver-directed T3 action offsets the diabetogenic liability of glucagon, and glucagon-mediated delivery spares the cardiovascular system from adverse T3 action. Our findings support the therapeutic utility of integrating these hormones into a single molecular entity that offers unique potential for treatment of obesity, type 2 diabetes, and cardiovascular disease.

NETs-Induced Thrombosis Impacts on Cardiovascular and Chronic Kidney Disease.

Arterial and venous thrombosis constitute a major source of morbidity and mortality worldwide. Association between thrombotic complications and cardiovascular and other chronic inflammatory diseases are well described. Inflammation and subsequent initiation of thrombotic events, termed immunothrombosis, also receive growing attention but are still incompletely understood. Nevertheless, the clinical relevance of aberrant immunothrombosis, referred to as thromboinflammation, is evident by an increased risk of thrombosis and cardiovascular events in patients with inflammatory or infectious diseases. Proinflammatory mediators released from platelets, complement activation, and the formation of NETs (neutrophil extracellular traps) initiate and foster immunothrombosis. In this review, we highlight and discuss prominent and emerging interrelationships and functions between NETs and other mediators in immunothrombosis in cardiovascular disease. Also, with patients with chronic kidney disease suffering from increased cardiovascular and thrombotic risk, we summarize current knowledge on neutrophil phenotype, function, and NET formation in chronic kidney disease. In addition, we elaborate on therapeutic targeting of NETs-induced immunothrombosis. A better understanding of the functional relevance of antithrombotic mediators which do not increase bleeding risk may provide opportunities for successful therapeutic interventions to reduce thrombotic risk beyond current treatment options.

Somatic RIT1 delins in arteriovenous malformations hyperactivate RAS-MAPK signaling amenable to MEK inhibition.

Arteriovenous malformations (AVM) are benign vascular anomalies prone to pain, bleeding, and progressive growth. AVM are mainly caused by mosaic pathogenic variants of the RAS-MAPK pathway. However, a causative variant is not identified in all patients. Using ultra-deep sequencing, we identified novel somatic RIT1 delins variants in lesional tissue of three AVM patients. RIT1 encodes a RAS-like protein that can modulate RAS-MAPK signaling. We expressed RIT1 variants in HEK293T cells, which led to a strong increase in ERK1/2 phosphorylation. Endothelial-specific mosaic overexpression of RIT1 delins in zebrafish embryos induced AVM formation, highlighting their functional importance in vascular development. Both ERK1/2 hyperactivation in vitro and AVM formation in vivo could be suppressed by pharmacological MEK inhibition. Treatment with the MEK inhibitor trametinib led to a significant decrease in bleeding episodes and AVM size in one patient. Our findings implicate RIT1 in AVM formation and provide a rationale for clinical trials with targeted treatments.

Targeting platelet-derived CXCL12 impedes arterial thrombosis.

The prevention and treatment of arterial thrombosis continue to be clinically challenging, and understanding the relevant molecular mechanisms in detail may facilitate the quest to identify novel targets and therapeutic approaches that improve protection from ischemic and bleeding events. The chemokine CXCL12 augments collagen-induced platelet aggregation by activating its receptor CXCR4. Here we show that inhibition of CXCR4 attenuates platelet aggregation induced by collagen or human plaque homogenate under static and arterial flow conditions by antagonizing the action of platelet-secreted CXCL12. We further show that platelet-specific CXCL12 deficiency in mice limits arterial thrombosis by affecting thrombus growth and stability without increasing tail bleeding time. Accordingly, neointimal lesion formation after carotid artery injury was attenuated in these mice. Mechanistically, CXCL12 activated via CXCR4 a signaling cascade involving Bruton's tyrosine kinase (Btk) that led to integrin αIIbß3 activation, platelet aggregation, and granule release. The heterodimeric interaction between CXCL12 and CCL5 can inhibit CXCL12-mediated effects as mimicked by CCL5-derived peptides such as [VREY]4. An improved variant of this peptide, i[VREY]4, binds to CXCL12 in a complex with CXCR4 on the surface of activated platelets, thereby inhibiting Btk activation and preventing platelet CXCL12-dependent arterial thrombosis. In contrast to standard antiplatelet therapies such as aspirin or P2Y12 inhibition, i[VREY]4 reduced CXCL12-induced platelet aggregation and yet did not prolong in vitro bleeding time. We provide evidence that platelet-derived CXCL12 is involved in arterial thrombosis and can be specifically targeted by peptides that harbor potential therapeutic value against atherothrombosis.

GIP receptor agonism improves dyslipidemia and atherosclerosis independently of body weight loss in preclinical mouse model for cardio-metabolic disease.

BACKGROUND: Agonism at the receptor for the glucose-dependent insulinotropic polypeptide (GIPR) is a key component of the novel unimolecular GIPR:GLP-1R co-agonists, which are among the most promising drugs in clinical development for the treatment of obesity and type 2 diabetes. The therapeutic effect of chronic GIPR agonism to treat dyslipidemia and thus to reduce the cardiovascular disease risk independently of body weight loss has not been explored yet. METHODS: After 8 weeks on western diet, LDL receptor knockout (LDLR-/-) male mice were treated with daily subcutaneous injections of long-acting acylated GIP analog (acyl-GIP; 10nmol/kg body weight) for 28 days. Body weight, food intake, whole-body composition were monitored throughout the study. Fasting blood glucose and intraperitoneal glucose tolerance test (ipGTT) were determined on day 21 of the study. Circulating lipid levels, lipoprotein profiles and atherosclerotic lesion size was assessed at the end of the study. Acyl-GIP effects on fat depots were determined by histology and transcriptomics. RESULTS: Herein we found that treatment with acyl-GIP reduced dyslipidemia and atherogenesis in male LDLR-/- mice. Acyl-GIP administration resulted in smaller adipocytes within the inguinal fat depot and RNAseq analysis of the latter revealed that acyl-GIP may improve dyslipidemia by directly modulating lipid metabolism in this fat depot. CONCLUSIONS: This study identified an unanticipated efficacy of chronic GIPR agonism to improve dyslipidemia and cardiovascular disease independently of body weight loss, indicating that treatment with acyl-GIP may be a novel approach to alleviate cardiometabolic disease.

Sex-specific and hormone-related differences in vascular remodelling in atherosclerosis.

Atherosclerosis, a lipid-driven inflammatory disease, is the main underlying cause of cardiovascular diseases (CVDs) both in men and women. Sex-related dimorphisms regarding CVDs and atherosclerosis were observed since more than a decade ago. Inflammatory mediators such as cytokines, but also endothelial dysfunction, vascular smooth muscle cell migration and proliferation lead to vascular remodelling but are differentially affected by sex. Each year a greater number of men die of CVDs compared with women and are also affected by CVDs at an earlier age (40-70 years old) while women develop atherosclerosis-related complications mainly after menopause (60+ years). The exact biological reasons behind this discrepancy are still not well-understood. From the numerous animal studies on atherosclerosis, only a few include both sexes and even less investigate and highlight the sex-specific differences that may arise. Endogenous sex hormones such as testosterone and oestrogen modulate the atherosclerotic plaque composition and the frequency of such plaques. In men, testosterone seems to act like a double-edged sword as its decrease with ageing correlates with an increased risk of atherosclerotic CVDs, while testosterone is also reported to promote inflammatory immune cell recruitment into the atherosclerotic plaque. In premenopausal women, oestrogen exerts anti-atherosclerotic effects, which decline together with its level after menopause resulting in increased CVD risk in ageing women. However, the interplay of sex hormones, sex-specific immune responses and other sex-related factors is still incompletely understood. This review highlights reported sex differences in atherosclerotic vascular remodelling and the role of endogenous sex hormones in this process.

Propionate attenuates atherosclerosis by immune-dependent regulation of intestinal cholesterol metabolism.

AIMS: Atherosclerotic cardiovascular disease (ACVD) is a major cause of mortality and morbidity worldwide, and increased low-density lipoproteins (LDLs) play a critical role in development and progression of atherosclerosis. Here, we examined for the first time gut immunomodulatory effects of the microbiota-derived metabolite propionic acid (PA) on intestinal cholesterol metabolism. METHODS AND RESULTS: Using both human and animal model studies, we demonstrate that treatment with PA reduces blood total and LDL cholesterol levels. In apolipoprotein E-/- (Apoe-/-) mice fed a high-fat diet (HFD), PA reduced intestinal cholesterol absorption and aortic atherosclerotic lesion area. Further, PA increased regulatory T-cell numbers and interleukin (IL)-10 levels in the intestinal microenvironment, which in turn suppressed the expression of Niemann-Pick C1-like 1 (Npc1l1), a major intestinal cholesterol transporter. Blockade of IL-10 receptor signalling attenuated the PA-related reduction in total and LDL cholesterol and augmented atherosclerotic lesion severity in the HFD-fed Apoe-/- mice. To translate these preclinical findings to humans, we conducted a randomized, double-blinded, placebo-controlled human study (clinical trial no. NCT03590496). Oral supplementation with 500 mg of PA twice daily over the course of 8 weeks significantly reduced LDL [-15.9 mg/dL (-8.1%) vs. -1.6 mg/dL (-0.5%), P = 0.016], total [-19.6 mg/dL (-7.3%) vs. -5.3 mg/dL (-1.7%), P = 0.014] and non-high-density lipoprotein cholesterol levels [PA vs. placebo: -18.9 mg/dL (-9.1%) vs. -0.6 mg/dL (-0.5%), P = 0.002] in subjects with elevated baseline LDL cholesterol levels. CONCLUSION: Our findings reveal a novel immune-mediated pathway linking the gut microbiota-derived metabolite PA with intestinal Npc1l1 expression and cholesterol homeostasis. The results highlight the gut immune system as a potential therapeutic target to control dyslipidaemia that may introduce a new avenue for prevention of ACVDs.

Endotoxinemia Accelerates Atherosclerosis Through Electrostatic Charge-Mediated Monocyte Adhesion.

BACKGROUND: Acute infection is a well-established risk factor of cardiovascular inflammation increasing the risk for a cardiovascular complication within the first weeks after infection. However, the nature of the processes underlying such aggravation remains unclear. Lipopolysaccharide derived from Gram-negative bacteria is a potent activator of circulating immune cells including neutrophils, which foster inflammation through discharge of neutrophil extracellular traps (NETs). Here, we use a model of endotoxinemia to link acute infection and subsequent neutrophil activation with acceleration of vascular inflammation Methods: Acute infection was mimicked by injection of a single dose of lipopolysaccharide into hypercholesterolemic mice. Atherosclerosis burden was studied by histomorphometric analysis of the aortic root. Arterial myeloid cell adhesion was quantified by intravital microscopy. RESULTS: Lipopolysaccharide treatment rapidly enhanced atherosclerotic lesion size by expansion of the lesional myeloid cell accumulation. Lipopolysaccharide treatment led to the deposition of NETs along the arterial lumen, and inhibition of NET release annulled lesion expansion during endotoxinemia, thus suggesting that NETs regulate myeloid cell recruitment. To study the mechanism of monocyte adhesion to NETs, we used in vitro adhesion assays and biophysical approaches. In these experiments, NET-resident histone H2a attracted monocytes in a receptor-independent, surface charge-dependent fashion. Therapeutic neutralization of histone H2a by antibodies or by in silico designed cyclic peptides enables us to reduce luminal monocyte adhesion and lesion expansion during endotoxinemia. CONCLUSIONS: Our study shows that NET-associated histone H2a mediates charge-dependent monocyte adhesion to NETs and accelerates atherosclerosis during endotoxinemia.

Endothelial ACKR3 drives atherosclerosis by promoting immune cell adhesion to vascular endothelium.

Atherosclerosis is the foundation of potentially fatal cardiovascular diseases and it is characterized by plaque formation in large arteries. Current treatments aimed at reducing atherosclerotic risk factors still allow room for a large residual risk; therefore, novel therapeutic candidates targeting inflammation are needed. The endothelium is the starting point of vascular inflammation underlying atherosclerosis and we could previously demonstrate that the chemokine axis CXCL12-CXCR4 plays an important role in disease development. However, the role of ACKR3, the alternative and higher affinity receptor for CXCL12 remained to be elucidated. We studied the role of arterial ACKR3 in atherosclerosis using western diet-fed Apoe-/- mice lacking Ackr3 in arterial endothelial as well as smooth muscle cells. We show for the first time that arterial endothelial deficiency of ACKR3 attenuates atherosclerosis as a result of diminished arterial adhesion as well as invasion of immune cells. ACKR3 silencing in inflamed human coronary artery endothelial cells decreased adhesion molecule expression, establishing an initial human validation of ACKR3's role in endothelial adhesion. Concomitantly, ACKR3 silencing downregulated key mediators in the MAPK pathway, such as ERK1/2, as well as the phosphorylation of the NF-kB p65 subunit. Endothelial cells in atherosclerotic lesions also revealed decreased phospho-NF-kB p65 expression in ACKR3-deficient mice. Lack of smooth muscle cell-specific as well as hematopoietic ACKR3 did not impact atherosclerosis in mice. Collectively, our findings indicate that arterial endothelial ACKR3 fuels atherosclerosis by mediating endothelium-immune cell adhesion, most likely through inflammatory MAPK and NF-kB pathways.

Neutrophil Extracellular Traps Participate in Cardiovascular Diseases: Recent Experimental and Clinical Insights.

Neutrophil extracellular traps (NETs) have recently emerged as a newly recognized contributor to venous and arterial thrombosis. These strands of DNA extruded by activated or dying neutrophils, decorated with various protein mediators, become solid-state reactors that can localize at the critical interface of blood with the intimal surface of diseased arteries and propagate and amplify the regional injury. NETs thus furnish a previously unsuspected link between inflammation, innate immunity, thrombosis, oxidative stress, and cardiovascular diseases. In response to disease-relevant stimuli, neutrophils undergo a specialized series of reactions that culminate in NET formation. DNA derived from either nuclei or mitochondria can contribute to NET formation. The DNA liberated from neutrophils forms a reticular mesh that resembles morphologically a net, rendering the acronym NETs particularly appropriate. The DNA backbone of NETs not only presents intrinsic neutrophil proteins (eg, MPO [myeloperoxidase] and various proteinases) but can gather other proteins found in blood (eg, tissue factor procoagulant). This review presents current concepts of neutrophil biology, the triggers to and mechanisms of NET formation, and the contribution of NETs to atherosclerosis and to thrombosis. We consider the use of markers of NETs in clinical studies. We aim here to integrate critically the experimental literature with the growing body of clinical information regarding NETs.

Chemokines and galectins form heterodimers to modulate inflammation.

Chemokines and galectins are simultaneously upregulated and mediate leukocyte recruitment during inflammation. Until now, these effector molecules have been considered to function independently. Here, we tested the hypothesis that they form molecular hybrids. By systematically screening chemokines for their ability to bind galectin-1 and galectin-3, we identified several interacting pairs, such as CXCL12 and galectin-3. Based on NMR and MD studies of the CXCL12/galectin-3 heterodimer, we identified contact sites between CXCL12 ß-strand 1 and Gal-3 F-face residues. Mutagenesis of galectin-3 residues involved in heterodimer formation resulted in reduced binding to CXCL12, enabling testing of functional activity comparatively. Galectin-3, but not its mutants, inhibited CXCL12-induced chemotaxis of leukocytes and their recruitment into the mouse peritoneum. Moreover, galectin-3 attenuated CXCL12-stimulated signaling via its receptor CXCR4 in a ternary complex with the chemokine and receptor, consistent with our structural model. This first report of heterodimerization between chemokines and galectins reveals a new type of interaction between inflammatory mediators that can underlie a novel immunoregulatory mechanism in inflammation. Thus, further exploration of the chemokine/galectin interactome is warranted.

Native, Intact Glucagon-Like Peptide 1 Is a Natural Suppressor of Thrombus Growth Under Physiological Flow Conditions.

OBJECTIVE: In patients with diabetes mellitus, increased platelet reactivity predicts cardiac events. Limited evidence suggests that DPP-4 (dipeptidyl peptidase 4) influences platelets via GLP-1 (glucagon-like peptide 1)-dependent effects. Because DPP-4 inhibitors are frequently used in diabetes mellitus to improve the GLP-1-regulated glucose metabolism, we characterized the role of DPP-4 inhibition and of native intact versus DPP-4-cleaved GLP-1 on flow-dependent thrombus formation in mouse and human blood. Approach and Results: An ex vivo whole blood microfluidics model was applied to approach in vivo thrombosis and study collagen-dependent platelet adhesion, activation, and thrombus formation under shear-flow conditions by multiparameter analyses. In mice, in vivo inhibition or genetic deficiency of DPP-4 (Dpp4-/-), but not of GLP-1-receptors (Glp1r-/-), suppressed flow-dependent platelet aggregation. In human blood, GLP-1(7-36), but not DPP-4-cleaved GLP-1(9-36), reduced thrombus volume by 32% and impaired whole blood thrombus formation at both low/venous and high/arterial wall-shear rates. These effects were enforced upon ADP costimulation and occurred independently of plasma factors and leukocytes. Human platelets did not contain detectable levels of GLP-1-receptor transcripts. Also, GLP-1(7-36) did not inhibit collagen-induced aggregation under conditions of stirring or stasis of platelets, pointing to a marked flow-dependent role. CONCLUSIONS: Native, intact GLP-1 is a natural suppressor of thrombus growth under physiological flow conditions, with DPP-4 inhibition and increased intact GLP-1 suppressing platelet aggregation under flow without a main relevance of GLP-1-receptor on platelets.

PCSK9 Imperceptibly Affects Chemokine Receptor Expression In Vitro and In Vivo.

Proprotein convertase subtilin/kexin type 9 (PCSK9) is a protease secreted mainly by hepatocytes and in lesser quantities by intestines, pancreas, and vascular cells. Over the years, this protease has gained importance in the field of cardiovascular biology due to its regulatory action on the low-density lipoprotein receptor (LDLR). However, recently, it has also been shown that PCSK9 acts independent of LDLR to cause vascular inflammation and increase the severity of several cardiovascular disorders. We hypothesized that PCSK9 affects the expression of chemokine receptors, major mediators of inflammation, to influence cardiovascular health. However, using overexpression of PCSK9 in murine models in vivo and PCSK9 stimulation of myeloid and vascular cells in vitro did not reveal influences of PCSK9 on the expression of certain chemokine receptors that are known to be involved in the development and progression of atherosclerosis and vascular inflammation. Hence, we conclude that the inflammatory effects of PCSK9 are not associated with the here investigated chemokine receptors and additional research is required to elucidate which mechanisms mediate PCSK9 effects independent of LDLR.

Hematopoietic ChemR23 (Chemerin Receptor 23) Fuels Atherosclerosis by Sustaining an M1 Macrophage-Phenotype and Guidance of Plasmacytoid Dendritic Cells to Murine Lesions-Brief Report.

Objective- Expression of the chemokine-like receptor ChemR23 (chemerin receptor 23) has been specifically attributed to plasmacytoid dendritic cells (pDCs) and macrophages and ChemR23 has been suggested to mediate an inflammatory immune response in these cells. Because chemokine receptors are important in perpetuating chronic inflammation, we aimed to establish the role of ChemR23-deficiency on macrophages and pDCs in atherosclerosis. Approach and Results- ChemR23-knockout/knockin mice expressing eGFP (enhanced green fluorescent protein) were generated and after crossing with apolipoprotein E-deficient ( Apoe-/- ChemR23 e/e) animals were fed a western-type diet for 4 and 12 weeks. Apoe-/- ChemR23 e/e mice displayed reduced lesion formation and reduced leukocyte adhesion to the vessel wall after 4 weeks, as well as diminished plaque growth, a decreased number of lesional macrophages with an increased proportion of M2 cells and a less inflammatory lesion composition after 12 weeks of western-type diet feeding. Hematopoietic ChemR23-deficiency similarly reduced atherosclerosis. Additional experiments revealed that ChemR23-deficiency induces an alternatively activated macrophage phenotype, an increased cholesterol efflux and a systemic reduction in pDC frequencies. Consequently, expression of the pDC marker SiglecH in atherosclerotic plaques of Apoe-/- ChemR23 e/e mice was declined. ChemR23-knockout pDCs also exhibited a reduced migratory capacity and decreased CCR (CC-type chemokine receptor)7 expression. Finally, adoptive transfer of sorted wild-type and knockout pDCs into Apoe-/- recipient mice revealed reduced accumulation of ChemR23-deficient pDCs in atherosclerotic lesions. Conclusions- Hematopoietic ChemR23-deficiency increases the proportion of alternatively activated M2 macrophages in atherosclerotic lesions and attenuates pDC homing to lymphatic organs and recruitment to atherosclerotic lesions, which synergistically restricts atherosclerotic plaque formation and progression.

Immunotherapy for cardiovascular disease.

The outcomes of the Canakinumab Anti-inflammatory Thrombosis Outcome Study (CANTOS) trial have unequivocally proven that inflammation is a key driver of atherosclerosis and that targeting inflammation, in this case by using an anti-interleukin-1ß antibody, improves cardiovascular disease (CVD) outcomes. This is especially true for CVD patients with a pro-inflammatory constitution. Although CANTOS has epitomized the importance of targeting inflammation in atherosclerosis, treatment with canakinumab did not improve CVD mortality, and caused an increase in infections. Therefore, the identification of novel drug targets and development of novel therapeutics that block atherosclerosis-specific inflammatory pathways and exhibit limited immune-suppressive side effects, as pursued in our collaborative research centre, are required to optimize immunotherapy for CVD. In this review, we will highlight the potential of novel immunotherapeutic targets that are currently considered to become a future treatment for CVD.

Neutrophil Extracellular Traps in Atherosclerosis and Atherothrombosis.

Neutrophil extracellular traps expelled from suicidal neutrophils comprise a complex structure of nuclear chromatin and proteins of nuclear, granular, and cytosolic origin. These net-like structures have also been detected in atherosclerotic lesions and arterial thrombi in humans and mice. Functionally, neutrophil extracellular traps have been shown to induce activation of endothelial cells, antigen-presenting cells, and platelets, resulting in a proinflammatory immune response. Overall, this suggests that they are not only present in plaques and thrombi but also they may play a causative role in triggering atherosclerotic plaque formation and arterial thrombosis. This review will focus on current findings of the involvement of neutrophil extracellular traps in atherogenesis and atherothrombosis.

Chronic Intake of the Selective Serotonin Reuptake Inhibitor Fluoxetine Enhances Atherosclerosis.

OBJECTIVE: Cardiovascular diseases and depression are the leading causes of disability in Western countries. Clinical data on potential cardiovascular effects of serotonin reuptake inhibitors (SSRIs), the most commonly used antidepressant drugs, are controversial. In addition to blocking serotonin reuptake transporter in the brain, SSRIs deplete the major peripheral serotonin (5-hydroxytryptamine [5-HT]) storage by inhibiting serotonin reuptake transporter-mediated uptake in platelets. In this study, we aimed to investigate the effect of chronic SSRI intake on the development of atherosclerosis. APPROACH AND RESULTS: Treatment of apolipoprotein E-deficient mice with the SSRI fluoxetine for 2, 4, or 16 weeks increased atherosclerotic lesion formation, with most pronounced effect during early plaque development. Intravital microscopy of carotid arteries revealed enhanced myeloid cell adhesion on fluoxetine treatment. Mechanistically, we found that fluoxetine augmented vascular permeability and increased chemokine-induced integrin-binding activity of circulating leukocytes. In vitro stimulation of murine blood demonstrated that fluoxetine, but not 5-HT, could directly promote ß1 and ß2 integrin activation provided C-C motif chemokine ligand 5 was also present. Similar effects were observed with the SSRI escitalopram. Enhanced C-C motif chemokine ligand 5-induced integrin activation by fluoxetine was also confirmed in a human neutrophil-like cell line. In contrast to the proatherogenic properties of fluoxetine, pharmacological inhibition of the peripheral 5-HT synthesizing enzyme tryptophan hydroxylase 1 did not promote atherosclerosis, suggesting that the proatherogenic effect of fluoxetine occurs independent of peripheral 5-HT depletion. CONCLUSIONS: SSRI intake may promote atherosclerosis and therefore potentially increase the risk for acute cardiovascular events by a mechanism that is independent of 5-HT depletion.

Vascular CXCR4 Limits Atherosclerosis by Maintaining Arterial Integrity: Evidence From Mouse and Human Studies.

BACKGROUND: The CXCL12/CXCR4 chemokine ligand/receptor axis controls (progenitor) cell homeostasis and trafficking. So far, an atheroprotective role of CXCL12/CXCR4 has only been implied through pharmacological intervention, in particular, because the somatic deletion of the CXCR4 gene in mice is embryonically lethal. Moreover, cell-specific effects of CXCR4 in the arterial wall and underlying mechanisms remain elusive, prompting us to investigate the relevance of CXCR4 in vascular cell types for atheroprotection. METHODS: We examined the role of vascular CXCR4 in atherosclerosis and plaque composition by inducing an endothelial cell (BmxCreERT2-driven)-specific or smooth muscle cell (SMC, SmmhcCreERT2- or TaglnCre-driven)-specific deficiency of CXCR4 in an apolipoprotein E-deficient mouse model. To identify underlying mechanisms for effects of CXCR4, we studied endothelial permeability, intravital leukocyte adhesion, involvement of the Akt/WNT/ß-catenin signaling pathway and relevant phosphatases in VE-cadherin expression and function, vascular tone in aortic rings, cholesterol efflux from macrophages, and expression of SMC phenotypic markers. Finally, we analyzed associations of common genetic variants at the CXCR4 locus with the risk for coronary heart disease, along with CXCR4 transcript expression in human atherosclerotic plaques. RESULTS: The cell-specific deletion of CXCR4 in arterial endothelial cells (n=12-15) or SMCs (n=13-24) markedly increased atherosclerotic lesion formation in hyperlipidemic mice. Endothelial barrier function was promoted by CXCL12/CXCR4, which triggered Akt/WNT/ß-catenin signaling to drive VE-cadherin expression and stabilized junctional VE-cadherin complexes through associated phosphatases. Conversely, endothelial CXCR4 deficiency caused arterial leakage and inflammatory leukocyte recruitment during atherogenesis. In arterial SMCs, CXCR4 sustained normal vascular reactivity and contractile responses, whereas CXCR4 deficiency favored a synthetic phenotype, the occurrence of macrophage-like SMCs in the lesions, and impaired cholesterol efflux. Regression analyses in humans (n=259 796) identified the C-allele at rs2322864 within the CXCR4 locus to be associated with increased risk for coronary heart disease. In line, C/C risk genotype carriers showed reduced CXCR4 expression in carotid artery plaques (n=188), which was furthermore associated with symptomatic disease. CONCLUSIONS: Our data clearly establish that vascular CXCR4 limits atherosclerosis by maintaining arterial integrity, preserving endothelial barrier function, and a normal contractile SMC phenotype. Enhancing these beneficial functions of arterial CXCR4 by selective modulators might open novel therapeutic options in atherosclerosis.

Resolving Lipid Mediators Maresin 1 and Resolvin D2 Prevent Atheroprogression in Mice.

RATIONALE: Atheroprogression is a consequence of nonresolved inflammation, and currently a comprehensive overview of the mechanisms preventing resolution is missing. However, in acute inflammation, resolution is known to be orchestrated by a switch from inflammatory to resolving lipid mediators. Therefore, we hypothesized that lesional lipid mediator imbalance favors atheroprogression. OBJECTIVE: To understand the lipid mediator balance during atheroprogression and to establish an interventional strategy based on the delivery of resolving lipid mediators. METHODS AND RESULTS: Aortic lipid mediator profiling of aortas from Apoe-/- mice fed a high-fat diet for 4 weeks, 8 weeks, or 4 months revealed an expansion of inflammatory lipid mediators, Leukotriene B4 and Prostaglandin E2, and a concomitant decrease of resolving lipid mediators, Resolvin D2 (RvD2) and Maresin 1 (MaR1), during advanced atherosclerosis. Functionally, aortic Leukotriene B4 and Prostaglandin E2 levels correlated with traits of plaque instability, whereas RvD2 and MaR1 levels correlated with the signs of plaque stability. In a therapeutic context, repetitive RvD2 and MaR1 delivery prevented atheroprogression as characterized by halted expansion of the necrotic core and accumulation of macrophages along with increased fibrous cap thickness and smooth muscle cell numbers. Mechanistically, RvD2 and MaR1 induced a shift in macrophage profile toward a reparative phenotype, which secondarily stimulated collagen synthesis in smooth muscle cells. CONCLUSIONS: We present evidence for the imbalance between inflammatory and resolving lipid mediators during atheroprogression. Delivery of RvD2 and MaR1 successfully prevented atheroprogression, suggesting that resolving lipid mediators potentially represent an innovative strategy to resolve arterial inflammation.