Targeting a cell-specific microRNA repressor of CXCR4 ameliorates atherosclerosis in mice.

The CXC chemokine receptor 4 (CXCR4) in endothelial cells (ECs) and vascular smooth muscle cells (VSMCs) is crucial for vascular integrity. The atheroprotective functions of CXCR4 in vascular cells may be counteracted by atherogenic functions in other nonvascular cell types. Thus, strategies for cell-specifically augmenting CXCR4 function in vascular cells are crucial if this receptor is to be useful as a therapeutic target in treating atherosclerosis and other vascular disorders. Here, we identified miR-206-3p as a vascular-specific CXCR4 repressor and exploited a target-site blocker (CXCR4-TSB) that disrupted the interaction of miR-206-3p with CXCR4 in vitro and in vivo. In vitro, CXCR4-TSB enhanced CXCR4 expression in human and murine ECs and VSMCs to modulate cell viability, proliferation, and migration. Systemic administration of CXCR4-TSB in Apoe-deficient mice enhanced Cxcr4 expression in ECs and VSMCs in the walls of blood vessels, reduced vascular permeability and monocyte adhesion to endothelium, and attenuated the development of diet-induced atherosclerosis. CXCR4-TSB also increased CXCR4 expression in B cells, corroborating its atheroprotective role in this cell type. Analyses of human atherosclerotic plaque specimens revealed a decrease in CXCR4 and an increase in miR-206-3p expression in advanced compared with early lesions, supporting a role for the miR-206-3p-CXCR4 interaction in human disease. Disrupting the miR-206-3p-CXCR4 interaction in a cell-specific manner with target-site blockers is a potential therapeutic approach that could be used to treat atherosclerosis and other vascular diseases.

The Amino Acid Homoarginine Inhibits Atherogenesis by Modulating T-Cell Function.

BACKGROUND: Amino acid metabolism is crucial for inflammatory processes during atherogenesis. The endogenous amino acid homoarginine is a robust biomarker for cardiovascular outcome and mortality with high levels being protective. However, the underlying mechanisms remain elusive. We investigated the effect of homoarginine supplementation on atherosclerotic plaque development with a particular focus on inflammation. METHODS: Female ApoE-deficient mice were supplemented with homoarginine (14 mg/L) in drinking water starting 2 weeks before and continuing throughout a 6-week period of Western-type diet feeding. Control mice received normal drinking water. Immunohistochemistry and flow cytometry were used for plaque- and immunological phenotyping. T cells were characterized using mass spectrometry-based proteomics, by functional in vitro approaches, for example, proliferation and migration/chemotaxis assays as well as by super-resolution microscopy. RESULTS: Homoarginine supplementation led to a 2-fold increase in circulating homoarginine concentrations. Homoarginine-treated mice exhibited reduced atherosclerosis in the aortic root and brachiocephalic trunk. A substantial decrease in CD3+ T cells in the atherosclerotic lesions suggested a T-cell-related effect of homoarginine supplementation, which was mainly attributed to CD4+ T cells. Macrophages, dendritic cells, and B cells were not affected. CD4+ T-cell proteomics and subsequent pathway analysis together with in vitro studies demonstrated that homoarginine profoundly modulated the spatial organization of the T-cell actin cytoskeleton and increased filopodia formation via inhibition of Myh9 (myosin heavy chain 9). Further mechanistic studies revealed an inhibition of T-cell proliferation as well as a striking impairment of the migratory capacities of T cells in response to relevant chemokines by homoarginine, all of which likely contribute to its atheroprotective effects. CONCLUSIONS: Our study unravels a novel mechanism by which the amino acid homoarginine reduces atherosclerosis, establishing that homoarginine modulates the T-cell cytoskeleton and thereby mitigates T-cell functions important during atherogenesis. These findings provide a molecular explanation for the beneficial effects of homoarginine in atherosclerotic cardiovascular disease.

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.

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.

The dimeric form of CXCL12 binds to atypical chemokine receptor 1.

The pleiotropic chemokine CXCL12 is involved in diverse physiological and pathophysiological processes, including embryogenesis, hematopoiesis, leukocyte migration, and tumor metastasis. It is known to engage the classical receptor CXCR4 and the atypical receptor ACKR3. Differential receptor engagement can transduce distinct cellular signals and effects as well as alter the amount of free, extracellular chemokine. CXCR4 binds both monomeric and the more commonly found dimeric forms of CXCL12, whereas ACKR3 binds monomeric forms. Here, we found that CXCL12 also bound to the atypical receptor ACKR1 (previously known as Duffy antigen/receptor for chemokines or DARC). In vitro nuclear magnetic resonance spectroscopy and isothermal titration calorimetry revealed that dimeric CXCL12 bound to the extracellular N terminus of ACKR1 with low nanomolar affinity, whereas the binding affinity of monomeric CXCL12 was orders of magnitude lower. In transfected MDCK cells and primary human Duffy-positive erythrocytes, a dimeric, but not a monomeric, construct of CXCL12 efficiently bound to and internalized with ACKR1. This interaction between CXCL12 and ACKR1 provides another layer of regulation of the multiple biological functions of CXCL12. The findings also raise the possibility that ACKR1 can bind other dimeric chemokines, thus potentially further expanding the role of ACKR1 in chemokine retention and presentation.

Age-related changes in the local milieu of inflamed tissues cause aberrant neutrophil trafficking and subsequent remote organ damage.

Aging is associated with dysregulated immune functions. Here, we investigated the impact of age on neutrophil diapedesis. Using confocal intravital microscopy, we found that in aged mice, neutrophils adhered to vascular endothelium in inflamed tissues but exhibited a high frequency of reverse transendothelial migration (rTEM). This retrograde breaching of the endothelium by neutrophils was governed by enhanced production of the chemokine CXCL1 from mast cells that localized at endothelial cell (EC) junctions. Increased EC expression of the atypical chemokine receptor 1 (ACKR1) supported this pro-inflammatory milieu in aged venules. Accumulation of CXCL1 caused desensitization of the chemokine receptor CXCR2 on neutrophils and loss of neutrophil directional motility within EC junctions. Fluorescent tracking revealed that in aged mice, neutrophils undergoing rTEM re-entered the circulation and disseminated to the lungs where they caused vascular leakage. Thus, neutrophils stemming from a local inflammatory site contribute to remote organ damage, with implication to the dysregulated systemic inflammation associated with aging.

Autophagy unleashes noncanonical microRNA functions.

MicroRNAs (miRNAs) are post-transcriptional regulators of gene expression which act by guiding AGO (argonaute) proteins to target RNA transcripts in the RNA-induced silencing complex (RISC). This macromolecular complex includes multiple additional components (e.g., TNRC6A) that allow for interaction with enzymes mediating inhibition of translation or RNA decay. However, miRNAs also reside in low-molecular weight complexes without being engaged in target repression, and their function in this context is largely unknown. Our recent findings show that endothelial cells exposed to protective high-shear stress or MTORC inhibition activate the macroautophagy/autophagy machinery to sustain viability by promoting differential trafficking of MIR126 strands and by enabling unconventional features of MIR126-5p. Whereas MIR126-3p is degraded upon autophagy activation, MIR126-5p interacts with the RNA-binding protein MEX3A to form a ternary complex with AGO2. This complex forms on the autophagosomal surface and facilitates its nuclear localization. Once in the nucleus, MIR126-5p dissociates from AGO2 and establishes aptamer-like interactions with the effector CASP3 (caspase 3). The binding to MIR126-5p prevents dimerization and proper active site formation of CASP3, thus inhibiting proteolytic activity and limiting apoptosis. Disrupting this pathway in vivo by genetic deletion of Mex3a or by specific deficiency of endothelial autophagy aggravates endothelial apoptosis and exacerbates the progression of atherosclerosis. The direct inhibition of CASP3 by MIR126-5p reveals a non-canonical mechanism by which miRNAs can modulate protein function and mediate the autophagy-apoptosis crosstalk.

Noncanonical inhibition of caspase-3 by a nuclear microRNA confers endothelial protection by autophagy in atherosclerosis.

MicroRNAs (miRNAs) are versatile regulators of gene expression with profound implications for human disease including atherosclerosis, but whether they can exert posttranslational functions to control cell adaptation and whether such noncanonical features harbor pathophysiological relevance is unknown. Here, we show that miR-126-5p sustains endothelial integrity in the context of high shear stress and autophagy. Bound to argonaute-2 (Ago2), miR-126-5p forms a complex with Mex3a, which occurs on the surface of autophagic vesicles and guides its transport into the nucleus. Mutational studies and biophysical measurements demonstrate that Mex3a binds to the central U- and G-rich regions of miR-126-5p with nanomolar affinity via its two K homology domains. In the nucleus, miR-126-5p dissociates from Ago2 and binds to caspase-3 in an aptamer-like fashion with its seed sequence, preventing dimerization of the caspase and inhibiting its activity to limit apoptosis. The antiapoptotic effect of miR-126-5p outside of the RNA-induced silencing complex is important for endothelial integrity under conditions of high shear stress promoting autophagy: ablation of Mex3a or ATG5 in vivo attenuates nuclear import of miR-126-5p, aggravates endothelial apoptosis, and exacerbates atherosclerosis. In human plaques, we found reduced nuclear miR-126-5p and active caspase-3 in areas of disturbed flow. The direct inhibition of caspase-3 by nuclear miR-126-5p reveals a noncanonical mechanism by which miRNAs can modulate protein function.

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.

PD-L1 expression on nonclassical monocytes reveals their origin and immunoregulatory function.

The role of nonclassical monocytes (NCMs) in health and disease is emerging, but their location and function within tissues remain poorly explored. Imaging of NCMs has been limited by the lack of an established single NCM marker. Here, we characterize the immune checkpoint molecule PD-L1 (CD274) as an unequivocal marker for tracking NCMs in circulation and pinpoint their compartmentalized distribution in tissues by two-photon microscopy. Visualization of PD-L1+ NCMs in relation to bone marrow vasculature reveals that conversion of classical monocytes into NCMs requires contact with endosteal vessels. Furthermore, PD-L1+ NCMs are present in tertiary lymphoid organs (TLOs) under inflammatory conditions in both mice and humans, and NCMs exhibit a PD-L1-dependent immunomodulatory function that promotes T cell apoptosis within TLOs. Our findings establish an unambiguous tool for the investigation of NCMs and shed light on their origin and function.

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.

Atypical Chemokine Receptors in Cardiovascular Disease.

Inflammation has been well recognized as one of the main drivers of atherosclerosis development and therefore cardiovascular diseases (CVDs). It has been shown that several chemokines, small 8 to 12 kDa cytokines with chemotactic properties, play a crucial role in the pathophysiology of atherosclerosis. Chemokines classically mediate their effects by binding to G-protein-coupled receptors called chemokine receptors. In addition, chemokines can also bind to atypical chemokine receptors (ACKRs). ACKRs fail to induce G-protein-dependent signalling pathways and thus subsequent cellular response, but instead are able to internalize, scavenge or transport chemokines. In this review, we will give an overview of the current knowledge about the involvement of ACKR1-4 in CVDs and especially in atherosclerosis development. In the recent years, several studies have highlighted the importance of ACKRs in CVDs, although there are still several controversies and unexplored aspects that have to be further elucidated. A better understanding of the precise role of these atypical receptors may pave the way towards novel and improved therapeutic strategies.

Targeting CD40-Induced TRAF6 Signaling in Macrophages Reduces Atherosclerosis.

BACKGROUND: Disrupting the costimulatory CD40-CD40L dyad reduces atherosclerosis, but can result in immune suppression. The authors recently identified small molecule inhibitors that block the interaction between CD40 and tumor necrosis factor receptor-associated factor (TRAF) 6 (TRAF-STOPs), while leaving CD40-TRAF2/3/5 interactions intact, thereby preserving CD40-mediated immunity. OBJECTIVES: This study evaluates the potential of TRAF-STOP treatment in atherosclerosis. METHODS: The effects of TRAF-STOPs on atherosclerosis were investigated in apolipoprotein E deficient (Apoe-/-) mice. Recombinant high-density lipoprotein (rHDL) nanoparticles were used to target TRAF-STOPs to macrophages. RESULTS: TRAF-STOP treatment of young Apoe-/- mice reduced atherosclerosis by reducing CD40 and integrin expression in classical monocytes, thereby hampering monocyte recruitment. When Apoe-/- mice with established atherosclerosis were treated with TRAF-STOPs, plaque progression was halted, and plaques contained an increase in collagen, developed small necrotic cores, and contained only a few immune cells. TRAF-STOP treatment did not impair "classical" immune pathways of CD40, including T-cell proliferation and costimulation, Ig isotype switching, or germinal center formation, but reduced CD40 and ß2-integrin expression in inflammatory monocytes. In vitro testing and transcriptional profiling showed that TRAF-STOPs are effective in reducing macrophage migration and activation, which could be attributed to reduced phosphorylation of signaling intermediates of the canonical NF-κB pathway. To target TRAF-STOPs specifically to macrophages, TRAF-STOP 6877002 was incorporated into rHDL nanoparticles. Six weeks of rHDL-6877002 treatment attenuated the initiation of atherosclerosis in Apoe-/- mice. CONCLUSIONS: TRAF-STOPs can overcome the current limitations of long-term CD40 inhibition in atherosclerosis and have the potential to become a future therapeutic for atherosclerosis.

Pericardial Adipose Tissue Regulates Granulopoiesis, Fibrosis, and Cardiac Function After Myocardial Infarction.

BACKGROUND: The pericardial adipose tissue (AT) contains a high density of lymphoid clusters. It is unknown whether these clusters play a role in post-myocardial infarction (MI) inflammatory responses and cardiac outcome. METHODS: Lymphoid clusters were examined in epicardial AT of humans with or without coronary artery disease. Murine pericardial lymphoid clusters were visualized in mice subjected to coronary artery ligation. To study the relevance of pericardial clusters during inflammatory responses after MI, we surgically removed the pericardial AT and performed B-cell depletion and granulocyte-macrophage colony-stimulating factor blockade. Leukocytes in murine hearts, pericardial AT, spleen, mediastinal lymph nodes, and bone marrow were quantified by flow cytometry. Cannabinoid receptor CB2 (CB2-/-) mice were used as a model for enhanced B-cell responses. The effect of impaired dendritic cell (DC) trafficking on pericardial AT inflammatory responses was tested in CCR7-/- mice subjected to MI. Cardiac fibrosis and ventricular function were assessed by histology and echocardiography. RESULTS: We identified larger B-cell clusters in epicardial AT of human patients with coronary artery disease in comparison with controls without coronary artery disease. Infarcted mice also had larger pericardial clusters and 3-fold upregulated numbers of granulocyte-macrophage colony-stimulating factor-producing B cells within pericardial AT, but not spleen or lymph nodes. This was associated with higher DC and T-cell counts in pericardial AT, which outnumbered DCs and T cells in lymph nodes. Analysis of DC maturation markers, tracking experiments with fluorescently labeled cells, and use of CCR7-deficient mice suggested that activated DCs migrate from infarcts into pericardial AT via CCR7. B-cell depletion or granulocyte-macrophage colony-stimulating factor neutralization inhibited DC and T-cell expansion within pericardial AT, and translated into reduced bone marrow granulopoiesis and cardiac neutrophil infiltration 3 days after MI. The relevance of the pericardial AT in mediating all these effects was confirmed by removal of pericardial AT and ex vivo coculture with pericardial AT and granulocyte progenitors. Finally, enhanced fibrosis and worsened ejection fraction in CB2-/- mice were limited by pericardial AT removal. CONCLUSIONS: Our findings unveil a new mechanism by which the pericardial AT coordinates immune cell activation, granulopoiesis, and outcome after MI.

Ly6Chigh Monocytes Oscillate in the Heart During Homeostasis and After Myocardial Infarction-Brief Report.

OBJECTIVE: Circadian regulation of neutrophil homeostasis affects myocardial infarction (MI) healing. It is unknown whether diurnal variations of monocyte counts exist in the heart and whether this affects their cardiac infiltration in response to MI. APPROACH AND RESULTS: Murine blood and organs were harvested at distinct times of day and analyzed by flow cytometry. Ly6Chigh monocyte surface expression levels of chemokine receptors (CCR) were ≈2-fold higher at the beginning of the active phase, Zeitgeber Time (ZT) 13 compared with ZT5. This was because of enhanced receptor surface expression at ZT13, whereas no significant changes in total cellular protein levels were found. Most blood Ly6Chigh monocytes were CCR2high, whereas only a minority was CCR1high and CCR5high. We also found diurnal changes of classical monocyte blood counts in humans, being higher in the evening, while exhibiting enhanced CCR2 surface expression in the morning. In support of monocyte oscillations between blood and tissue, murine cardiac Ly6Chigh monocyte counts were highest at ZT13, accompanied by an upregulation of cardiac CC chemokine ligand 2 mRNA. Mice subjected to MI at ZT13 had an even higher upregulation of CCR2 surface expression on circulating monocytes compared with noninfarcted mice and more elevated cardiac CC chemokine ligand 2 protein expression and more pronounced Ly6Chigh monocyte infiltration compared with ZT5-infarcted mice. Concomitantly, CCR2 antagonism only inhibited the excessive cardiac Ly6Chigh monocyte infiltration after ZT13 MI but not ZT5 MI. CONCLUSIONS: CCR2 surface expression on Ly6Chigh monocytes changes in a time-of-day-dependent manner, which crucially affects cardiac monocyte recruitment after an acute ischemic event.