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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.

All leukocytes express chemokine receptor CXCR4 on their surface and efficiently migrate in response to its sole ligand CXCL12. Here we report for the first time that CXCR4 is also prominently expressed in erythroblasts, the bone marrow erythroid precursor cells but, instead of chemotaxis, CXCR4 signaling promoted erythroblast enucleation, a unique mammalian process of their asymmetrical division into an anuclear reticulocyte and a nucleus-containing pyrenocyte. Mechanistically, CXCL12 ligation of erythroblast CXCR4 triggered its endocytosis and translocation into multiple intracellular compartments. These included, surprisingly, the nuclear envelope and nucleus where CXCL12 and CXCR4 colocalized with either Gαi or phosphorylated β-arrestin1, respectively, suggesting an ongoing differential CXCR4 signaling around and within the erythroblast nucleus. Supporting the notion of intracellular signaling, stimulation of erythroblasts with CXCL12 caused a persistent phosphorylation of intracellular and intranuclear CXCR4 and a delayed activation of multiple protein kinases. Furthermore, CXCL12 induced erythroblast elongation, a condensation and ex-centric positioning of their nuclei and triggered rapid perinuclear Ca2+ transients, which led directly to erythroblast enucleation. The CXCR4-mediated sequential erythroblast responses taking place during ultimate steps of their development and culminating in enucleation pinpoint a new physiological role of CXCR4 and the bone marrow-derived CXCL12 in regulating erythrocyte production.

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 αIIbβ3 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.

Basic research in cardiology 117(1), 30 (2022). doi:10.1007/s00395-022-00937-4, Published by Springer, Berlin

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. published

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.

Summary 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., Graphical abstract, Highlights • Aged mice show high levels of neutrophil reverse transendothelial migration (rTEM) • Mast cells (MC) and MC-derived CXCL1 drive neutrophil rTEM in inflamed aged tissues • Intensified endothelial ACKR1-CXCL1 axis promotes neutrophil CXCR2 internalization • Aged lungs program rTEM neutrophils toward an activated and noxious phenotype, Aging is a critical risk factor for inflammatory disorders. Barkaway, Rolas et al. show that inflamed aged tissues present a high frequency of neutrophil reverse transendothelial migration (rTEM) back into the circulation in a mast cell-dependent manner. rTEM neutrophils are retained in aged lungs and programmed toward an activated phenotype, capable of inducing tissue damage.

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., SCOPUS: ar.j, info:eu-repo/semantics/published

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 β‐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., Chemokines and galectins are simultaneously upregulated during inflammation and mediate leukocyte recruitment. A systematic screen now demonstrates their physical interaction as heterodimers, identifying several novel interacting pairs.

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., SCOPUS: ar.j, info:eu-repo/semantics/published

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.

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. Ly6C high 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 Ly6C high monocytes were CCR2 high , whereas only a minority was CCR1 high and CCR5 high . 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 Ly6C high 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 Ly6C high monocyte infiltration compared with ZT5-infarcted mice. Concomitantly, CCR2 antagonism only inhibited the excessive cardiac Ly6C high monocyte infiltration after ZT13 MI but not ZT5 MI. Conclusions— CCR2 surface expression on Ly6C high monocytes changes in a time-of-day–dependent manner, which crucially affects cardiac monocyte recruitment after an acute ischemic event.

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.

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-1β 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.

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 β2-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., Central Illustration

Mechanotransduction pathways are activated in response to biophysical stimuli during the development or homeostasis of organs and tissues. In zebrafish, the blood-flow-sensitive transcription factor Klf2a promotes VEGF-dependent angiogenesis. However, the means by which the Klf2a mechanotransduction pathway is regulated to prevent continuous angiogenesis remain unknown. Here we report that the upregulation of klf2 mRNA causes enhanced egfl7 expression and angiogenesis signaling, which underlies cardiovascular defects associated with the loss of cerebral cavernous malformation (CCM) proteins in the zebrafish embryo. Using CCM-protein-depleted human umbilical vein endothelial cells, we show that the misexpression of KLF2 mRNA requires the extracellular matrix-binding receptor beta 1 integrin and occurs in the absence of blood flow. Downregulation of beta 1 integrin rescues ccm mutant cardiovascular malformations in zebrafish. Our work reveals a beta 1 integrin-Klf2-Egfl7-signaling pathway that is tightly regulated by CCM proteins. This regulation prevents angiogenic overgrowth and ensures the quiescence of endothelial cells.

Healthy individuals of African ancestry have neutropenia that has been linked with the variant rs2814778(G) of the gene encoding atypical chemokine receptor 1 (ACKR1). This polymorphism selectively abolishes the expression of ACKR1 in erythroid cells, causing a Duffy-negative phenotype. Here we describe an unexpected fundamental role for ACKR1 in hematopoiesis and provide the mechanism that links its absence with neutropenia. Nucleated erythroid cells had high expression of ACKR1, which facilitated their direct contact with hematopoietic stem cells. The absence of erythroid ACKR1 altered mouse hematopoiesis including stem and progenitor cells, which ultimately gave rise to phenotypically distinct neutrophils that readily left the circulation, causing neutropenia. Individuals with a Duffy-negative phenotype developed a distinct profile of neutrophil effector molecules that closely reflected the one observed in the ACKR1-deficient mice. Thus, alternative physiological patterns of hematopoiesis and bone marrow cell outputs depend on the expression of ACKR1 in the erythroid lineage, findings with major implications for the selection advantages that have resulted in the paramount fixation of the ACKR1 rs2814778(G) polymorphism in Africa.

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.Murine blood and organs were harvested at distinct times of day and analyzed by flow cytometry. Ly6CCCR2 surface expression on Ly6C

The renin-angiotensin (Ang) system is involved in maintaining cardiovascular function by regulating blood pressure and electrolyte homeostasis. More recently, alternative pathways within the renin-angiotensin system have been described, such as the ACE-2/Ang-(1-7)/Mas axis, with opposite effects to the ones of the ACE/Ang-II/AT1 axis. Correspondingly, our previous work reported that Ang-(1-7) via its receptor Mas inhibits the mRNA expression of the proinflammatory cytokines interleukin 6 (IL-6) and tumor necrosis factor-α increased by lipopolysaccharide (LPS) in mouse peritoneal macrophages. These data led us to investigate the functional role of the Ang-(1-7)/Mas axis in an in vivo LPS model. In this work, we present evidence that Ang-(1-7) via Mas significantly reduced the LPS-increased production of circulating cytokines, such as IL-6, IL-12, and CXCL-1. This inhibitory effect was mediated by Mas because it was not detectable in Mas-deficient (Mas) mice. Accordingly, IL-6, CXCL-1, and CXCL-2 levels were higher after LPS treatment in the absence of Mas. Mas mice were less resistant to LPS-induced endotoxemia, their survival rate being 50% compared with 95% in wild-type mice. Telemetric analyses showed that Mas mice presented more pronounced LPS-induced hypothermia with a 3°C lower body temperature compared with wild-type mice. Altogether, our findings suggest that Ang-(1-7) and Mas inhibit LPS-induced cytokine production and hypothermia and thereby protect mice from the fatal consequences of endotoxemia.

The ELR(+)-CXCL chemokines have been described typically as potent chemoattractants and activators of neutrophils during the acute phase of inflammation. Their role in atherosclerosis, a chronic inflammatory vascular disease, has been largely unexplored. Using a mouse model of atherosclerosis, we found that CXCL5 expression was upregulated during disease progression, both locally and systemically, but was not associated with neutrophil infiltration. Unexpectedly, inhibition of CXCL5 was not beneficial but rather induced a significant macrophage foam cell accumulation in murine atherosclerotic plaques. Additionally, we demonstrated that CXCL5 modulated macrophage activation, increased expression of the cholesterol efflux regulatory protein ABCA1, and enhanced cholesterol efflux activity in macrophages. These findings reveal a protective role for CXCL5, in the context of atherosclerosis, centered on the regulation of macrophage foam cell formation.

CXCR4 identifies an immobilized BM precursor (i.e., transitional premonocyte [TpMo]) that proliferates and replenishes mature Ly6Chi monocytes in mice and humans. Upon entering the circulation, CXCR4 governs monocyte margination in the lung vasculature., It is well established that Ly6Chi monocytes develop from common monocyte progenitors (cMoPs) and reside in the bone marrow (BM) until they are mobilized into the circulation. In our study, we found that BM Ly6Chi monocytes are not a homogenous population, as current data would suggest. Using computational analysis approaches to interpret multidimensional datasets, we demonstrate that BM Ly6Chi monocytes consist of two distinct subpopulations (CXCR4hi and CXCR4lo subpopulations) in both mice and humans. Transcriptome studies and in vivo assays revealed functional differences between the two subpopulations. Notably, the CXCR4hi subset proliferates and is immobilized in the BM for the replenishment of functionally mature CXCR4lo monocytes. We propose that the CXCR4hi subset represents a transitional premonocyte population, and that this sequential step of maturation from cMoPs serves to maintain a stable pool of BM monocytes. Additionally, reduced CXCR4 expression on monocytes, upon their exit into the circulation, does not reflect its diminished role in monocyte biology. Specifically, CXCR4 regulates monocyte peripheral cellular activities by governing their circadian oscillations and pulmonary margination, which contributes toward lung injury and sepsis mortality. Together, our study demonstrates the multifaceted role of CXCR4 in defining BM monocyte heterogeneity and in regulating their function in peripheral tissues.

The chemokine CXCL12/SDF-1 is crucial for heart development and affects cardiac repair processes due to its ability to attract leukocytes and stem cells to injured myocardium. However, there is a great controversy whether CXCL12 is beneficial or detrimental after myocardial infarction (MI). The divergence in the reported CXCL12 actions may be due to the cellular source and time of release of the chemokine after MI. This study was designed to evaluate the role of cardiomyocyte-derived CXCL12 for cardiogenesis and heart repair after MI. We generated two rodent models each targeting CXCL12 in only one cardiac cell type: cardiomyocyte-specific CXCL12-overexpressing transgenic (Tg) rats and CXCL12 conditional knockout (cKO) mice. Animals of both models did not show any signs of cardiac abnormalities under baseline conditions. After induction of MI, cKO mice displayed preserved cardiac function and remodeling. Moreover, fibrosis was less pronounced in the hearts of cKO mice after MI. Accordingly, CXCL12 Tg rats revealed impaired cardiac function post-MI accompanied by enhanced fibrosis. Furthermore, we observed decreased numbers of infiltrating Th1 cells in the hearts of cKO mice. Collectively, our findings demonstrate that cardiomyocyte-derived CXCL12 is not involved in cardiac development but has adverse effects on the heart after injury via promotion of inflammation and fibrosis. • CXCL12 in cardiomyocytes is not involved in cardiac development. • CXCL12 deficiency in cardiomyocytes improves outcome of myocardial infarction. • CXCL12 overexpression in cardiomyocytes worsens outcome of myocardial infarction. • CXCL12 increases fibrosis and invasion of Th1 cells in the heart after infarction.

BACKGROUND AND PURPOSE Pre-menopausal females have a lower incidence of cardiovascular disease compared with age-matched males, implying differences in the mechanisms and pathways regulating vasoactivity. In small arteries, myogenic tone (constriction in response to raised intraluminal pressure) is a major determinant of vascular resistance. Endothelium-derived dilators, particularly NO, tonically moderate myogenic tone and, because the endothelium is an important target for female sex hormones, we investigated whether NO-mediated moderation of myogenic tone differed between the sexes. EXPERIMENTAL APPROACH Pressure–diameter or relaxation concentration–response curves to the NO donor spermine-NO or soluble guanylate cyclase (sGC) stimulation (BAY41-2272) were constructed before and following drug intervention in murine mesenteric resistance arteries. Hypotensive responses to activators of the NO-sGC pathway were determined. Quantitative PCR and Western blotting were used for expression analysis. KEY RESULTS NO synthase inhibition enhanced myogenic tone of arteries of both sexes while block of endothelium-derived hyperpolarizing factor (EDHF) enhanced responses in arteries of females only. Spermine-NO concentration-dependently relaxed mesenteric arteries isolated from either sex. However, while inhibition of sGC activity attenuated responses of arteries from male mice only, endothelial denudation attenuated responses of arteries from females only. BAY41-2272 and spermine-NO-induced vasodilatation and hypotension were greater in males than in females. CONCLUSIONS AND IMPLICATIONS NO moderated myogenic tone in arteries of male mice by a sGC-dependent pathway while EDHF was the predominant endothelial regulator in arteries of females. This is a potentially important sexual dimorphism in NO-mediated reactivity and further implicates EDHF as the predominant endothelial vasodilator in female resistance arteries.

Hypercholesterolemia is associated with decreased nitric oxide (NO) bioavailability and endothelial dysfunction, a phenomenon thought to have a major role in the altered cerebral blood flow evident in stroke. Therefore, strategies that increase endothelial NO production have potential utility. Vascular reactivity of the middle cerebral artery (MCA) from C57BL/6J wild-type (WT) mice, apolipoprotein-E knockout (ApoE−/−) mice, and mice treated with the phosphodiesterase inhibitor cilostazol (100 mg/kg) was analyzed using the tension myograph. Contractile responses to endothelin-1 were significantly enhanced in MCA from ApoE−/− mice compared with WT mice ( P−/− mice. Acetylcholine-induced relaxation (which is entirely NO-dependent) was significantly impaired in MCA of ApoE−/− mice compared with WT mice ( P1179 was decreased in the aorta of ApoE−/− mice compared with WT mice ( P

Objective— The proinflammatory phenotype induced by low laminar shear stress (LSS) is implicated in atherogenesis. The kinin B1 receptor (B1R), known to be induced by inflammatory stimuli, exerts many proinflammatory effects including vasodilatation and leukocyte recruitment. We investigated whether low LSS is a stimulus for endothelial B1R expression and function. Methods and Results— Human and mouse atherosclerotic plaques expressed high level of B1R mRNA and protein. In addition, B1R expression was upregulated in the aortic arch (low LSS region) of ApoE −/− mice fed a high-fat diet compared to vascular regions of high LSS and animals fed normal chow. Of interest, a greater expression of B1R was noticed in endothelial cells from regions of low LSS in aortic arch of ApoE −/− mice. B1R was also upregulated in human umbilical vein endothelial cells (HUVECs) exposed to low LSS (0 to 2 dyn/cm 2 ) compared to physiological LSS (6 to 10 dyn/cm 2 ): an effect similarly evident in murine vascular tissue perfused ex vivo. Functionally, B1R activation increased prostaglandin and CXCL5 expression in cells exposed to low, but not physiological, LSS. IL-1β and ox-LDL induced B1R expression and function in HUVECs, a response substantially enhanced under low LSS conditions and inhibited by blockade of NFκB activation. Conclusion— Herein, we show that LSS is a major determinant of functional B1R expression in endothelium. Furthermore, whereas physiological high LSS is a powerful repressor of this inflammatory receptor, low LSS at sites of atheroma is associated with substantial upregulation, identifying this receptor as a potential therapeutic target.

Beyond the classical actions of the renin-angiotensin system on the regulation of cardiovascular homeostasis, several studies have shown its involvement in acute and chronic inflammation. The G protein-coupled receptor Mas is a functional binding site for the angiotensin-(1-7); however, its role in the immune system has not been fully elucidated. In this study, we evaluated the effect of genetic deletion of Mas receptor in lipopolysaccharide (LPS)-induced systemic and cerebral inflammation in mice. Inflammatory response was triggered in Mas deficient (Mas(-/-)) and C57BL/6 wild-type (WT) mice (8-12 weeks-old) by intraperitoneal injection of LPS (5 mg/kg). Mas(-/-) mice presented more intense hypothermia compared to WT mice 24 h after LPS injection. Systemically, the bone marrow of Mas(-/-) mice contained a lower number of neutrophils and monocytes 3 h and 24 h after LPS injection, respectively. The plasma levels of inflammatory mediators KC, MCP-1 and IL-10 were higher in Mas(-/-) mice 24 h after LPS injection in comparison to WT. In the brain, Mas(-/-) animals had a significant increase in the number of adherent leukocytes to the brain microvasculature compared to WT mice, as well as, increased number of monocytes and neutrophils recruited to the pia-mater. The elevated number of adherent leukocytes on brain microvasculature in Mas(-/-) mice was associated with increased expression of CD11b - the alpha-subunit of the Mac-1 integrin - in bone marrow neutrophils 3h after LPS injection, and with increased brain levels of chemoattractants KC, MIP-2 and MCP-1, 24 h later. In conclusion, we demonstrated that Mas receptor deficiency results in exacerbated inflammation in LPS-challenged mice, which suggest a potential role for the Mas receptor as a regulator of systemic and brain inflammatory response induced by LPS.

Mitogenic G protein-coupled receptor (GPCR) signaling has been extensively studied. In contrast, little is known about anti-mitogenic GPCR signaling. We show here that anti-mitogenic signaling of a GPCR, the bradykinin B2 receptor, involves a novel direct protein-protein interaction. The antiproliferative effect of bradykinin was accompanied by a transient increase in protein-tyrosine phosphatase activity. Using surface plasmon resonance analysis, we observed that an immunoreceptor tyrosine-based inhibitory motif (ITIM) located in the C-terminal part of the B2 receptor interacted specifically with the protein-tyrosine phosphatase SHP-2. The interaction was confirmed in primary culture renal mesangial cells by co-immunoprecipitation of a B2 receptor.SHP-2 complex. The extent of the interaction was transiently increased by stimulation with bradykinin, which was accompanied by an increase in specific SHP-2 phosphatase activity. Mutational analysis of the key ITIM residue confirmed that the B2 receptor ITIM sequence is required for interaction with SHP-2, SHP-2 activation, and the anti-mitogenic effect of bradykinin. Finally, in mesangial cells transfected with a dominant-negative form of SHP-2, bradykinin lost the ability to inhibit cell proliferation. These observations demonstrate that bradykinin inhibits cell proliferation by a novel mechanism involving a direct protein-protein interaction between a GPCR (the B2 receptor) and SHP-2.

Several experimental data document an activation of the mitogen-activated protein kinases Erk1 and Erk2 by bradykinin (BK), an agonist of the kinin B2receptor (B2R). In contrast, other reports showed an inhibitory modulation of mitogenesis by BK. Therefore, we explored in the isolated glomeruli the effect of B2R activation on the signaling of insulin-like growth factor-1 (IGF-1), platelet-derived growth factor-BB (PDGF-BB), and high glucose (HG), three factors that are believed to be involved in the development of glomerulosclerosis via the phosphorylation of Erk1 and Erk2. We observed that the activation of B2R negatively modulates the phosphorylation of Erk1 and Erk2 induced by IGF-1, PDGF-BB, and HG in the glomerulus. These effects are consistent with the hypothesis of a protective role for BK in the kidney during development of glomerulosclerosis and renal pathologies associated with a hyperproliferative state.Key words: bradykinin, kinin B2receptors, growth factors, hyperglycemia, glomerulosclerosis.

Nature Communications 7: Article number: 12597 (2016); Published: 31 August 2016; Updated: 3 May 2017 The authors inadvertently omitted Christine Häger, who was involved in the initial characterization of Notch mutant mice presented in this Article, from the author list and Author contributions statement.

Atherosclerosis is a chronic inflammatory disease of large arteries and, among others, characterized by continuous influx of monocytes into the subendothelial space, subsequent macrophage accumulation, and foam cell formation. Chemokines and their receptors tightly orchestrate monocyte trafficking and fate from birth to death. This brief review summarizes our current understanding of the interplay between monocytes and chemokines entertaining crucial processes in atherosclerosis development, progression, and regression.

SummaryIn atherosclerosis, activated platelets have been recently recognised not only to participate in thrombotic events but also to play an essential role in the development of atherosclerotic lesions. Upon their activation, platelets release several pro-inflammatory mediators including chemokines. Chemokines are key molecules in inflammation as they are able to recruit leukocytes, modulate their activation/differentiation and control their proliferation/apoptosis.In this review we will discuss recent findings regarding the specific roles of chemokines released by platelets on leukocytes and their effects on atherosclerosis.

Most end-stage renal disease kidneys display accumulation of extracellular matrix (ECM) in the renal tubular compartment (tubular interstitial fibrosis - TIF) which is strongly correlated with the future loss of renal function. Although inflammation is a key event in the development of TIF, it can also have a beneficial anti-fibrotic role depending in particular on the stage of the pathology. Chemokines play an important role in monocyte extravasation in the inflammatory process. CCL2 has already been shown to be involved in the development of TIF but CCL7, a close relative of CCL2 and able to bind to similar receptors, has not been studied in renal disease. We therefore studied chemokine CCL7 in a model of unilateral ureteral obstruction (UUO)-induced TIF. We observed that the role of CCL7 differs depending on the stage of the pathology. In early stages (0-8 days), CCL7 deficient (CCL7-KO) mice displayed attenuated TIF potentially involving two mechanisms: an early (0-3 days) decrease of inflammatory cell infiltration followed (3-8 days) by a decrease in tubular ECM production independent of inflammation. In contrast, during later stages of obstruction (10-14 days), CCL7-KO mice displayed increased TIF which was again associated with reduced inflammation. Interestingly, the switch between this anti- to profibrotic effect was accompanied by an increased influx of immunosuppressive regulatory T cells. In conclusion, these results highlight for the first time a dual role for CCL7 in the development of renal TIF, deleterious in early stages but beneficial during later stages. (C) 2013 Elsevier Inc. All rights reserved.

Aims Acute myocardial infarction (MI) is the leading cause of mortality worldwide. Anti-inflammatory strategies to reduce neutrophil-driven acute post-MI injury have been shown to limit acute cardiac tissue damage. On the other hand, whether neutrophils are required for resolving post-MI inflammation and repair is unknown. Methods and results We show that neutrophil-depleted mice subjected to MI had worsened cardiac function, increased fibrosis, and progressively developed heart failure. Flow cytometry of blood, lymphoid organs and digested hearts revealed reduced numbers of Ly6Chigh monocytes in infarcts of neutrophil-depleted mice, whereas the number of macrophages increased, which was paralleled by reduced splenic Ly6Chigh monocyte mobilization but enhanced proliferation of cardiac macrophages. Macrophage subtype analysis revealed reduced cardiac expression of M1 markers, whereas M2 markers were increased in neutrophil-depleted mice. Surprisingly, we found reduced expression of phagocytosis receptor myeloid-epithelial-reproductive tyrosine kinase, a marker of reparative M2c macrophages which mediate clearance of apoptotic cells. In agreement with this finding, neutrophil-depleted mice had increased numbers of TUNEL-positive cells within infarcts. We identified neutrophil gelatinase-associated lipocalin (NGAL) in the neutrophil secretome as a key inducer of macrophages with high capacity to engulf apoptotic cells. The cardiac macrophage phenotype in neutrophil-depleted mice was restored by administration of neutrophil secretome or NGAL. Conclusion Neutrophils are crucially involved in cardiac repair after MI by polarizing macrophages towards a reparative phenotype. Therapeutic strategies to reduce acute neutrophil-driven inflammation after MI should be carefully balanced as they might interfere with the healing response and cardiac remodelling.

Rationale : Atherosclerotic lesions express matrix metalloproteinase (MMP)8, which possesses proteolytic activity on matrix proteins particularly fibrillar collagens and on nonmatrix proteins such as angiotensin (Ang) I. Objective : We studied whether MMP8 plays a role in atherogenesis. Methods and Results : In atherosclerosis-prone apolipoprotein E–deficient mice, inactivating MMP8 resulted in a substantial reduction in atherosclerotic lesion formation. Immunohistochemical examinations showed that atherosclerotic lesions in MMP8-deficient mice had significantly fewer macrophages but increased collagen content. In line with results of in vitro assays showing that Ang I cleavage by MMP8 generated Ang II, MMP8 knockout mice had lower Ang II levels and lower blood pressure. In addition, we found that products of Ang I cleavage by MMP8 increased vascular cell adhesion molecule (VCAM)-1 expression and that MMP8-deficient mice had reduced VCAM-1 expression in atherosclerotic lesions. Intravital microscopy analysis showed that leukocyte rolling and adhesion on vascular endothelium was reduced in MMP8 knockout mice. Furthermore, we detected an association between MMP8 gene variation and extent of coronary atherosclerosis in patients with coronary artery disease. A relationship among MMP8 gene variation, plasma VCAM-1 level, and atherosclerosis progression was also observed in a population-based, prospective study. Conclusions : These results indicate that MMP8 is an important player in atherosclerosis.

International audience; Renal fibrosis is the common histological feature of advanced glomerular and tubulointerstitial disease leading to end-stage renal disease (ESRD). However, specific antifibrotic therapies to slow down the evolution to ESRD are still absent. Because persistent inflammation is a key event in the development of fibrosis, we hypothesized that the proinflammatory kinin B1 receptor (B1R) could be such a new target. Here we show that, in the unilateral ureteral obstruction model of renal fibrosis, the B1R is overexpressed and that delayed treatment with an orally active nonpeptide B1R antagonist blocks macrophage infiltration, leading to a reversal of the level of renal fibrosis. In vivo bone marrow transplantation studies as well as in vitro studies on renal cells show that part of this antifibrotic mechanism of B1R blockade involves a direct effect on resident renal cells by inhibiting chemokine CCL2 and CCL7 expression. These findings suggest that blocking the B1R is a promising antifibrotic therapy.

The kinin B1 receptor is an inducible receptor not normally expressed but induced by inflammatory stimuli and plays a major role in neutrophil recruitment, particularly in response to the cytokine IL-1β. However, the exact mechanism involved in this response is unclear. The aim of this study was to dissect the molecular mechanism involved, in particular to determine whether specific ELR-CXCL chemokines (specific neutrophil chemoattractants) played a role. Using intravital microscopy, we demonstrated that IL-1β-induced leukocyte rolling, adherence, and emigration in mesenteric venules of wild-type (WT) mice, associated with an increase in B1 receptor mRNA expression, were substantially attenuated (>80%) in B1 receptor knockout mice (B1KO). This effect in B1KO mice was correlated with a selective down-regulation of IL-1β-induced CXCL5 mRNA and protein expression compared with WT mice. Furthermore a selective neutralizing CXCL5 Ab caused profound suppression of leukocyte emigration in IL-1β-treated WT mice. Finally, treatment of human endothelial cells with IL-1β enhanced mRNA expression of the B1 receptor and the human (h) CXCL5 homologues (hCXCL5 and hCXCL6). This response was suppressed by ∼50% when cells were pretreated with the B1 receptor antagonist des-Arg9-[Leu8]-bradykinin while treatment with des-Arg9-bradykinin, the B1 receptor agonist, caused a concentration-dependent increase in hCXCL5 and hCXCL6 mRNA expression. This study unveils a proinflammatory pathway centered on kinin B1 receptor activation of CXCL5 leading to leukocyte trafficking and highlights the B1 receptor as a potential target in the therapeutics of inflammatory disease.