The CXCL12/CXCR4 chemokine ligand/receptor axis in cardiovascular disease

The chemokine receptor CXCR4 and its ligand CXCL12 play an important homeostatic function by mediating the homing of progenitor cells in the bone marrow and regulating their mobilization into peripheral tissues upon injury or stress. Although the CXCL12/CXCR4 interaction has long been regarded as a monogamous relation, the identification of the pro-inflammatory chemokine macrophage migration inhibitory factor (MIF) as an important second ligand for CXCR4, and of CXCR7 as an alternative receptor for CXCL12, has undermined this interpretation and has considerably complicated the understanding of CXCL12/CXCR4 signaling and associated biological functions. This review aims to provide insight into the current concept of the CXCL12/CXCR4 axis in myocardial infarction (MI) and its underlying pathologies such as atherosclerosis and injury-induced vascular restenosis. It will discuss main findings from in vitro studies, animal experiments and large-scale genome-wide association studies. The importance of the CXCL12/CXCR4 axis in progenitor cell homing and mobilization will be addressed, as will be the function of CXCR4 in different cell types involved in atherosclerosis. Finally, a potential translation of current knowledge on CXCR4 into future therapeutical application will be discussed

Systematic RNA-interference in primary human monocyte-derived macrophages: A high-throughput platform to study foam cell formation

Macrophage-derived foam cells are key regulators of atherogenesis. They accumulate in atherosclerotic plaques and support inflammatory processes by producing cytokines and chemokines. Identifying factors that regulate macrophage lipid uptake may reveal therapeutic targets for coronary artery disease (CAD). Here, we establish a high-throughput screening workflow to systematically identify genes that impact the uptake of DiI-labeled low-density lipoprotein (LDL) into monocyte-derived primary human macrophages. For this, monocytes isolated from peripheral blood were seeded onto 384-well plates, solid-phase transfected with siRNAs, differentiated in vitro into macrophages, and LDL-uptake per cell was measured by automated microscopy and quantitative image analysis. We applied this workflow to study how silencing of 89 genes impacts LDL-uptake into cells from 16 patients with CAD and 16 age-matched controls. Silencing of four novel genes (APOC1, CMTM6, FABP4, WBP5) reduced macrophage LDL-uptake. Additionally, knockdown of the chemokine receptor CXCR4 reduced LDL-uptake, most likely through a G-protein coupled mechanism that involves the CXCR4 ligand macrophage-induced factor (MIF), but is independent of CXCL12. We introduce a high-throughput strategy to systematically study gene function directly in primary CAD-patient cells. Our results propose a function for the MIF/CXCR4 signaling pathway, as well as several novel candidate genes impacting lipid uptake into human macrophages

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/beta-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=1215) 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/beta-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

Diversity and inter-connections in the CXCR4 chemokine receptor/ligand family: molecular perspectives

CXCR4 and its ligand CXCL12 mediate the homing of progenitor cells in the bone marrow and their recruitment to sites of injury, as well as affect processes such as cell arrest, survival, and angiogenesis. CXCL12 was long thought to be the sole CXCR4 ligand, but more recently the atypical chemokine macrophage migration inhibitory factor (MIF) was identified as an alternative, non cognate ligand for CXCR4 and shown to mediate chemotaxis and arrest of CXCR4-expressing T-cells. This has complicated the understanding of CXCR4-mediated signaling and associated biological processes. Compared to CXCL12/CXCR4-induced signaling, only few details are known on MIF/CXCR4-mediated signaling and it remains unclear to which extent MIF and CXCL12 reciprocally influence CXCR4 binding and signaling. Furthermore, the atypical chemokine receptor 3 (ACKR3) (previously CXCR7) has added to the complexity of CXCR4 signaling due to its ability to bind CXCL12 and MIF, and to evoke CXCL12-and MIF-triggered signaling independently of CXCR4. Also, extracellular ubiquitin (eUb) and the viral protein gp120 (HIV) have been reported as CXCR4 ligands, whereas viral chemokine vMIP-11 (Herpesvirus) and human beta 3-defensin (HBD-3) have been identified as CXCR4 antagonists. This review will provide insight into the diversity and inter-connections in the CXCR4 receptor/ligand family. We will discuss signaling pathways initiated by binding of CXCL12 vs. MIF to CXCR4, elaborate on how ACKR3 affects CXCR4 signaling, and summarize biological functions of CXCR4 signaling mediated by CXCL12 or MIF. Also, we will discuss eUb and gp120 as alternative ligands for CXCR4, and describe vMIP-11 and HBD-3 as antagonists for CXCR4. Detailed insight into biological effects of CXCR4 signaling und underlying mechanisms, including diversity of CXCR4 ligands and inter connections with other (chemokine) receptors, is clinically important, as the CXCR4 antagonist AMD3100 has been approved as stem cell mobilizer in specific disease settings

Bone Marrow-Specific Knock-In of a Non-Activatable Ikkα Kinase Mutant Influences Haematopoiesis but Not Atherosclerosis in Apoe-Deficient Mice

Background: The Ikkα kinase, a subunit of the NF-kappa B-activating IKK complex, has emerged as an important regulator of inflammatory gene expression. However, the role of Ikkα-mediated phosphorylation in haematopoiesis and atherogenesis remains unexplored. In this study, we investigated the effect of a bone marrow (BM)-specific activation-resistant Ikk alpha mutant knock-in on haematopoiesis and atherosclerosis in mice. Methods and Results: Apolipoprotein E (Apoe)-deficient mice were transplanted with BM carrying an activation-resistant Ikkα gene (Ikkα(AA/AA) Apoe(-/-)) or with Ikkα(+/+) Apoe(-/-) BM as control and were fed a high-cholesterol diet for 8 or 13 weeks. Interestingly, haematopoietic profiling by flow cytometry revealed a significant decrease in B-cells, regulatory T-cells and effector memory T-cells in Ikkα(AA/AA) Apoe(-/-) BM-chimeras, whereas the naive T-cell population was increased. Surprisingly, no differences were observed in the size, stage or cellular composition of atherosclerotic lesions in the aorta and aortic root of Ikkα(AA/AA) Apoe(-/-) vs Ikkα(+/+) Apoe(-/-) BM-transplanted mice, as shown by histological and immunofluorescent stainings. Necrotic core sizes, apoptosis, and intracellular lipid deposits in aortic root lesions were unaltered. In vitro, BM-derived macrophages from Ikkα(AA/AA) Apoe(-/-) vs Ikkα(+/+) Apoe(-/-) mice did not show significant differences in the uptake of oxidized low-density lipoproteins (oxLDL), and, with the exception of Il-12, the secretion of inflammatory proteins in conditions of Tnf-α or oxLDL stimulation was not significantly altered. Furthermore, serum levels of inflammatory proteins as measured with a cytokine bead array were comparable. Conclusion: Our data reveal an important and previously unrecognized role of haematopoietic Ikkα kinase activation in the homeostasis of B-cells and regulatory T-cells. However, transplantation of Ikkα AA mutant BM did not affect atherosclerosis in Apoe(-/-) mice. This suggests that the diverse functions of Ikkα in haematopoietic cells may counterbalance each other or may not be strong enough to influence atherogenesis, and reveals that targeting haematopoietic Ikkα kinase activity alone does not represent a therapeutic approach

NON-ACTIVATABLE MUTANT OF INHIBITOR OF KAPPA B KINASE alpha (IKK alpha) EXERTS SITE-SPECIFIC EFFECTS ON ATHEROSCLEROSIS

53rd ERA-EDTA Congress, Vienna, AUSTRIA, MAY 21-24, 2016International audienceno abstrac

Leukocyte numbers and frequencies of leukocyte subsets in <i>Apoe^−/−</i> mice transplanted with <i>Ikkα^AA/AAApoe^−/−</i> or <i>Ikkα^+/+Apoe^−/−</i> BM.

<p>Peripheral blood leukocyte counts were determined after 8 weeks of high-fat diet (n = 8). Leukocyte subset frequencies were quantified by flow cytometry after 13 weeks of high-fat diet (n = 18–19).</p