C-X-C Motif Chemokine Receptor 4 Blockade Promotes Tissue Repair After Myocardial Infarction by Enhancing Regulatory T Cell Mobilization and Immune-Regulatory Function.

BACKGROUND: Acute myocardial infarction (MI) elicits an inflammatory response that drives tissue repair and adverse cardiac remodeling. Inflammatory cell trafficking after MI is controlled by C-X-C motif chemokine ligand 12 (CXCL12) and its receptor, C-X-C motif chemokine receptor 4 (CXCR4). CXCR4 antagonists mobilize inflammatory cells and promote infarct repair, but the cellular mechanisms are unclear. METHODS: We investigated the therapeutic potential and mode of action of the peptidic macrocycle CXCR4 antagonist POL5551 in mice with reperfused MI. We applied cell depletion and adoptive transfer strategies using lymphocyte-deficient Rag1 knockout mice; DEREG mice, which express a diphtheria toxin receptor-enhanced green fluorescent protein fusion protein under the control of the promoter/enhancer region of the regulatory T (Treg) cell-restricted Foxp3 transcription factor; and dendritic cell-depleted CD11c-Cre iDTR mice. Translational potential was explored in a porcine model of reperfused MI using serial contrast-enhanced magnetic resonance imaging. RESULTS: Intraperitoneal POL5551 injections in wild-type mice (8 mg/kg at 2, 4, 6, and 8 days) enhanced angiogenesis in the infarct border zone, reduced scar size, and attenuated left ventricular remodeling and contractile dysfunction at 28 days. Treatment effects were absent in splenectomized wild-type mice, Rag1 knockout mice, and Treg cell-depleted DEREG mice. Conversely, treatment effects could be transferred into infarcted splenectomized wild-type mice by transplanting splenic Treg cells from POL5551-treated infarcted DEREG mice. Instructive cues provided by infarct-primed dendritic cells were required for POL5551 treatment effects. POL5551 injections mobilized Treg cells into the peripheral blood, followed by enhanced Treg cell accumulation in the infarcted region. Neutrophils, monocytes, and lymphocytes displayed similar mobilization kinetics, but their cardiac recruitment was not affected. POL5551, however, attenuated inflammatory gene expression in monocytes and macrophages in the infarcted region via Treg cells. Intravenous infusion of the clinical-stage POL5551 analogue POL6326 (3 mg/kg at 4, 6, 8, and 10 days) decreased infarct volume and improved left ventricular ejection fraction in pigs. CONCLUSIONS: These data confirm CXCR4 blockade as a promising treatment strategy after MI. We identify dendritic cell-primed splenic Treg cells as the central arbiters of these therapeutic effects and thereby delineate a pharmacological strategy to promote infarct repair by augmenting Treg cell function in vivo.

[Expression and significance of chemokine CXCL12 and receptor CXCR4 in adenomyosis].

Objective: To observe the expression, correlation and significance of chemokine (C-X-C motif) ligand 12 (CXCL12) and chemokine (C-X-C motif) receptor 4 (CXCR4) in endometrium and myometrium of adenomyosis. Methods: Totally 38 patients were selected in this study, who underwent hysterectomy for adenomyosis at Beijing Obstetrics and Gynecology Hospital from October 2017 to December 2018 as the adenomyosis group, and, in the same period, selected 31 patients with cervical intraepithelial neoplasia Ⅲ or cervical cancer undergoing hysterectomy served as control group. The expression levels of mRNA and protein for CXCL12, CXCR4 in the endometrium and myometrium of the two groups were detected by immunohistochemistry and real-time PCR. Results: (1) The protein levels of CXCL12 and CXCR4 in endometrium in uterus with adenomyosis (0.229±0.025 and 0.226±0.016) were significantly higher than those in endometrium in uterus without adenomyosis (0.153±0.018 and 0.178±0.026); compared with each other, the differences were statistically significant (all P<0.05). And the expressions of CXCL12 and CXCR4 proteins in uterine myometrium of adenomyosis were 0.222±0.045 and 0.126±0.058, respectively, which were higher than those in the control group (0.091±0.029 and 0.099±0.020); compared with each other, the differences were statistically significant (all P<0.05). (2) The expression levels of CXCL12 and CXCR4 mRNA in endometrium of patients with adenomyosis were 6.31±0.12 and 8.49±0.21, respectively, which were higher than those in the control group (1.23±0.10 and 1.36±0.13); compared with each other, the differences were statistically significant (all P<0.05). Moreover, the expression levels of CXCL12 and CXCR4 mRNA in myometrium of patients with adenomyosis were 9.11±0.12 and 8.45±0.16, respectively, which were higher than those in the control group (1.18±0.08 and 1.46±0.13); compared with each other, the differences were statistically significant (all P<0.05). (3) In endometrium and myometrium of uterus with adenomyosis, CXCL12 and CXCR4 mRNA expression levels were positively associated (r=0.478, 0.542, all P<0.05). Conclusions: The levels of CXCL12 and CXCR4 in the endometrium and myometrium of adenomyosis are increased and positively correlated. The two chemokine may be involved in the development of adenomyosis.

The macrophage migration inhibitory factor pathway in human B cells is tightly controlled and dysregulated in multiple sclerosis.

In MS, B cells survive peripheral tolerance checkpoints to mediate local inflammation, but the underlying molecular mechanisms are relatively underexplored. In mice, the MIF pathway controls B-cell development and the induction of EAE. Here, we found that MIF and MIF receptor CD74 are downregulated, while MIF receptor CXCR4 is upregulated in B cells from early onset MS patients. B cells were identified as the main immune subset in blood expressing MIF. Blocking of MIF and CD74 signaling in B cells triggered CXCR4 expression, and vice versa, with separate effects on their proinflammatory activity, proliferation, and sensitivity to Fas-mediated apoptosis. This study reveals a new reciprocal negative regulation loop between CD74 and CXCR4 in human B cells. The disturbance of this loop during MS onset provides further insights into how pathogenic B cells survive peripheral tolerance checkpoints to mediate disease activity in MS.

The Effects of the CXCR4 Antagonist, AMD3465, on Human Retinal Vascular Endothelial Cells (hRVECs) in a High Glucose Model of Diabetic Retinopathy.

BACKGROUND High blood glucose levels in diabetes result in retinal angiogenesis, which is the key feature of diabetic retinopathy. This study aimed to investigate the effects of the CXCR4 antagonist, AMD3465, on human retinal vascular endothelial cells (hRVECs) [i]in vitro[/i]. MATERIAL AND METHODS Cell viability and the protein expression levels of CXCR4 and stromal cell-derived factor 1 (SDF-1) were evaluated in high glucose (HG)-treated human retinal vascular endothelial cells (hRVECs). The cell counting kit 8 (CCK-8) assay, the colony formation assay, immunofluorescence, and Western blot were used to investigate the effects of AMD3465 on hRVEC cell viability, colony formation, cell proliferation, and expression of CXCR4 and SDF-1. Cell apoptosis and angiogenesis were assessed by flow cytometry and Western blot. RESULTS Treatment with high glucose reduced the viability of hRVECs and increased the protein expression levels of CXCR4 and SDF-1. Following treatment with AMD3465, the colony formation capacity and cell proliferation in hRVECs increased, and there was a significant reduction in apoptosis rate compared with the untreated cells. AMD3465 significantly reduced the expression of angiogenesis-associated proteins, including ICAM1, VCAM1, VEGF, and AngII. AMD3465 significantly reduced the protein expression levels of TNF-α, IL-1ß, NF-κB, and p-p65. CONCLUSIONS The CXCR4 antagonist, AMD3465, reduced apoptosis of HG-treated hRVECs in an [i]in vitro[/i] model of diabetic retinopathy.

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.

Stimulatory Effects of Mesenchymal Stem Cells on cKit+ Cardiac Stem Cells Are Mediated by SDF1/CXCR4 and SCF/cKit Signaling Pathways.

RATIONALE: Culture-expanded cells originating from cardiac tissue that express the cell surface receptor cKit are undergoing clinical testing as a cell source for heart failure and congenital heart disease. Although accumulating data support that mesenchymal stem cells (MSCs) enhance the efficacy of cardiac cKit(+) cells (CSCs), the underlying mechanism for this synergistic effect remains incompletely understood. OBJECTIVE: To test the hypothesis that MSCs stimulate endogenous CSCs to proliferate, migrate, and differentiate via the SDF1/CXCR4 and stem cell factor/cKit pathways. METHODS AND RESULTS: Using genetic lineage-tracing approaches, we show that in the postnatal murine heart, cKit(+) cells proliferate, migrate, and form cardiomyocytes, but not endothelial cells. CSCs exhibit marked chemotactic and proliferative responses when cocultured with MSCs but not with cardiac stromal cells. Antagonism of the CXCR4 pathway with AMD3100 (an SDF1/CXCR4 antagonist) inhibited MSC-induced CSC chemotaxis but stimulated CSC cardiomyogenesis (P<0.0001). Furthermore, MSCs enhanced CSC proliferation via the stem cell factor/cKit and SDF1/CXCR4 pathways (P<0.0001). CONCLUSIONS: Together these findings show that MSCs exhibit profound, yet differential, effects on CSC migration, proliferation, and differentiation and suggest a mechanism underlying the improved cardiac regeneration associated with combination therapy using CSCs and MSCs. These findings have important therapeutic implications for cell-based therapy strategies that use mixtures of CSCs and MSCs.

Misregulation of SDF1-CXCR4 signaling impairs early cardiac neural crest cell migration leading to conotruncal defects.

RATIONALE: Cardiac neural crest cells (NCs) contribute to heart morphogenesis by giving rise to a variety of cell types from mesenchyme of the outflow tract, ventricular septum, and semilunar valves to neurons of the cardiac ganglia and smooth muscles of the great arteries. Failure in cardiac NC development results in outflow and ventricular septation defects commonly observed in congenital heart diseases. Cardiac NCs derive from the vagal neural tube, which also gives rise to enteric NCs that colonize the gut; however, so far, molecular mechanisms segregating these 2 populations and driving cardiac NC migration toward the heart have remained elusive. OBJECTIVE: Stromal-derived factor-1 (SDF1) is a chemokine that mediates oriented migration of multiple embryonic cells and mice deficient for Sdf1 or its receptors, Cxcr4 and Cxcr7, exhibit ventricular septum defects, raising the possibility that SDF1 might selectively drive cardiac NC migration toward the heart via a chemotactic mechanism. METHODS AND RESULTS: We show in the chick embryo that Sdf1 expression is tightly coordinated with the progression of cardiac NCs expressing Cxcr4. Cxcr4 loss-of-function causes delayed migration and enhanced death of cardiac NCs, whereas Sdf1 misexpression results in their diversion from their normal pathway, indicating that SDF1 acts as a chemoattractant for cardiac NCs. These alterations of SDF1 signaling result in severe cardiovascular defects. CONCLUSIONS: These data identify Sdf1 and its receptor Cxcr4 as candidate genes responsible for cardiac congenital pathologies in human.

Deficiency of endothelial CXCR4 reduces reendothelialization and enhances neointimal hyperplasia after vascular injury in atherosclerosis-prone mice.

OBJECTIVE: The Cxcl12/Cxcr4 chemokine ligand/receptor axis mediates the mobilization of smooth muscle cell progenitors, driving injury-induced neointimal hyperplasia. This study aimed to investigate the role of endothelial Cxcr4 in neointima formation. APPROACH AND RESULTS: ß-Galactosidase staining using bone marrow x kinase (Bmx)-CreER(T2) reporter mice and double immunofluorescence revealed an efficient and endothelial-specific deletion of Cxcr4 in Bmx-CreER(T2+) compared with Bmx-CreER(T2-) Cxcr4-floxed apolipoprotein E-deficient (Apoe(-/-)) mice (referred to as Cxcr4(EC-KO)ApoE(-/-) and Cxcr4(EC-WT) ApoE(-/-), respectively). Endothelial Cxcr4 deficiency significantly increased wire injury-induced neointima formation in carotid arteries from Cxcr4(EC-KO)ApoE(-/-) mice. The lesions displayed a higher number of macrophages, whereas the smooth muscle cell and collagen content were reduced. This was associated with a significant reduction in reendothelialization and endothelial cell proliferation in injured Cxcr4(EC-KO)ApoE(-/-) carotids compared with Cxcr4(EC-WT)ApoE(-/-) controls. Furthermore, stimulation of human aortic endothelial cells with chemokine (C-X-C motif) ligand 12 (CXCL12) significantly enhanced their wound-healing capacity in an in vitro scratch assay, an effect that could be reversed with the CXCR4 antagonist AMD3100. Also, flow cytometric analysis showed a reduced mobilization of Sca1(+)Flk1(+)Cd31(+) and of Lin(-)Sca1(+) progenitors in Cxcr4(EC-KO) ApoE(-/-) mice after vascular injury, although Cxcr4 surface expression was unaltered. No differences could be detected in plasma concentrations of Cxcl12, vascular endothelial growth factor, sphingosine 1-phosphate, or Flt3 (fms-related tyrosine kinase 3) ligand, all cytokines with an established role in progenitor cell mobilization. Nonetheless, double immunofluorescence revealed a significant reduction in local endothelial Cxcl12 staining in injured carotids from Cxcr4(EC-KO)ApoE(-/-) mice. CONCLUSIONS: Endothelial Cxcr4 is crucial for efficient reendothelialization after vascular injury through endothelial wound healing and proliferation, and through the mobilization of Sca1(+)Flk1(+)Cd31(+) cells, often referred to as circulating endothelial progenitor cells.

Clinical Significance of C-X-C Motif Chemokine Receptor 4 and Integrin αvß6 Expression in Breast Cancer.

PURPOSE: C-X-C motif chemokine receptor 4 (CXCR4) and integrin αvß6 play important roles in the malignant progression of multiple cancers. However, it remains unclear whether the expression of one or both proteins in breast cancer (BC) is of clinical significance. In this study, we investigated the expression of CXCR4 and integrin αvß6 in BC tissues and their correlation with clinicopathological characteristics, including survival. METHODS: CXCR4 and αvß6 expression in 111 BC tissues was examined by immunocytochemistry. Correlations between the expression of the 2 proteins and patient clinicopathological characteristic were investigated using the Kaplan-Meier method and the Cox proportional hazards model. RESULTS: CXCR4 and αvß6 were overexpressed in BC tissue compared with normal breast tissue. Overexpression of both molecules was related to lymph node status (p = 0.013 and p = 0.022, respectively). αvß6 overexpression was also associated with tumor size (p = 0.044). A positive correlation was detected between the expression of CXCR4 and αvß6 (r = 0.649, p = 0.001), and co-overexpression of both molecules was associated with tumor size (p = 0.018) and lymph node metastasis (p = 0.015). Kaplan-Meier analysis revealed that overexpression of CXCR4, αvß6, or both molecules was associated with short overall survival (OS; p < 0.001, p < 0.001, and p = 0.009, respectively) and disease-free survival (DFS; p < 0.001, p = 0.005, and p = 0.019, respectively). Multivariate analysis indicated that lymph node metastasis was an independent prognostic factor for unfavorable OS and DFS (p = 0.002 and p = 0.005, respectively), whereas co-overexpression of CXCR4 and αvß6 was an independent prognostic factor only for OS (p = 0.043). CONCLUSION: CXCR4 and αvß6 may play synergistic roles in the progression of BC, and co-targeting of CXCR4 and αvß6 could be a potential strategy for the prevention and treatment of BC.

Expression and Functional Analysis of CXCL12 and Its Receptors in Human Term Trophoblast Cells.

Chemokine CXCL12 and its receptors CXCR4/CXCR7 play a pivotal role in many physiological and pathological situations, while the expression and function in human term trophoblast cells remain largely unknown. In the study, the expression and function of CXCL12 and its receptors CXCR4/CXCR7 in human term trophoblast cells were investigated. Immunocytochemistry and flow cytometry showed that the expression of CXCL12/CXCR4/CXCR7 could be detected in term trophoblast cells while expression level differed. The secretion of CXCL12 in human term trophoblast cells was confirmed by enzyme-linked immunosorbent assay (ELISA). In order to reveal the function of CXCL12, exogenetic recombinant human CXCL12 protein (rhCXCL12) was added to the cultured term trophoblast cells; results showed that cell proliferation ability was increased while cell apoptosis rate was decreased. Moreover, the effects of rhCXCL12 on term trophoblast cells could be diminished or attenuated by antibodies against CXCL12, CXCR4, or CXCR7, respectively. Therefore, these results revealed the important role of CXCL12 on human term trophoblast cells. Our study will provide new insights into understanding the role of CXCL12 on human term trophoblast cells.