The expression and biological function of chemokine CXCL12 and receptor CXCR4/CXCR7 in placenta accreta spectrum disorders.

OBJECTIVES: Investigation of mechanism related to excessive invasion of trophoblast cells in placenta accreta spectrum disorders (PAS) provides more strategies and ideas for clinical diagnosis and treatment. MATERIALS AND METHODS: Blood and placental samples were collected from included patients. The distribution and expression of CXCL12, CXCR4 and CXCR7 proteins in the paraffin of placental tissue in the included cases were analysed, and we analyse the downstream pathways or key proteins involved in cell invasion. RESULTS: Firstly, our results determined that CXCL12 and CXCR4/CXCR7 were increased in extravillous trophoblastic cell (CXCL12: P < .001; CXCR4: P < .001; CXCR7: P < .001), and the expression levels were closely related to the invasion depth of trophoblastic cells. Secondly, CXCL12 has the potential to become a biochemical indicator of PAS since the high expression of placental trophoblast CXCL12 may be an important source of blood CXCL12. Using lentivirus-mediated RNA interference and overexpression assay, it was found that both chemokine CXCL12 and receptor CXCR4/CXCR7 are associated with regulation of trophoblast cell proliferation, migration and invasion. Further results proved that through the activating the phosphorylation and increasing the expression of MLC and AKT proteins in the Rho/rock, PI3K/AKT signalling pathway, CXCL12, CXCR4 and CXCR7 could up-regulate the expression of RhoA, Rac1 and Cdc42 proteins to promote the migration and invasion of extravillous trophoblastic cell and ultimately formate the placenta accrete compare to the normal placenta. CONCLUSIONS: Our research proved that trophoblasts may contribute to a PAS-associated increase in CXCL12 levels in maternal blood. CXCL12 is not only associated with biological roles of PAS, but may also be potential for prediction of PAS.

Platelet-derived CXCL12 regulates monocyte function, survival, differentiation into macrophages and foam cells through differential involvement of CXCR4-CXCR7.

Platelets store and release CXCL12 (SDF-1), which governs differentiation of hematopoietic progenitors into either endothelial or macrophage-foam cells. CXCL12 ligates CXCR4 and CXCR7 and regulates monocyte/macrophage functions. This study deciphers the relative contribution of CXCR4-CXCR7 in mediating the effects of platelet-derived CXCL12 on monocyte function, survival, and differentiation. CXCL12 and macrophage migration inhibitory factor (MIF) that ligate CXCR4-CXCR7 induced a dynamic bidirectional trafficking of the receptors, causing CXCR4 internalization and CXCR7 externalization during chemotaxis, thereby influencing relative receptor availability, unlike MCP-1. In vivo we found enhanced accumulation of platelets and platelet-macrophage co-aggregates in peritoneal fluid following induction of peritonitis in mice. The relative surface expression of CXCL12, CXCR4, and CXCR7 among infiltrated monocytes was also enhanced as compared with peripheral blood. Platelet-derived CXCL12 from collagen-adherent platelets and recombinant CXCL12 induced monocyte chemotaxis specifically through CXCR4 engagement. Adhesion of monocytes to immobilized CXCL12 and CXCL12-enriched activated platelet surface under static and dynamic arterial flow conditions were mediated primarily through CXCR7 and were counter-regulated by neutralizing platelet-derived CXCL12. Monocytes and culture-derived-M1-M2 macrophages phagocytosed platelets, with the phagocytic potential of culture-derived-M1 macrophages higher than M2 involving CXCR4-CXCR7 participation. CXCR7 was the primary receptor in promoting monocyte survival as exerted by platelet-derived CXCL12 against BH3-mimetic induced apoptosis (phosphatidylserine exposure, caspase-3 activation, loss of mitochondrial transmembrane potential). In co-culture experiments with platelets, monocytes predominantly differentiated into CD163(+) macrophages, which was attenuated upon CXCL12 neutralization and CXCR4/CXCR7 blocking antibodies. Moreover, OxLDL uptake by platelets induced platelet apoptosis, like other platelet agonists TRAP and collagen-related peptide (CRP). CXCL12 facilitated phagocytosis of apoptotic platelets by monocytes and M1-M2 macrophages, also promoted their differentiation into foam cells via CXCR4 and CXCR7. Thus, platelet-derived CXCL12 could regulate monocyte-macrophage functions through differential engagement of CXCR4 and CXCR7, indicating an important role in inflammation at site of platelet accumulation.

Platelet surface expression of stromal cell-derived factor-1 receptors CXCR4 and CXCR7 is associated with clinical outcomes in patients with coronary artery disease.

BACKGROUND: Surface expression of stromal cell-derived factor-1 (SDF-1, CXCL12) on platelets is enhanced during ischemic events and plays an important role in peripheral homing of stem cells and myocardial repair mechanisms. SDF-1 effects are mediated through CXCR4 and CXCR7. Both CXCR4 and CXCR7 are surface expressed on human platelets and to a higher degree in patients with coronary artery disease (CAD) compared with healthy controls. In this study, we investigated the prognostic role of platelet CXCR4- and CXCR7 surface expression in patients with symptomatic CAD. METHODS AND RESULTS: In a cohort study, platelet surface expression of CXCR4 and CXCR7 was measured by using flow cytometry in 284 patients with symptomatic CAD at the time of percutaneous coronary intervention (PCI). The primary combined end point was defined as all-cause death and/or myocardial infarction (MI) during 12-month follow-up. Secondary end points were defined as the single events of all-cause death and MI. We found significant differences of CXCR4 values in patients who developed a combined end point compared with event-free patients (mean MFIAUTHOR: Please define MFI at first use. 3.17 vs. 3.44, 95% confidence interval [CI] 0.09-0.45) and in patients who subsequently died (mean MFI 3.10 vs. 3.42, 95% CI 0.09-0.56). In multivariate Cox regression analysis, lower platelet CXCR4 levels were independently and significantly associated with all-cause mortality (hazard ratio 0.24, 95% CI 0.07-0.87) and the primary combined end point of all-cause death and/or MI (hazard ratio 0.30, 95% CI 0.13-0.72). CONCLUSION: These findings highlight a potential prognostic value of platelet expression CXCR4 on clinical outcomes in patients with CAD.

CXCR7 is highly expressed in acute lymphoblastic leukemia and potentiates CXCR4 response to CXCL12.

Recently, a novel CXCL12-binding receptor, has been identified. This CXCL12-binding receptor commonly known as CXCR7 (CXC chemokine receptor 7), has lately, based on a novel nomenclature, has received the name ACKR3 (atypical chemokine receptor 3). In this study, we aimed to investigate the expression of CXCR7 in leukemic cells, as well as its participation in CXCL12 response. Interesting, we clearly demonstrated that CXCR7 is highly expressed in acute lymphoid leukemic cells compared with myeloid or normal hematopoietic cells and that CXCR7 contributed to T-acute lymphoid leukemic cell migration induced by CXCL12. Moreover, we showed that the cellular location of CXCR7 varied among T-lymphoid cells and this finding may be related to their migration capacity. Finally, we hypothesized that CXCR7 potentiates CXCR4 response and may contribute to the maintenance of leukemia by initiating cell recruitment to bone marrow niches that were once occupied by normal hematopoietic stem cells.

Mechanisms affecting neutrophil migration capacity in breast cancer patients before and after chemotherapy.

PURPOSE: To investigate the mechanisms affecting neutrophil migration capacity in breast cancer patients before and after chemotherapy. METHODS: Peripheral venous blood was collected at the time of diagnosis and immediately prior to the 4th cycle of an anthracycline-based chemotherapy regimen for patients diagnosed with different stages of breast cancer (n = 30), for experimental assays. Blood samples were also collected from a healthy control group (n = 17). RESULTS: IL-8 serum concentrations were higher in the patient group than in the control group (p = 0.02), and chemotherapy did not further affect this increase. Levels of TNF-α, IL-6, and IL-10 did not differ between controls and patients, or in relation to chemotherapy. Serum levels of nitric oxide (NO) metabolites were elevated following chemotherapy compared to levels detected prior to treatment (p = 0.01). When the supernatants of lipopolysaccharide-stimulated mononuclear cells and neutrophils obtained from the patients were assayed for levels of nitrite, these levels were significantly higher and unchanged, respectively, compared with controls. Expression levels of the chemokine receptors, CXCR1 and CXCR2, were significantly reduced in patients compared to controls, and chemotherapy did not further affect these differences. Furthermore, filamentous actin content for IL-8-activated neutrophils was reduced with chemotherapy (median 8.85; range 3.38-13.43) compared to the content detected prior to treatment (median 9.23; range 2.86-22.16) (p = 0.001). CONCLUSION: Elevated systemic levels of IL-8 and NO, desensitization to CXCR activation, and reduction in actin polymerization may affect neutrophil motility in patients before and after chemotherapy.

A role for the CXCL12 receptor, CXCR7, in the pathogenesis of human pulmonary vascular disease.

Given the critical role that endothelial cell dysfunction plays in the pathogenesis of pulmonary hypertensive diseases, we set out to establish if CXCR7, a receptor for the pro-angiogenic ligand CXCL12, is expressed in the vasculature of human lung diseases and examine its role in mediating CXCL12-induced responses in primary pulmonary human microvascular endothelial cells. Receptor and ligand expression was examined in control and explanted human hypertensive lungs, in human plasma and in hypoxic rodent lungs, by ELISA and immunohistochemical studies. Functional in vitro experiments examined the role of CXCR7 in CXCL12-induced lung microvascular endothelial cell proliferation, migration, and wound regeneration and repair. CXCR7 is elevated in the endothelium of explanted human hypertensive lungs and circulating CXCL12 concentrations are significantly elevated in disease. We demonstrate that alveolar hypoxia similar to that found in lung disease increases CXCR7 expression in the pulmonary endothelium. Furthermore, CXCR7 is the receptor through which endothelial cell regeneration and repair, and proliferation, is mediated, whereas signalling via CXCR4 is essential for chemotactic cell migration. Our findings demonstrate that CXCR7 has a critical but previously unrecognised role to play in endothelial cell proliferation, suggesting that CXCR7-mediated signalling may be functionally important in pulmonary vascular diseases.

CXCL12-binding receptors expression in non-small cell lung cancer relates to tumoral microvascular density and CXCR4 positive circulating tumoral cells in lung draining venous blood.

OBJECTIVES: Lung cancer is the main cause of cancer-related death in Western countries. Despite early diagnosis, approximately 40% of patients have undergone surgical resection for localized non-small cell lung cancer relapse within 24 months after surgery. Current prognostic criteria for patients with non-small cell lung cancer are gradually enriched by the discovery of critical biological markers in surgical samples to better stratify patients with high risk for recurrent and metastatic disease after surgical manipulation. In fact, specific biological features are needed to drive metastasis development and, among these chemokine receptors, when activated, seem to play a relevant role, promoting both neovessels formation and tumoral cell migration. METHODS: To this purpose, blood samples from the closed stumps of the pulmonary veins were drawn immediately after major pulmonary surgery in 45 patients with resectable non-small cell lung cancer to evaluate the expression of chemokine CXCL12 receptor, CXCR4, in circulating tumor cells. In addition, primary tumor sections have been used to assess microvascular density (MVD) and vessels invasion and build prognostic tissue micro-array to investigate the expression of CXCL12 receptors CXCR4 and CXCR7. RESULTS: Cells positive for cytokeratins from tumor draining pulmonary venous blood were detectable in 11 cases (23.9%). In 8 out of 11 cases, CK positive cells coexpressed CXCR4. Moreover, in tumoral tissue high CXCR4 expression was significantly associated to high mMVD (p = 0.046), high CXCR7 expression (p = 0.001), adenocarcinoma histotype (p = 0.023), and to the presence of circulating tumoral cells in pulmonary veins (p = 0.001). Finally, vessel invasions relate to high MVD. CONCLUSION: In conclusion, the results of our study underline the significant potential role of CXCL12 receptors in determining both vessel formation and tumoral cell migration to blood stream, favoring metastasis development.

Modulation of CXC chemokine receptor expression and function in human neutrophils during aging in vitro suggests a role in their clearance from circulation.

In mice, differential regulation of CXC chemokine receptor expression in circulating polymorphonuclear neutrophils (PMNs) undergoing senescence results in homing to the bone marrow. However, the role of this compartment and of the chemokine receptor CXCR4 is still under discussion, and only scarce data exist about CXCR4 function in human PMN. In our study, we provide evidence that also in human neutrophils, expression (cell surface and mRNA), chemotactic and signaling functions of the homing-related chemokine receptor CXCR4 are upregulated during aging in vitro, independent of addition of stimulatory cytokines (TNF, IL-1, IL-8, G-CSF). In contrast, interleukin-8 receptors are downmodulated (CXCR2) or remain unchanged (CXCR1), suggesting that human PMNs undergoing senescence acquire a phenotype that impairs inflammatory extravasation and favors homing to the bone marrow or other tissues involved in sequestration. Partially retained responsiveness to interleukin-8 may be important for neutrophil function when senescence occurs after extravasation in inflamed tissues.

The possible role of hematopoietic cell kinase in the pathophysiology of COPD.

BACKGROUND: Hematopoietic cell kinase (Hck) is a myeloid cell-specific tyrosine kinase, which is known to induce neutrophil infiltration to the lungs. Although the overexpression of Hck causes emphysema-like histologic changes in mice, its expression and activity in patients with COPD are unclear. METHOD: The aim of this study was to clarify the expression and activity of Hck in neutrophils from COPD patients, and to investigate the association between the degree of Hck expression and the lung function parameters in COPD patients. Peripheral blood neutrophils were isolated from 22 patients with COPD and 9 healthy subjects (HSs). The protein levels of Hck and phosphorylated Hck were assessed, and the correlation with various background characteristics was evaluated. RESULTS: The Hck protein level was significantly higher in neutrophils from COPD patients compared with HSs (COPD patients, 1.094; HSs, 0.801; p < 0.05). A significant positive correlation was observed between the protein level of Hck and the surface expression of the integrin molecule CD-11b (r = 0.540; p < 0.01) or CXC chemokine receptor-1 (r = 0.432; p < 0.05). In contrast, there was no difference in the phosphorylation of the Hck protein between COPD patients and HSs. CONCLUSION: The Hck protein level in peripheral blood neutrophils was increased in COPD patients, suggesting that Hck might have an important role in the neutrophil function and play a key role in the pathophysiology of COPD.

Molecular pathways involved in loss of kidney graft function with tubular atrophy and interstitial fibrosis.

Loss of kidney graft function with tubular atrophy (TA) and interstitial fibrosis (IF) causes most kidney allograft losses. We aimed to identify the molecular pathways involved in IF/TA progression. Kidney biopsies from normal kidneys (n = 24), normal allografts (n = 6), and allografts with IF/TA (n = 17) were analyzed using high-density oligonucleotide microarray. Probe set level tests of hypotheses tests were conducted to identify genes with a significant trend in gene expression across the three groups using Jonckheere-Terpstra test for trend. Interaction networks and functional analysis were used. An unsupervised hierarchical clustering analysis showed that all the IF/TA samples were associated with high correlation. Gene ontology classified the differentially expressed genes as related to immune response, inflammation, and matrix deposition. Chemokines (CX), CX receptor (for example, CCL5 and CXCR4), interleukin, and interleukin receptor (for example, IL-8 and IL10RA) genes were overexpressed in IF/TA samples compared with normal allografts and normal kidneys. Genes involved in apoptosis (for example, CASP4 and CASP5) were importantly overexpressed in IF/TA. Genes related to angiogenesis (for example, ANGPTL3, ANGPT2, and VEGF) were downregulated in IF/TA. Genes related to matrix production-deposition were upregulated in IF/TA. A distinctive gene expression pattern was observed in IF/TA samples compared with normal allografts and normal kidneys. We were able to establish a trend in gene expression for genes involved in different pathways among the studied groups. The top-scored networks were related to immune response, inflammation, and cell-to-cell interaction, showing the importance of chronic inflammation in progressive graft deterioration.

Expression of CXC and CC type of chemokines and its receptors in tuberculous and non-tuberculous effusions.

Chemokines mediate their biological functions by transmigration of various immune cells to the site of infection. Tuberculous pleurisy provides an effective model to study the role of chemokines in the recruitment of immune cells to the pleura. Our aim was to understand the cumulative effect of chemokines (IP-10, MIG, IL-8, MCP-1, MIP-1alpha and RANTES) and its receptors (CXCR2, CXCR3, CCR1, CCR2, CCR5 and CCR7) in the recruitment of CD4(+) T cells obtained from blood (BL) and pleural fluid (PF) of tuberculous (TB) and non-tuberculous (NTB) patients. We observed significant increase in CD4(+) T cells in TB PF indicating lymphocytic rich effusion. All chemokines except RANTES were significantly high in PF compared to BL in TB group, whereas IL-8 and MCP-1 showed significant increase only in NTB PF. The significantly high levels of IFN-gamma and TauNuF-alpha in TB PF and their positive correlation with IP-10 and MIP-1alpha indicated their synergistic action to elicit a strong protective Th1 response. In spite of high levels of Th1 cytokines and chemokines in TB PF, significantly lower levels of RANTES indicated its limited role at the site. The CXC receptors in PF of both the groups and CC receptors except CCR5 in TB PF were significantly high compared to BL. Only CXCR2, CCR5 and CCR7 showed significant increase in TB compared to NTB. Thus a selective concentration of chemokines, cytokines and abundant expression of chemokine receptors confirm the accumulation of activated and memory T cells at the site of infection and help in polarizing Th1 immune response.