Angiostatic Chemokines Research Articles

Spontaneous abortion is associated with differentially expressed angiogenic chemokines in placenta and decidua.

Miscarriage is one of the most common complications of pregnancy. Although chromosomal abnormalities of the embryo is a well-known cause of miscarriage, a lot of cases remain unexplained, with immunologic and vascular growth alterations being considered as probable causes. Chemokines are produced by a variety of cells and exhibit several functions including both pro and anti-angiogenic properties. In this study, we investigated the role of the angiogenic and angiostatic chemokines in placenta and decidua tissues from spontaneous and induced abortions. Total RNA was extracted from the placenta and decidua tissues, which was then purified and converted into cDNA. Real-time PCR was then performed for the expression of the angiogenic CCL2, CCL5, CCL20, CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL7, CXCL8 and CXCL4, and the angiostatic CXCL9, CXCL10, CXCL11, CXCL12 and CXCL14 and results were then statistically analyzed. Regarding the placenta, CXCL7 (2.29-fold, 2.16-2.38, p < 0.05), CXCL4 (1.01-fold, 0.74-4.447, p < 0.05), CXCL9 (0.87-fold, 0.43-1.34, p < 0.05) and CXCL11 (0.31-fold, 0.22-0.45, p < 0.05) were altered in spontaneous abortions. CCL2, CCL5, CXCL2-3, CXCL8, CXCL10, CXCL12 and CXCL14 were not statistically significant altered. Regarding the decidua, CXCL7 (7.13-fold, 6.32-7.54, p < 0.01), CXCL8 (11.02-fold, 8.58-13.45, p < 0.05), CCL20 (1.21-fold, 0.29-1.89, p < 0.05) and CXCL9 (5.49-fold, 3.67-6.39, p < 0.05) were overexpressed in spontaneous abortions. CXCL2-4, CCL2, CCL5, CXCL10-12 and CXCL14 did not show any differences. The expression of the chemokines CXCL1, CXCL5-6 was absent in either tissue or group. Our results show that the overexpression of angiostatic and diminished expression of angiogenic chemokines takes place in the placenta and decidua of spontaneous abortions, suggesting that dysregulation of angiogenesis could be a contributive factor to the pathogenesis of miscarriage.

Angiodrastic Chemokines Production by Colonic Cancer Cell Lines

Purpose: To study the production of angiodrastic chemokines by colonic cancer cell lines. Methods: A pro-angiogenic factor (VEGF), two angiogenic chemokines (CXCL8, CXCL6), and one angiostatic (CXCL4) chemokine were measured by ELISA in the supernatants of the colon cancer cell lines HT-29 and Caco-2. Cells were cultured for 24 h in the presence of serum from cancer patients or healthy individuals. Results were analyzed by one-way ANOVA and the General Linear Model for repeated measures. Results: Colonic epithelial cells are potent producers of angiodrastic chemokines. HT-29 and Caco-2 cells produce all four chemokines under basal conditions and 24 h after incubation with human serum. The secretion response, however, was completely different. HT-29 cells produce more CXCL8 and VEGF irrespective of culture conditions, while Caco-2 cells seem unresponsive with respect to CXCL6 and CXCL4. Moreover, HT-29 cells produce more CXCL8 and VEGF when incubated with cancer serum, contrary to Caco-2 cells which produce more CXCL4 under the same conditions. Conclusions: The two colon cancer cell lines were producers of all chemokines studied, but their responses were not uniform under similar culture conditions. CXCL8 and VEGF are differently regulated compared to CXCL4 and CXCL6 in these two cell lines

Endometriosis pain and epithelial neutrophil activating peptide-78 levels

Endometriosis is a chronic gynecological disorder involved in the pathogenesis of chronic pelvic pain, based on a probable up regulation of the inflammatory system. The objective of the study is to investigate the peritoneal and serum levels of ENA-78 with the severity of endometriosis symptoms (dysmenorrhea, chronic pelvic pain and dyspareunia) using the visual analogue scale (VAS). This is a prospective case–control study that included 53 symptomatic women with evidence of endometriosis and 53 age-matched controls who underwent elective laparoscopic surgery for benign diseases. The concentration of ENA-78 was assessed in blood and peritoneal fluid samples in the follicular phase. In peritoneal fluid and plasma, the concentration of ENA-78 was significantly higher in cases than in controls (p < 0.001). A significant correlation was observed between peritoneal fluid ENA-78 levels and the severity of dysmenorrhea (Spearman Rho = 0.237; p = 0.014), and chronic pelvic pain (Spearman Rho = 0.220; p = 0.022) in endometriosis patients. Plasma levels ENA-78 showed a significant correlation with the severity (VAS score) of chronic pelvic pain (Spearman Rho = 0.270, p = 0.005 for cases), though a weak correlation was evident between plasma levels of ENA-78 and severity of dysmenorrhea (Spearman Rho = 0.083, p = 0.399 for cases). In conclusion, chronic pelvic pain in endometriosis is caused by changes of local and systemic activated chemokine patterns. These modifications involve the relationship between pro-inflammatory, angiogenic and angiostatic chemokines that modulate the severity of endometriosis associated symptoms.

Programmed death (PD)-1/PD-ligand 1 blockade mediates antiangiogenic effects by tumor-derived CXCL10/11 as a potential predictive biomarker.

Immune checkpoint inhibitor (ICI) programmed death (PD)‐1/PD‐ligand 1 (PD‐L1) blockade has been approved for various cancers. However, the underlying antitumor mechanisms mediated by ICIs and the predictive biomarkers remain unclear. We report the effects of anti‐PD‐L1/PD‐1 Ab in tumor angiogenesis. In syngeneic mouse models, anti‐PD‐L1 Ab inhibited tumor angiogenesis and induces net‐like hypoxia only in ICI‐sensitive cell lines. In tumor tissue and serum of ICI‐sensitive cell line‐bearing mice, interferon‐γ (IFN‐γ) inducible angiostatic chemokines CXCL10/11 were upregulated by PD‐L1 blockade. In vitro, CXCL10/11 gene upregulation by IFN‐γ stimulation in tumor cell lines correlated with the sensitivity of PD‐L1 blockade. The CXCL10/11 receptor CXCR3‐neutralizing Ab or CXCL11 silencing in tumor cells inhibited the antiangiogenic effect of PD‐L1 blockade in vivo. In pretreatment serum of lung carcinoma patients receiving anti‐PD‐1 Ab, the concentration of CXCL10/11 significantly correlated with the clinical outcome. Our results indicate the antiangiogenic function of PD‐1/PD‐L1 blockade and identify tumor‐derived CXCL10/11 as a potential circulating biomarker of therapeutic sensitivity.

Pirfenidone Inhibits Cytokines/chemokines Release from Alveolar Macrophages

Objective To investigate the effect of pirfenidone on cytokine/chemokine production by alveolar macrophages(AMs)in patients with idiopathic nonspecific interstitial pneumonia(iNSIP)or idiopathic pulmonary fibrosis(IPF).Methods We prospectively enrolled 10 iNSIP patients,11 IPF patients,and 8 non-interstitial lung disease(non-ILD)patients(control group)from our center from January 2015 to December 2018.AMs from bronchoalveolar lavage fluid(BALF)were cultured with or without lipopolysaccharide(LPS)stimulation.The production of Th1 cytokines [soluble tumor necrosis factor receptor(sTNFR)-1,sTNFR-2,and interleukin(IL)-1β],Th2 cytokines [IL-10 and granulocyte-macrophage colony-stimulating factor(GM-CSF)],angiogenic chemokines [IL-18 and macrophage inflammatory protein(MIP)-1β],and angiostatic chemokines [interferon-gama inducible monokines(MIG)and interferon-gama inducible protein(IP-10)] in the culture supernatants were measured by a bead-based assay,Luminex.The effect of pirfenidone on the cytokine/chemokine production was tested at various concentrations(0,0.03,0.10,0.30 mg/ml).Results The spontaneous and LPS-stimulated release of TNF-α,sTNFR-1,sTNFR-2,IL-1β,IL-10,MIP-1β,MIG,and IP-10 by AMs were significantly increased in iNSIP and IPF groups compared with control group(all P<0.05),but no difference in IL-18 was seen among three groups(all P>0.05).MIG and IP-10 were significantly higher in iNSIP group than in IPF group(both P<0.05).Pirfenidone suppressed the spontaneous and LPS-stimulated AMs release of all studied cytokine/chemokine in iNSIP and IPF in a dose-dependent manner at concentrations of 0.10 and 0.30 mg/ml,and no difference was observed between iNSIP and IPF groups(both P>0.05).Conclusion Pirfenidone can markedly suppress cytokine/chemokine expression in iNSIP and IPF patients,but the difference is not significant between these two groups of patients.

Genetic perturbation of IFN-α transcriptional modulators in human endothelial cells uncovers pivotal regulators of angiogenesis

Interferon-α (IFN-α) comprises a family of 13 cytokines involved in the modulation of antiviral, immune, and anticancer responses by orchestrating a complex transcriptional network. The activation of IFN-α signaling pathway in endothelial cells results in decreased proliferation and migration, ultimately leading to suppression of angiogenesis. In this study, we knocked-down the expression of seven established or candidate modulators of IFN-α response in endothelial cells to reconstruct a gene regulatory network and to investigate the antiangiogenic activity of IFN-α. This genetic perturbation approach, along with the analysis of interferon-induced gene expression dynamics, highlighted a complex and highly interconnected network, in which the angiostatic chemokine C-X-C Motif Chemokine Ligand 10 (CXCL10) was a central node targeted by multiple modulators. IFN-α-induced secretion of CXCL10 protein by endothelial cells was blunted by the silencing of Signal Transducer and Activator of Transcription 1 (STAT1) and of Interferon Regulatory Factor 1 (IRF1) and it was exacerbated by the silencing of Ubiquitin Specific Peptidase 18 (USP18). In vitro sprouting assay, which mimics in vivo angiogenesis, confirmed STAT1 as a positive modulator and USP18 as a negative modulator of IFN-α-mediated sprouting suppression. Our data reveal an unprecedented physiological regulation of angiogenesis in endothelial cells through a tonic IFN-α signaling, whose enhancement could represent a viable strategy to suppress tumor neoangiogenesis.

Adipose tissue gene expression of CXCL10 and CXCL11 modulates inflammatory markers in obesity: implications for metabolic inflammation and insulin resistance.

Background:The CXCL subfamily of chemokines (CXCL9, CXCL10, and CXCL11; angiostatic chemokines) plays a key role in many inflammatory diseases. However, the expression of CXCLs in adipose tissue (AT) during obesity and association of these CXCLs with inflammatory markers and insulin resistance are poorly understood. Therefore, this study aimed to investigate the effects of CXCL gene expression on subcutaneous AT inflammatory markers and insulin resistance.Methods:Subcutaneous-fat biopsies were collected from 59 nondiabetic (lean/overweight/obese) individuals for RNA isolation. Expression levels of AT CXCL and inflammatory markers were determined by quantitative reverse transcriptase polymerase chain reaction (RT-qPCR). Biomedical parameters in the plasma were measured by enzyme-linked immunosorbent assay (ELISA). Insulin resistance was estimated using homeostatic model assessment (HOMA-IR).Results:AT CXCL expression was higher in obese compared with lean individuals (p < 0.05) and positively correlated with body mass index (BMI; r ⩾ 0.269, p < 0.05). Expression of CXCL9, CXCL10, and CXCL11 correlated significantly with various pro-inflammatory markers, including family members of interleukins, chemokines, and their prospective receptors (r ⩾ 0.339, p ⩽ 0.009), but not anti-inflammatory markers. CXCL11 expression correlated specifically with the expression of CCL5, CCL18, TLR3, TLR4, TLR8, IRF5, and NF-κB (r ⩾ 0.279, p ⩽ 0.039). Notably, CXCL11 was correlated with C-reactive protein (CRP), fasting blood glucose (FBG), and HOMA-IR. In multiple regression analysis, CXCL11 was identified as an independent predictor of CCL19, CCL5, IL-6, and TLR3.Conclusion:These data suggest that the CXCL family members, specifically CXCL10 and CXCL11, are potential biomarkers for the onset of AT inflammation during obesity.

IL‑17A promotes CXCR2‑dependent angiogenesis in a mouse model of liver cancer.

Serum interleukin (IL)-17A level is associated with higher microvessel density and poor prognosis in liver cancer. However, the specific mechanism underlying the role of IL-17A in liver cancer remains controversial. In the present study, the effect of IL-17A on liver cancer cells was examined. IL-17A had no evident impact on vascular endothelial growth factor A (VEGFA) production in HepG2 and Huh7.5 cells as determined by reverse transcription-quantitative PCR and ELISA, but it did stimulate angiogenic CXC chemokine secretion, including chemokine (C-X-C motif) ligand 1 (CXCL1), CXCL2, CXCL3, CXCL5, CXCL6 and CXCL8 in Huh7.5 cells and CXCL2 in HepG2 cells. In addition, the production of angiostatic chemokines such as CXCL10 was not affected. The supernatant of Huh7.5-IL17A cells promoted endothelial cell chemotaxis, which was attenuated by the C-X-C chemokine receptor type 2 (CXCR2) inhibitor SB225002. Although there was no role of IL-17A in promoting in vitro cell proliferation, IL-17A markedly increased the tumor growth of Huh7.5 cells in both subcutaneous and orthotopic xenograft models with increased vascularization. Taken together, these results demonstrated that IL-17A may stimulate chemokine-induced angiogenesis and promote tumor progression, independent of VEGF signaling. The CXCL-CXCR2 axis may be a novel target for the anti-angiogenesis treatment of liver cancer.

Support of Tumor Endothelial Cells by Chemokine Receptors.

Tumor-associated vascular endothelium comprises a specialized and diverse group of endothelial cells that, although not cancer themselves, are integral to cancer progression. Targeting the tumor vasculature can have significant efficacy in reducing tumor burden, although loss of efficacy due to acquisition of resistance mechanisms is common. Here we review mechanisms by which tumor endothelial cells (TEC) utilize chemokine receptors to support tumor progression. We illustrate how chemokine receptors support and may serve as functional markers of the diverse TEC population. We focus on ACKR1 (DARC), ACKR3 (CXCR7), CXCR4, and CCR2, as these are the best studied chemokine receptors in TEC; and suggest that targeting these receptors on the tumor vasculature may prove efficacious in slowing or reversing tumor growth. We also mention CXCR2 and CXCR3 as important mediators or tumor angiogenesis, given their distinct roles with angiogenic and angiostatic chemokines, respectively.

Abstract A37: Epigenetic modification of ovarian cancer immunogenicity

Abstract Background: Ovarian high-grade serous carcinoma (HGSC) remains a poor prognosis disease due to its late presentation and lack of “actionable” mutations to target pharmacologically. The host immune system, however, plays a pivotal role with the presence of cytotoxic T lymphocytes and chemokines that regulate T-cell trafficking both having positive effect on survival in preclinical and clinical studies. HGSC cells can achieve immune escape via epigenetic silencing of key immune-related genes, including via DNA methylation; when DNA methylation is pharmacologically reversed, genes involved in antigen presentation, receptor-dependent immune cell stimulation, and chemokine release are reactivated. One of these genes encodes the angiostatic chemokine Cxcl10, which is a T-lymphocyte attractant. In order to elucidate other potential epigenetic mechanisms directly or indirectly involved in Cxcl10 gene silencing, we will screen a library of novel epigenetic probes that target bromodomains, methyltransferases, demethylases, and others epigenetic pathways (Structural Genomic Consortium library). Aims: We will use Trp53-/- ID8 ovarian cancer cells to perform a medium-throughput screening of 40 novel epigenetic probes, measuring cxcl10 production, with the demethylating drug decitabine as positive control. Methods: Trp53-/- ID8 cells were treated with decitabine for up to 72 hours. Cell-cycle analysis was assessed via BrdU incorporation assay. Cytokine/chemokine transcription was assessed using RT2 profiler 84 gene chemo/cytokine PCR array (Qiagen), and verified using qRT-PCR. Acid-hydrolysed DNA was subjected to mass spectrometry/liquid chromatography to quantify the mean ratio of methyl-cytosine/cytosine. Western immunoblotting was performed to test DNMT1 protein levels in protein lysates after decitabine treatment. Cxcl10 levels in supernatant were quantified by ELISA. Results: Treatment of Trp53-/- ID8 cells with decitabine (DAC), at non-cytostatic/cytotoxic doses causes upregulation of multiple chemokines, specifically Cxcl10. 200nM DAC for 48 hours increased Cxcl10 transcription 22-fold (95% CI 20.6-21.1-fold, p=0.0001, values normalized to Gapdh control), and protein levels in supernatant three-fold (t test p&amp;lt;0.0001). At this dose, DNTM1, the target enzyme of DAC, is downregulated on Western-blot analysis, and the methyl-cytosine/cytosine ratio decreases significantly (means compared with t test, p =0.002). Other statistically significant upregulated chemokine genes are Cxcl1 (p=0.002), Cxcl3 (p&amp;lt;0.001), Cxcl4 (p&amp;lt;0.001), Cxcl5 (p=0.03), and Ccl4 (p=0.01). Conclusions: Abrogation of DNA methylation with DAC in Trp53-/- ID8 cells induces an increase in the chemokine cxcl10, at both transcriptional and protein level. This provides a robust control for a medium-throughput screening of other epigenetic modifiers that are still at an early drug development stage, such as probes against bromodomains, methyltransferases, and demethylases. Such screening can unmask novel epigenetic mechanisms involved in immune-related gene transcription and offer new ways to potentiate immunotherapy in ovarian cancer. Citation Format: Pavlina Spiliopoulou, Josephine Walton, Suzanne Dowson, Alex Binks, Oliver Maddocks, Peter Adams, Iain McNeish. Epigenetic modification of ovarian cancer immunogenicity. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr A37.

Upregulation of angiostatic chemokines IP-10/CXCL10 and I-TAC/CXCL11 in human obesity and their implication for adipose tissue angiogenesis.

Impaired angiogenesis is linked to adipose tissue (AT) dysfunction, inflammation, and insulin resistance in human obesity. Chemokine (C-X-C motif) receptor. (CXCR3) ligands are important regulators of angiogenesis in different disease contexts such as cancer; however, their role in human morbid obesity is unknown. We investigated the role of the CXCR3 axis in AT angiogenesis in morbidly obese patients. The study group comprised 50 morbidly obese patients (mean age 44 ± 1 years, body mass index 44 ± 1 kg/m2) who had undergone laparoscopic Roux-Y-gastric bypass surgery, and 25 age-matched non-obese control subjects. We measured the circulating levels of the CXCR3 ligands monokine induced by interferon-γ (MIG/CXCL9), interferon-γ inducible protein 10 (IP-10/CXCL10), and interferon-γ-inducible T-cell alpha chemoattractant (I-TAC/CXCL11) in all studied subjects. Additionally, the expression of CXCR3 ligands was analyzed in paired biopsies of subcutaneous and visceral AT obtained during the laparoscopic procedure in morbidly obese patients. Additionally, we explored the functional role of CXCR3 ligands on angiogenesis in AT from morbidly obese patients using an ex vivo assay. Plasma levels of CXCL10 and CXCL11 were significantly higher in morbidly obese patients than in controls (p < 0.01). In ex vivo assays, angiogenic growth was markedly lower in visceral AT than in subcutaneous AT (p < 0.05), which was related to significant tissue upregulation of CXCL10, CXCL11 and CXCR3 (p < 0.05). CXCL10 or CXCL11 inhibited AT angiogenesis (p < 0.05), and blockade of CXCR3 function significantly increased capillary sprouting in visceral fat deposits (p < 0.05). Western blot analysis showed that the p38 mitogen-activated protein kinase signaling pathway was implicated in the angiostatic effects of CXCR3 in AT. CXCL10 and CXCL11 may play. deleterious role in obesity as potential inhibitors of AT angiogenesis. Accordingly, pharmacological blockade of CXCR3 could represent. therapy to prevent AT dysfunction in obesity.

Upregulation of angiostatic chemokines CXCL10 and CXCL11 in morbid obese patients and their implication in adipose tissue angiogenesis

Upregulation of angiostatic chemokines CXCL10 and CXCL11 in morbid obese patients and their implication in adipose tissue angiogenesis

Angiodrastic Chemokines in Colorectal Cancer: Clinicopathological Correlations.

Aim To study the expression of angiodrastic chemokines in colorectal tumors and correlate findings with clinicopathological parameters and survival. Methods The proangiogenic factor VEGF, the angiogenic chemokines CXCL8 and CXCL6, and the angiostatic chemokine CXCL4 were measured by ELISA in tumor and normal tissue of 35 stage II and III patients and correlated with the histopathology markers Ki67, p53, p21, bcl2, EGFR, and MLH1 and 5-year survival. The Wilcoxon and chi-square tests were used for statistical comparisons. Results There was a significant increase of CXCL6 (p = 0.005) and VEGF (p = 0.003) in cancerous tissue compared to normal. Patients with lower levels of CXCL8 and CXCL4 lived significantly longer. Patients with loss of EGFR expression had higher levels of CXCL8 while p21 loss was associated with higher levels of CXCL6. Chemokine levels were not correlated with TNM or Dukes classification. Strong expression of p53 was accompanied by decreased survival. Conclusions (1) The angiogenic factors CXCL6 and VEGF are increased in colorectal cancer tissue with no association with the clinical stage of the disease or survival. (2) However, increased levels of tissue CXCL8 and CXCL4 are associated with poor survival. (3) Strong expression of p53 is found in patients with poor survival.

Relative distribution and biological characterization of CXCL4L1 isoforms in platelets from healthy donors

CXCL4L1, a platelet-derived ELR-negative CXC chemokine, is a powerful angiostatic and anti-tumoral chemokine. We developed a mass spectrometric assay for the detection of different natural CXCL4L1 isoforms. Using this assay, we identified 4 different CXCL4L1 isoforms in the supernatant of thrombin-stimulated platelets from healthy volunteers: the classical isoform CXCL4L1(1-70), CXCL4L1(-4-70), which probably arises through alternative signal peptide removal and two COOH-terminally truncated isoforms CXCL4L1(1-69) and CXCL4L1(-4-69). CXCL4L1(1-70) was the most abundant isoform, whereas CXCL4L1(-4-70) was detected in 50% of the platelet preparations. Since alterations to the NH2-terminus of chemokines can have severe biological consequences, we investigated the impact of the extension with 4 NH2-terminal amino acids on the biological activity of CXCL4L1. In vitro, CXCL4L1(-4-70) was as potent as CXCL4L1(1-70) in inhibiting signal transduction and migration of human microvascular endothelial cells towards vascular endothelial growth factor (VEGF) and fibroblast growth factor-2 (FGF-2). In a FITC-conjugated dextran cell permeability assay, both splice variants showed a strong but comparable anti-permeable effect upon VEGF stimulation of the endothelial cell monolayer. In vivo angiogenesis induced by FGF-2 was equally reduced by CXCL4L1(1-70) and CXCL4L1(-4-70). In chemotaxis assays with CXCR3A-transfected cells the CXCL4L1 isoforms both induced migration from 125ng/ml onward. Finally, CXCL4L1(1-70) and CXCL4L1(-4-70) showed the same affinity for heparin. In conclusion, the investigated biological activities of CXCL4L1 are not influenced by the four extra NH2-terminal residues present in the alternatively spliced isoform CXCL4L1(-4-70). Therefore, our results suggest that both isoforms equally interact with the CXCR3A and CXCR3B receptor.

Increased Levels of Interferon-Inducible Protein 10 (IP-10) in 22q11.2 Deletion Syndrome.

The 22q11.2 deletion syndrome (22q11.2 DS), also known as DiGeorge syndrome, is a genetic disorder with an estimated incidence of 1:4000 births. These patients may suffer from affection of many organ systems with cardiac malformations, thymic hypoplasia or aplasia, hypoparathyroidism, palate anomalies and psychiatric disorders being the most frequent. The incidence of autoimmune diseases is increased in older patients. The aim of the present study was to examine a cytokine profile in patients with 22q11.2 DS by measuring a broad spectrum of serum cytokines. Patients with a proven deletion of chromosome 22q11.2 (n=55) and healthy individuals (n=54) recruited from an age- and sex-comparable group were included in the study. Serum levels of 27 cytokines, including chemokines and growth factors, were analysed using multiplex technology. Interferon-inducible protein 10 (IP-10) was also measured by ELISA to confirm the multiplex results. The 22q11.2 DS patients had distinctly and significantly raised levels of pro-inflammatory and angiostatic chemokine IP-10 (P<0.001) compared to controls. The patients with congenital heart defects (n=31) had significantly (P=0.018) raised serum levels of IP-10 compared to patients born without heart defects (n=24). The other cytokines investigated were either not detectable or did not differ between patients and controls.

Role of chemokines in diabetic retinopathy

SummaryWe have detected altered expression of chemokines in the vitreous fluid of patients with proliferative diabetic retinopathy (PDR). In the early phase of disease chemokines (CXCL8, CCL2) are involved in leukocyte attraction. Later, CXCL12 may attract progenitor cells (endothelial cell precursors and fibrocytes) involved in establishment of neovessels and in formation of fibrovascular membranes. The myofibroblasts producing ECM in the fibrovascular membranes of the diabetic patients can originate from endothelial (precursor) cells via endoMT, from fibrocytes or from leukocyte‐like precursors. During the active disease stage also angiostatic chemokines (CXCL4, CXCL4L1 and CXCL10) are upregulated, probably in an attempt to counteract the stimulatory effects (angiogenesis and increased vascular permeability) of VEGF. Finally, CCL2 and CXCL10 have been reported to induce fibrosis and might play a role in the later stages of the pathology. Recently, we have demonstrated that CXCL4L1, a most potent angiostatic chemokine can be applied therapeutically as VEGF‐inhibitor in diabetic rats. Indeed, intraocular injections of CXCL4L1 early after the onset of diabetes protected animals against diabetes‐induced blood‐retinal barrier breakdown.

Intratumoral myeloid cells regulate responsiveness and resistance to antiangiogenic therapy.

Antiangiogenic therapy is commonly used in the clinic, but its beneficial effects are short-lived, leading to tumor relapse within months. Here, we found that the efficacy of angiogenic inhibitors targeting the VEGF/VEGFR pathway was dependent on induction of the angiostatic and immune-stimulatory chemokine CXCL14 in mouse models of pancreatic neuroendocrine and mammary tumors. In response, tumors reinitiated angiogenesis and immune suppression by activating PI3K signaling in all CD11b+ cells, rendering tumors nonresponsive to VEGF/VEGFR inhibition. Adaptive resistance was also associated with an increase in Gr1+CD11b+ cells, but targeting Gr1+ cells was not sufficient to further sensitize angiogenic blockade because tumor-associated macrophages (TAMs) would compensate for the lack of such cells and vice versa, leading to an oscillating pattern of distinct immune-cell populations. However, PI3K inhibition in CD11b+ myeloid cells generated an enduring angiostatic and immune-stimulatory environment in which antiangiogenic therapy remained efficient.

LEVELS OF ANGIOGENESIS-REGULATORY CHEMOKINES IN THE SYNOVIAL FLUID OF PATIENTS WITH RHEUMATOID ARTHRITIS

The role of chemokines in the immunopathogenesis of rheumatoid arthritis (RA) has been actively investigated in recent years. Angiogenic and angiostatic chemokines are important mediators of angiogenesis in the development and extent of pannus. Peripheral blood and synovial fluid (SF) is a major biomaterial in clinical and immunological studies. At the same time, it is the SF test that may yield the most informative results since that gives an idea of the processes that occur locally within a joint. Objective: to perform a comparative analysis of the levels of a number of CXC, CC, and CX3C chemokines in the SF of patients with RA, osteoarthritis (OA), and joint injuries. Subjects and methods. The multiplex analysis using xMAP technology (Luminex, USA) was used to analyze levels of CXC, CC, and CX3C chemokines in SF and serum of patients with RA (n = 20), OA (n = 9) and controls (n = 9). Results and discussion. The SF levels of CCL24/eotaxin-2, as well as those of the angiostatic chemokines CXCL9/MIG, CXCL10/IP10, CXCL11/ITAC, and CXCL13/BCA-1 were higher in the RA group than in the control and OA groups. There was a direct correlation between SF levels of CCL5/RANTES and DAS28, as well as patient global disease activity assessment on visual analogue scale, and that between the level of CCL2/MCP-1 in the SF and that of anticyclic citrullinated peptide (anti-CCP) antibodies in the serum. The SF concentrations of CXCL5/ENA78 and CXCL7/NAP-2 were shown to depend on the presence of serum anti-CCP. Serum CXCL13/BCA-1 levels were higher in RA than those in OA, as that of CXCL7/NAP-2 than in the control group.

CXCL4L1 and CXCL4 signaling in human lymphatic and microvascular endothelial cells and activated lymphocytes: involvement of mitogen-activated protein (MAP) kinases, Src and p70S6 kinase

CXC chemokines influence a variety of biological processes, such as angiogenesis, both in a physiological and pathological context. Platelet factor-4 (PF-4)/CXCL4 and its variant PF-4var/CXCL4L1 are known to favor angiostasis by inhibiting endothelial cell proliferation and chemotaxis. CXCL4L1 in particular is a potent inhibitor of angiogenesis with anti-tumoral characteristics, both through regulation of neovascularization and through attraction of activated lymphocytes. However, its underlying signaling pathways remain to be elucidated. Here, we have identified various intracellular pathways activated by CXCL4L1 in comparison with other CXCR3 ligands, including CXCL4 and interferon-γ-induced protein 10/CXCL10. Signaling experiments show involvement of the mitogen-activated protein kinase (MAPK) family in CXCR3A-transfected cells, activated lymphocytes and human microvascular endothelial cells (HMVEC). In CXCR3A transfectants, CXCL4 and CXCL4L1 activated p38 MAPK, as well as Src kinase within 30 and 5 min, respectively. Extracellular signal-regulated kinase (ERK) phosphorylation occurred in activated lymphocytes, yet was inhibited in microvascular and lymphatic endothelial cells. CXCL4L1 and CXCL4 counterbalanced the angiogenic chemokine stromal cell-derived factor-1/CXCL12 in both endothelial cell types. Notably, inhibition of ERK signaling by CXCL4L1 and CXCL4 in lymphatic endothelial cells implies that these chemokines might also regulate lymphangiogenesis. Furthermore, CXCL4, CXCL4L1 and CXCL10 slightly enhanced forskolin-stimulated cAMP production in HMVEC. Finally, CXCL4, but not CXCL4L1, induced activation of p70S6 kinase within 5 min in HMVEC. Our findings confirm that the angiostatic chemokines CXCL4L1 and CXCL4 activate both CXCR3A and CXCR3B and bring new insights into the complexity of their signaling cascades.

CXC and CC chemokines as angiogenic modulators in nonhaematological tumors.

Chemokines are a superfamily of structurally homologous heparin-binding proteins that includes potent inducers and inhibitors of angiogenesis. The imbalance between angiogenic and angiostatic chemokine activities can lead to abnormalities, such as chronic inflammation, dysplastic transformation, and even tumor development and spreading. In this review, we summarize the current literature regarding the role of chemokines as modulators of tumor angiogenesis and their potential role as therapeutic targets in patients with nonhaematological tumors.