produce pharmacological effects on Diabetes and Inflammatory Diseases
The CXC chemokines, CXCL4, -9, -10, -11, CXCL4L1, and the CC chemokine CCL21, activate CXC chemokine receptor 3 (CXCR3), a cell-surface G protein-coupled receptor expressed mainly by Th1 cells, cytotoxic T (Tc) cells and NK cells that have a key role in immunity and inflammation. However, CXCR3 is also expressed by vascular smooth muscle and endothelial cells, and appears to be important in controlling physiological vascular function. In the last decade, evidence from pre-clinical and clinical studies has revealed the participation of CXCR3 and its ligands in multiple cardiovascular diseases (CVDs) of different aetiologies including atherosclerosis, hypertension, cardiac hypertrophy and heart failure, as well as in heart transplant rejection and transplant coronary artery disease (CAD). CXCR3 ligands have also proven to be valid biomarkers for the development of heart failure and left ventricular dysfunction, suggesting an underlining pathophysiological relation between levels of these chemokines and the development of adverse cardiac remodelling. The observation that several of the above-mentioned chemokines exert biological actions independent of CXCR3 provides both opportunities and challenges for developing effective drug strategies. In this review, we provide evidence to support our contention that CXCR3 and its ligands actively participate in the development and progression of CVDs, and may additionally have utility as diagnostic and prognostic biomarkers.
BACKGROUND: The CXCL12/CXCR4 pathway regulates tumor cell proliferation, metastasis, angiogenesis and the tumor-microenvironment cross-talk in several solid tumors, including glioblastoma (GBM), the most common and fatal brain cancer. In the present study, we evaluated the effects of peptide R, a new specific CXCR4 antagonist that we recently developed by a ligand-based approach, in an in vitro and in vivo model of GBM. The well-characterized CXCR4 antagonist Plerixafor was also included in the study.
METHODS: The effects of peptide R on CXCR4 expression, cell survival and migration were assessed on the human glioblastoma cell line U87MG exposed to CXCL12, by immunofluorescence and western blotting, MTT assay, flow cytometry and transwell chamber migration assay. Peptide R was then tested in vivo, by using U87MG intracranial xenografts in CD1 nude mice. Peptide R was administered for 23 days since cell implantation and tumor volume was assessed by magnetic resonance imaging (MRI) at 4.7 T. Glioma associated microglia/macrophage (GAMs) polarization (anti-tumor M1 versus pro-tumor M2 phenotypes) and expressions of vascular endothelial growth factor (VEGF) and CD31 were assessed by immunohistochemistry and immunofluorescence.
RESULTS: We found that peptide R impairs the metabolic activity and cell proliferation of human U87MG cells and stably reduces CXCR4 expression and cell migration in response to CXCL12 in vitro. In the orthotopic U87MG model, peptide R reduced tumor cellularity, promoted M1 features of GAMs and astrogliosis, and hindered intra-tumor vasculature.
CONCLUSIONS: Our findings suggest that targeting CXCR4 by peptide R might represent a novel therapeutic approach against GBM, and contribute to the rationale to further explore in more complex pre-clinical settings the therapeutic potential of peptide R, alone or in combination with standard therapies of GBM.
The chemokine CXCL12 activates CXCR4, initiating multiple pathways that control immune cell trafficking, angiogenesis and embryogenesis; CXCR4 is also overexpressed in multiple tumors affecting metastatic dissemination. While there has been great enthusiasm for exploiting the CXCR4-CXCL12 axis as a target in cancer therapy, to date the promise has yet to be fulfilled. A new class of CXCR4-antagonist cyclic peptides was recently developed and the compound named Peptide R was identified as the most active. With the intent to improve the efficacy and biodistribution of Peptide R, stealth liposomes decorated with Peptide R were developed (PL-Peptide R). In vitro PL-Peptide R efficiently inhibited CXCR4-dependent migration and in vivo it significantly reduced lung metastases and increased overall survival in B16-CXCR4 injected C57BL/6 mice. To evaluate if PL-Peptide R could also be a drug delivery system for CXCR4 expressing tumors, the PL-Peptide R was loaded with doxorubicin (DOX) (PL-Peptide R-DOX). PL-Peptide R-DOX efficiently delivered DOX to CXCR4 expressing cell lines with a consequent decrease in the DOX IC50 efficient dose. In vivo, B16-CXCR4 injected C57BL/6 mice treated with PL-Peptide R-DOX developed fewer lung metastases compared to PL-DOX treated mice. This work provides the proof-of-concept to prevent metastasis by using combined nanomedicine.
Chronic rhinosinusitis (CRS) is a heterogeneous disease, with varying immunological and histopathological features. The CC chemokine ligand 19 (CCL19) can stimulate T cells and antigen-presenting cells into secondary lymphoid node formation, as observed in allergic rhinitis, inflammatory bowel disease and other inflammatory disorders. The purpose of this study was to investigate the expression and significance of CCL19 in CRS. Samples of uncinate process mucosa or nasal polyps were taken from patients with CRS (without or with nasal polyps) and normal controls during surgery. Hematoxylin and eosin, periodic acid Schiff and Masson trichrome staining were used for analysis of the nasal polyps. Western blot and immunofluorescence were used to detect CCL19 expression in the nasal polyps and normal nasal mucosa tissues. Spearman correlation analysis was used to analyze the association between CCL19 and blood eosinophil counts. The results showed that CCL19 protein levels in CRS (with or without nasal polyps) were significantly upregulated compared with those in controls. CCL19 expression in eosinophilic CRS was significantly higher than in non-eosinophilic CRS. CCL19 expression in fibroinflammatory and edematous type CRS with nasal polyps was higher than in controls; the upregulation was greater in the edematous type. Immunofluorescence assays showed that CCL19 was mainly expressed by CD68+ macrophages. Spearman correlation analysis demonstrated a positive correlation between CCL19 and blood eosinophils. The upregulation of CCL19 in CRS may play a protective role by limiting eosinophil infiltration and the extent of edema to exert anti-inflammatory and immunomodulatory effects.
Chemokines attract leukocytes to sites of infection in a G protein-coupled receptor (GPCR) and glycosaminoglycan (GAG) dependent manner. Therefore, chemokines are crucial molecules for proper functioning of our antimicrobial defense mechanisms. In addition, some chemokines haveGPCR-independent defensin-like antimicrobial activities against bacteria and fungi. Recently, high affinity for GAGs has been reported for the positively charged COOH-terminal region of the chemokine CXCL9. In addition to CXCL9, also CXCL12? has such a positively charged COOH-terminal region with about 50% positively charged amino acids. In this report, we compared the affinity of COOH-terminal peptides of CXCL9 and CXCL12? for GAGs and KD values in the low nM range were detected. Several enveloped viruses such as herpesviruses, hepatitis viruses, human immunodeficiency virus (HIV), dengue virus (DENV), etc. are known to bind to GAGs such as the negatively charged heparan sulfate (HS). In this way GAGs are important for the initial contacts between viruses and host cells and for the infection of the cell. Thus, inhibiting the virus-cell interactions, by blocking GAG-binding sites on the host cell, might be a way to target multiple virus families and resistant strains. This article reports that the COOH-terminal peptides of CXCL9 (Cat.# 045-62) and CXCL12? (Cat.# 030-23) have antiviral activity against DENV serotype 2, clinical and laboratory strains of herpes simplex virus (HSV)-1 and respiratory syncytial virus (RSV). Moreover, we show that CXCL9(74-103) competes with DENV envelope protein domain III for binding to heparin. These short chemokine-derived peptides may be lead molecules for the development of novel antiviral agents.
The ELR(-)CXC chemokine CXCL9 is characterized by a long, highly positively charged COOH-terminal region, absent in most other chemokines. Several natural leukocyte- and fibroblast-derived COOH-terminally truncated CXCL9 forms missing up to 30 amino acids were identified. To investigate the role of the COOH-terminal region of CXCL9, several COOH-terminal peptides were chemically synthesized. These peptides display high affinity for glycosaminoglycans (GAGs) and compete with functional intact chemokines for GAG binding, the longest peptide (CXCL9(74-103)) being the most potent. The COOH-terminal peptide CXCL9(74-103) does not signal through or act as an antagonist for CXCR3, the G protein-coupledCXCL9 receptor, and does not influence neutrophil chemotactic activity of CXCL8 in vitro. Based on the GAG binding data, an anti-inflammatory role for CXCL9(74-103) was further evidenced in vivo. Simultaneous intravenous injection of CXCL9(74-103) with CXCL8 injection in the joint diminished CXCL8-induced neutrophil extravasation. Analogously, monosodium urate crystal-induced neutrophil migration to the tibiofemural articulation, a murine model of gout, is highly reduced by intravenous injection of CXCL9(74-103). These data show that chemokine-derived peptides with high affinity for GAGs may be used as anti-inflammatory peptides; by competing with active chemokines for binding and immobilization on GAGs, these peptides may lower chemokine presentation on the endothelium and disrupt the generation of a chemokine gradient, thereby preventing a chemokine from properly performing its chemotactic function. The CXCL9 peptide may serve as a lead molecule for further development of inhibitors of inflammation based on interference with chemokine-GAG interactions.
Over the last 5 years, X-ray structures of CXCR4 in complex with three different ligands (the small-molecule antagonist IT1t, the polypeptide antagonist CVX15 and the viral chemokine antagonist vMIP-II) have been released. In addition to the inherent scientific value of these specific X-ray structures, they provide a reliable structural foundation for studies of the molecular interactions between CXCR4 and its key peptide ligands (CXCL12 and HIV-1 gp120), and serve as valuable templates for further development of small-molecule CXCR4 antagonists with therapeutic potential. We here review recent computational studies of the molecular interactions between CXCR4 and its peptide ligands - based on the X-ray structures of CXCR4 - and the current status of small-molecule peptide and peptidomimetic CXCR4 antagonists.
BACKGROUND: T-cell-mediated immune responses play important roles in the progression of atherosclerotic disease. Studies have linked various inflammatory biomarkers with the burden of coronary artery calcification, but the significance of T-cell-specific chemokines in coronary artery calcification has not been confirmed. We aimed to examine the association between serum levels of the monokine induced by gamma interferon (MIG) and the coronary artery calcium score (CACS).
METHODS: We enrolled 456 individuals (285 men, 66.5±5.8 years) who were registered in the Mapo-gu public health center cohort. We selected 228 individuals with a CACS of more than 100 and 228 age-matched and sex-matched individuals with a CACS of less than 100. All participants underwent coronary computed tomography for CACS measuring. Clinical and laboratory variables including serum MIG levels were analyzed at the time of enrollment.
RESULTS: The serum level of MIG was significantly higher in participants with a CACS of more than 100 (152.1±119.1 vs. 130.3±112.9, P=0.046). Serum MIG levels correlated significantly with CACS (r=0.113, P=0.016), and higher levels of MIG were associated with severe plaque burden (CACS>400, P=0.025). Multiple linear regression analysis showed that serum MIG levels were associated independently with CACS after controlling for confounding factors and medications (?=0.114, P=0.026).
CONCLUSION: Serum MIG levels were associated independently with CACS after adjusting for traditional cardiovascular risk factors. These findings suggest that MIG may be used as a novel biomarker for T-cell inflammation and atherosclerotic plaque burden in humans.
Chemokine gradient formation requires multiple processes that include ligand secretion and diffusion, receptor binding and internalization, and immobilization of ligand to surfaces. To understand how these events dynamically shape gradients and influence ensuing cell chemotaxis, we built a multi-scale hybrid agent-based model linking gradient formation, cell responses, and receptor-level information. The CXCL12/CXCR4/CXCR7 signaling axis is highly implicated in metastasis of many cancers. We model CXCL12 gradient formation as it is impacted by CXCR4 and CXCR7, with particular focus on the three most highly expressed isoforms of CXCL12. We trained and validated our model using data from an in vitro microfluidic source-sink device. Our simulations demonstrate how isoform differences on the molecular level affect gradient formation and cell responses. We determine that ligand properties specific to CXCL12 isoforms (binding to the migration surface and to CXCR4) significantly impact migration and explain differences in in vitro chemotaxis data. We extend our model to analyze CXCL12 gradient formation in a tumor environment and find that short distance, steep gradients characteristic of the CXCL12-? isoform are effective at driving chemotaxis. We highlight the importance of CXCL12-γ in cancer cell migration: its high effective affinity for both extracellular surface sites and CXCR4 strongly promote CXCR4+ cell migration. CXCL12-γ is also more difficult to inhibit, and we predict that co-inhibition of CXCR4 and CXCR7 is necessary to effectively hinderCXCL12-?-induced migration. These findings support the growing importance of understanding differences in protein isoforms, and in particular their implications for cancer treatment.
PURPOSE: To investigate the expression of platelet factor-4 variant (PF-4var/CXCL4L1) in epiretinal membranes from patients with proliferative diabetic retinopathy (PDR) and the role of PF-4var/CXCL4L1 in the regulation of blood-retinal barrier (BRB) breakdown in diabetic rat retinas and human retinal microvascular endothelial cells (HRMEC).
METHODS: Rats were treated intravitreally with PF-4var/CXCL4L1 or the anti-vascular endothelial growth factor (VEGF) agent bevacizumab on the first day after diabetes induction. Blood-retinal barrier breakdown was assessed in vivo with fluorescein isothiocyanate (FITC)-conjugated dextran and in vitro in HRMEC by transendothelial electrical resistance and FITC-conjugated dextran cell permeability assay. Occludin, vascular endothelial (VE)-cadherin, hypoxia-inducible factor (HIF)-1α, VEGF, tumor necrosis factor (TNF)-α, receptor for advanced glycation end products (RAGE), caspase-3 levels, and generation of reactive oxygen species (ROS) were assessed by Western blot, enzyme-linked immunosorbent assays, or spectrophotometry.
RESULTS: In epiretinal membranes, vascular endothelial cells and stromal cells expressed PF-4var/CXCL4L1. In vitro, HRMEC produced PF-4var/CXCL4L1 after stimulation with a combination of interleukin (IL)-1β and TNF-α, and PF-4var/CXCL4L1 inhibited VEGF-mediated hyperpermeability in HRMEC. In rats, PF-4var/CXCL4L1 was as potent as bevacizumab in attenuating diabetes-induced BRB breakdown. This effect was associated with upregulation of occludin and VE-cadherin and downregulation of HIF-1α, VEGF, TNF-α, RAGE, and caspase-3, whereas ROS generation was not altered.
CONCLUSIONS: Our findings suggest that increasing the intraocular PF-4var/CXCL4L1 levels early after the onset of diabetes protects against diabetes-induced BRB breakdown.
In cystic fibrosis (CF), colonization of the airways with Pseudomonas aeruginosa is associated with disease deterioration. The mechanism behind the disease progression is not fully understood. The present work shows that the antibacterial chemokine MIG/CXCL9 is present in the airways and in sputum of CF patients. MIG/CXCL9 showed high bactericidal activity against. P. aeruginosa, including some strains from the airways of CF patients. Full-length MIG/CXCL9 was detected in sputum from healthy controls and CF patients colonized with P. aeruginosa. However, degraded MIG/CXCL9 was only found in CF sputum. In vitro, elastase of P. aeruginosa cleaved off a fragment of similar size and two additional fragments from MIG/CXCL9. The fragments showed less bactericidal activity against P. aeruginosa compared with the full-length protein. The fragments did not activate the MIG/CXCL9 receptor CXCR3 (expressed e.g. by NK cells, mast cells, and activated T cells) but instead displayed noncompetitive inhibition. In vitro, a decrease in CXCR3-bearing cells was found within and in the proximity of the bronchial epithelium of CF lung tissue compared with controls. Taken together, both bactericidal and cell-recruiting activities of MIG/CXCL9 are corrupted by P. aeruginosa through release of elastase, and this may contribute to impaired airway host defense in CF.
CXC chemokines are involved in chemotaxis, regulation of cell growth, induction of apoptosis and modulation of angiostatic effects. CXCL9, CXCL10, CXCL11, CXCL4 and its variant CXCL4L1 are members of the CXC chemokine family, which bind to the CXCR3 receptor to exert their biological effects. These chemokines are associated with a variety o f human diseases including chronic inflammation, immune dysfunction, cancer and metastasis. In this review, we focus on accumulating evidence demonstrating the pivotal role of CXCR3 in tumor progression. Its effects are mediated directly in tumor cells or indirectly through the regulation of angiogenesis and tumor immunity. Understanding the emerging role of CXCR3 and its signaling mechanisms further validates this receptor as a biomarker and therapeutic target for tumor progression and tumor angiogenesis.
BACKGROUND: Platelet-derived chemokines are implicated in several aspects of vascular biology. However, for the chemokine platelet factor 4 variant (PF-4var/CXCL4L1), released by platelets during thrombosis and with different properties as compared to PF-4/CXCL4, its role in heart disease is not yet studied. We evaluated the determinants and prognostic value of the platelet-derived chemokines PF-4var, PF-4 and RANTES/CCL5 in patients with stable coronary artery disease (CAD).
METHODOLOGY/PRINCIPAL FINDINGS: From 205 consecutive patients with stable CAD and preserved left ventricular (LV) function, blood samples were taken at inclusion and were analyzed for PF-4var, RANTES, platelet factor-4 and N-terminal pro-B-type natriuretic peptide (NT-proBNP). Patients were followed (median follow-up 2.5 years) for the combined endpoint of cardiac death, non-fatal acute myocardial infarction, stroke or hospitalization for heart failure. Independent determinants of PF-4var levels (median 10 ng/ml; interquartile range 8-16 ng/ml) were age, gender and circulating platelet number. Patients who experienced cardiac events (n = 20) during follow-up showed lower levels of PF-4var (8.5 [5.3-10] ng/ml versus 12 [8-16] ng/ml, p = 0.033). ROC analysis for events showed an area under the curve (AUC) of 0.82 (95% CI 0.73-0.90, p<0.001) for higher NT-proBNP levels and an AUC of 0.32 (95% CI 0.19-0.45, p = 0.009) for lower PF-4var levels. Cox proportional hazard analysis showed that PF-4var has an independent prognostic value on top of NT-proBNP.
CONCLUSIONS: We conclude that low PF-4var/CXCL4L1 levels are associated with a poor outcome in patients with stable CAD and preserved LV function. This prognostic value is independent of NT-proBNP levels, suggesting that both neurohormonal and platelet-related factors determine outcome in these patients.
Chemokines influence tumor growth directly or indirectly via both angiogenesis and tumor-leukocyte interactions. Platelet factor-4 (CXCL4/PF-4), which is released from alpha-granules of activated platelets, is the first described angiostatic chemokine. Recently, it was found that the variant of CXCL4/PF-4 (CXCL4L1/PF-4var) could exert a more pronounced angiostatic and antitumoral effect than CXCL4/PF-4. However, the molecular mechanisms of the angiostatic activities of the PF-4 forms remain partially elusive. Here, we studied the biological properties of the chemically synthesized COOH-terminal peptides of CXCL4/PF-4 (CXCL4/PF-4(47-70)) and CXCL4L1/PF-4var (CXCL4L1/PF-4var(47-70)). Both PF-4 peptides lacked monocyte and lymphocyte chemotactic activity but equally well inhibited (25 nmol/L) endothelial cell motility and proliferation in the presence of a single stimulus (i.e., exogenous recombinant fibroblast growth factor-2). In contrast, when assayed in more complex angiogenesis test systems characterized by the presence of multiple mediators, including in vitro wound-healing (2.5 nmol/L versus 12.5 nmol/L), Matrigel (60 nmol/L versus 300 nmol/L), and chorioallantoic membrane assays, CXCL4L1/PF-4var(47-70) was found to be significantly (5-fold) more angiostatic than CXCL4/PF-4(47-70). In addition, low (7 microg total) doses of intratumoral CXCL4L1/PF-4var (47-70) inhibited B16 melanoma growth in mice more extensively than CXCL4/PF-4(47-70). This antitumoral activity was predominantly mediated through inhibition of angiogenesis (without affecting blood vessel stability) and induction of apoptosis, as evidenced by immunohistochemical and fluorescent staining of B16 tumor tissue. In conclusion, CXCL4L1/PF-4var(47-70) is a potent antitumoral and antiangiogenic peptide. These results may represent the basis for the design of CXCL4L1/PF-4var COOH-terminal-derived peptidomimetic anticancer drugs.
BACKGROUND: CXCL12alpha, a chemokine that importantly promotes the oriented cell migration and tissue homing of many cell types, regulates key homeostatic functions and pathological processes through interactions with its cognate receptor (CXCR4) and heparan sulfate (HS). The alternative splicing of the cxcl12 gene generates a recently identified isoform, CXCL12gamma, which structure/function relationships remain unexplored. The high occurrence of basic residues that characterize this isoform suggests however that it could feature specific regulation by HS.
METHODOLOGY/PRINCIPAL FINDINGS: Using surface plasmon resonance and NMR spectroscopy, as well as chemically and recombinantly produced chemokines, we show here that CXCL12gamma first 68 amino acids adopt a structure closely related to the well described alpha isoform, followed by an unfolded C-terminal extension of 30 amino acids. Remarkably, 60% of these residues are either lysine or arginine, and most of them are clustered in typical HS binding sites. This provides the chemokine with the highest affinity for HP ever observed (Kd = 0.9 nM), and ensures a strong retention of the chemokine at the cell surface. This was due to the unique combination of two cooperative binding sites, one strictly required, found in the structured domain of the protein, the other one being the C-terminus which essentially functions by enhancing the half life of the complexes. Importantly, this peculiar C-terminus also regulates the balance between HS and CXCR4 binding, and consequently the biological activity of the chemokine.
CONCLUSIONS/SIGNIFICANCE: Together these data describe an unusual binding process that gives rise to an unprecedented high affinity between a chemokine and HS. This shows that the gamma isoform of CXCL12, which features unique structural and functional properties, is optimized to ensure its strong retention at the cell surface. Thus, depending on the chemokine isoform to which it binds, HS could differentially orchestrate the CXCL12 mediated directional cell kinesis.
CXCL12/stromal cell-derived factor 1 is a member of the CXC family of chemokines that plays an important role in hematopoiesis and signals through CXCR4 and CXCR7. Two splice variants of human CXCL12 (CXCL12alpha and CXCL12beta) induce chemotaxis of CXCR4(+) cells and inhibit X4 infection. Recent studies described four other novel splice variants of human CXCL12; however, their antiviral activities were not investigated. We constructed and expressed all of the CXCL12 splice variants in Escherichia coli. Recombinant proteins were purified through a His affinity column, and their biological properties were analyzed. All six CXCL12 variants induced chemotaxis of CXCR4(+) and CXCR7(+) cell lines. Enhancement of survival and replating capacity of human hematopoietic progenitor cells were observed with CXCL12alpha, CXCL12beta, and CXCL12epsilon but not with the other variants. CXCL12gamma showed the greatest antiviral activity in X4 inhibition assays and the weakest chemotaxis activity through CXCR4. The order of potency in X4 inhibition assays was as follows: CXCL12gamma > CXCL12beta > CXCL12alpha > CXCL12theta > CXCL12epsilon > CXCL12delta. The order of anti-human immunodeficiency virus (HIV) activity was associated with the number of BBXB motifs present in each variant; the most potent inhibitor was CXCL12gamma, with five BBXB domains. The results suggest that the different C-termini of CXCL12 variants may contain important molecular determinants for the observed differences in antiviral effects and other biological functions. These studies implicate CXCL12gamma as a potent HIV-1 entry inhibitor with significantly reduced chemotaxis activity and small or absent effects on progenitor cell survival or replating capacity, providing important insight into the structure-function relationships of CXCL12.