Differential Effects of Platelet Factor 4 (CXCL4) and Its Non-Allelic Variant (CXCL4L1) on Cultured Human Vascular Smooth Muscle Cells.

Platelet factor 4 (CXCL4) is a chemokine abundantly stored in platelets. Upon injury and during atherosclerosis, CXCL4 is transported through the vessel wall where it modulates the function of vascular smooth muscle cells (VSMCs) by affecting proliferation, migration, gene expression and cytokine release. Variant CXCL4L1 is distinct from CXCL4 in function and expression pattern, despite a minor three-amino acid difference. Here, the effects of CXCL4 and CXCL4L1 on the phenotype and function of human VSMCs were compared in vitro. VSMCs were found to constitutively express CXCL4L1 and only exogenously added CXCL4 was internalized by VSMCs. Pre-treatment with heparin completely blocked CXCL4 uptake. A role of the putative CXCL4 receptors CXCR3 and DARC in endocytosis was excluded, but LDL receptor family members appeared to be involved in the uptake of CXCL4. Incubation of VSMCs with both CXCL4 and CXCL4L1 resulted in decreased expression of contractile marker genes and increased mRNA levels of KLF4 and NLRP3 transcription factors, yet only CXCL4 stimulated proliferation and calcification of VSMCs. In conclusion, CXCL4 and CXCL4L1 both modulate gene expression, yet only CXCL4 increases the division rate and formation of calcium-phosphate crystals in VSMCs. CXCL4 and CXCL4L1 may play distinct roles during vascular remodeling in which CXCL4 induces proliferation and calcification while endogenously expressed CXCL4L1 governs cellular homeostasis. The latter notion remains a subject for future investigation.

CXCL4 and CXCL4L1 in cancer.

Chemokines regulate leukocyte migration during physiological and pathological conditions. It is currently accepted that these chemotactic cytokines are also important in the development and progression of cancer. CXCL4 and its non-allelic variant CXCL4L1 are two platelet-associated chemokines that have been attributed anti-tumoral activity as a result of their angiostatic potential and the chemotactic activity for anti-tumoral leukocytes. Here we review the role of CXCL4 and CXCL4L1 in cancer, the use of both chemokines as cancer biomarkers and discuss some possible therapeutic opportunities.

Elevated expression of platelet-derived chemokines in patients with antiphospholipid syndrome.

OBJECTIVE: Platelet factor 4 tetramers (CXCL4 chemokine) form complexes with ß2glycoprotein I (ß2GPI), recognized by anti-ß2GPI antibodies leading to platelet activation in antiphospholipid syndrome (APS), either primary (PAPS) or secondary (SAPS). Increased plasma levels of CXCL4 may favor this process; therefore we measured plasma levels of CXCL4, a CXCL4 variant (CXCL4L1) and as controls, platelet-derived chemokines CXCL7 (NAP-2) and CCL5 (RANTES), in APS, and disease controls such as patients with systemic lupus erythematosus (SLE) coronary artery disease (CAD) and healthy donors (HDs). METHODS: Plasma samples and platelets were isolated from patients with APS (n = 87), SLE (n = 29), CAD (n = 14) and 54 HDs. Plasma levels of CXCL4, CXCL4L1, CXCL7 and CCL5 as well as intracellular platelet CXCL4 and CXCL4L1 were measured using ELISA. Platelet CXCL4 and CXCL4L1 RNA levels were determined by RT-PCR. RESULTS: CXCL4, CXCL7 (NAP-2) and CCL5 (RANTES) plasma levels were significantly higher in patients with APS compared to both control groups (SLE, CAD) and HDs. CXCL4L1 plasma levels were also significantly higher in APS than in SLE and HDs, but lower from that of CAD patients. Statistically significant concordance was detected between CXCL4 and CXCL7 (p < 0.0001) or CCL5 (p < 0.0001) plasma levels in patients with APS, either PAPS or SAPS. CXCL4L1 plasma levels were inversely correlated with CXCL4 (P = 0.0027), CXCL7 (p = 0.012) and CCL5 (p = 0.023) in PAPS and positively with CXCL4 (p = 0.0191), CCL5 (p < 0.0001) and CXCL7 (P < 0.0001), in SAPS. Levels of CXCL4, CXCL4L1, CXCL7 and CCL5 were divided in "high" (exceeding a level defined as the mean of HDs and 3 SD) and "low" (below this level); The "CXCL4L1 high" group was characterized by increased IgG aCL, (p = 0.0215), double antibody positivity (either aCL or anti-ß2GPI plus LA), (p = 0.0277), triple antibody positivity (aCL plus anti-ß2GPI plus LA), (p = 0.0073) and thrombocytopenia (p = 0.0061), as well as with at least 1 thrombotic event or the last 5 years (p = 0.0001), or more than 3 thrombotic events ever (p = 0.0151). CONCLUSIONS: Chemokines associated with platelet activation and immune cell chemotaxis were found to be elevated in APS patients' plasma and may contribute to the pathogenesis of the syndrome. High CXCL4L1 plasma levels are associated with the clinical expression of APS and should be prospectively evaluated as a biomarker.

Alternative C-terminal helix orientation alters chemokine function: structure of the anti-angiogenic chemokine, CXCL4L1.

BACKGROUND: CXCL4L1 is a highly potent anti-angiogenic and anti-tumor chemokine, and its structural information is unknown. RESULTS: CXCL4L1 x-ray structure is determined, and it reveals a previously unrecognized chemokine structure adopting a novel C-terminal helix conformation. CONCLUSION: The alternative helix conformation enhances the anti-angiogenic activity of CXCL4L1 by reducing the glycosaminoglycan binding ability. SIGNIFICANCE: Chemokine C-terminal helix orientation is critical in regulating their functions. Chemokines, a subfamily of cytokines, are small, secreted proteins that mediate a variety of biological processes. Various chemokines adopt remarkable conserved tertiary structure comprising an anti-parallel ß-sheet core domain followed by a C-terminal helix that packs onto the ß-sheet. The conserved structural feature has been considered critical for chemokine function, including binding to cell surface receptor. The recently isolated variant, CXCL4L1, is a homologue of CXCL4 chemokine (or platelet factor 4) with potent anti-angiogenic activity and differed only in three amino acid residues of P58L, K66E, and L67H. In this study we show by x-ray structural determination that CXCL4L1 adopts a previously unrecognized structure at its C terminus. The orientation of the C-terminal helix protrudes into the aqueous space to expose the entire helix. The alternative helix orientation modifies the overall chemokine shape and surface properties. The L67H mutation is mainly responsible for the swing-out effect of the helix, whereas mutations of P58L and K66E only act secondarily. This is the first observation that reports an open conformation of the C-terminal helix in a chemokine. This change leads to a decrease of its glycosaminoglycan binding properties and to an enhancement of its anti-angiogenic and anti-tumor effects. This unique structure is recent in evolution and has allowed CXCL4L1 to gain novel functional properties.

CXCL4L1 May Help Differentiate Benign from Malignant Pulmonary Lesions and Predicts Prognosis of Patients with Lung Cancer.

Background: Lung cancer (LC) is the leading type of cancer worldwide, yet it's challenging to detect early LC. Therefore, it is valuable to explore diagnostic biomarker that can distinguish malignant pulmonary lesions from benign diseases. The potential role of plate factor-4 variant (CXCL4L1) will be investigated in detecting early LC. Methods: A consecutive of 174 patients with single pulmonary nodule and 50 healthy controls were enrolled. Serum CXCL4L1 expression level was evaluated using ELISA. Survival curves were generated to analyze survival outcomes. Receiver operating characteristic curves were used to calculate diagnostic accuracy. Results: Serum CXCL4L1 was downregulated in patients with LC when compared with those with lung benign lesions (LBL) or healthy controls. Meanwhile, lower serum CXCL4L1 expression was associated with advanced TNM stage and lymph node metastasis. Furthermore, a low expression of CXCL4L1 resulted in worse survival outcomes in LC patients. Serum CXCL4L1 expression obtained an area under curve (AUC) of 0.81 (95% CI: 0.74-0.88), a sensitivity of 70.6%, and a specificity of 85.8% for discriminating patients with LC form patients with LBL. In addition, serum CXCL4L1 expression achieved an AUC of 0.82 (95% CI, 0.74-0.90), a sensitivity of 72.0%, and a specificity of 85.9% for distinguishing patients with LC form healthy controls. Conclusion: This study suggests that CXCL4L1 may prove to be a potential non-invasive diagnostic and prognostic biomarker for early LC patients.

New Autoantibody Specificities in Systemic Sclerosis and Very Early Systemic Sclerosis.

Chemokine (C-X-C motif) ligand 4 (CXCL4) is a biomarker of unfavorable prognosis in Systemic Sclerosis (SSc), a potentially severe autoimmune condition, characterized by vasculitis, fibrosis and interferon (IFN)-I-signature. We recently reported that autoantibodies to CXCL4 circulate in SSc patients and correlate with IFN-α. Here, we used shorter versions of CXCL4 and CXCL4-L1, the CXCL4 non-allelic variant, to search for autoantibodies exclusively reacting to one or the other CXCL4 form. Moreover, to address whether anti-CXCL4/CXCL4-L1 antibodies were present before SSc onset and predicted SSc-progression, we longitudinally studied two VEDOSS (Very Early Diagnosis of Systemic Sclerosis) patient cohorts, separating SSc-progressors from SSc-non-progressors. We found that anti-CXCL4-specific autoantibodies were present in both SSc and VEDOSS patients (both SSc-progressors and SSc-non-progressors). Anti-CXCL4-L1-specific autoantibodies were especially detected in long-standing SSc (lsSSc). Anti-CXCL4/CXCL4-L1 antibodies correlated with IFN-α and with specific SSc-skin features but only in lsSSc and not in early SSc (eaSSc) or VEDOSS. Thus, a broader antibody response, with reactivity spreading to CXCL4-L1, is characteristic of lsSSc. The early anti-CXCL4 autoantibody response seems qualitatively different from, and likely less pathogenic than, that observed in advanced SSc. Lastly, we confirm that anti-CXCL4 autoantibodies are SSc-biomarkers and uncover that also CXCL4-L1 becomes an autoantigen in lsSSc.

Downregulation of serum CXCL4L1 predicts progression and poor prognosis in prostate cancer patients treated by radical prostatectomy.

Our previous study found that plate factor-4 variant (CXCL4L1) was downregulated in the serum of patients with prostate cancer (PCa). The aim of the present study was to investigate the prognostic value of CXCL4L1 in PCa. In total, 213 PCa patients treated with radical prostatectomy were enrolled and peripheral blood samples of all patients were collected. Expression of serum CXCL4L1 in patients with different tumor stages and grades were measured by enzyme-linked immunosorbent assay (ELISA). The Kaplan-Meier method was applied to estimate the progression to castration-resistant prostate cancer (CRPC), metastasis, biochemical recurrence (BCR)-free survival, and overall survival (OS). Prognostic factors for BCR-free survival and OS were determined by univariate and multivariate analyses using the Cox proportional hazards regression model. The expression of CXCL4L1 was significantly lower in PCa patients with advanced pathological tumor stage, high-grade Gleason score, and metastasis. Moreover, downregulation of CXCL4L1 not only strongly correlated with aggressive clinicopathological features, but also predicted tumor progression and unfavorable outcomes. Finally, multivariate Cox regression analyses identified CXCL4L1 as an independent prognostic factor for both BCR-free survival (hazard ratio [HR]: 2.03, 95% confidence interval [CI]: 1.26-3.27; P = 0.004) and OS (HR: 2.26, 95% CI: 1.07-4.79; P = 0.033). In conclusion, our results indicate that CXCL4L1 might serve as a novel and promising prognostic biomarker for patients with PCa and potential therapeutic target in the future.

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.

Recombinant Parvoviruses Armed to Deliver CXCL4L1 and CXCL10 Are Impaired in Their Antiangiogenic and Antitumoral Effects in a Kaposi Sarcoma Tumor Model Due To the Chemokines' Interference with the Virus Cycle.

Application of oncolytic viruses is a valuable option to broaden the armament of anticancer therapies, as these combine specific cytotoxic effects and immune-stimulating properties. The self-replicating H-1 parvovirus (H-1PV) is a prototypical oncolytic virus that, besides targeting tumor cells, also infects endothelial cells, thus combining oncolytic and angiostatic traits. To increase its therapeutic value, H-1PV can be armed with cytokines or chemokines to enhance the immunological response. Some chemokines-more specifically, the CXCR3 ligands CXCL4L1 and CXCL10-combine immune-stimulating properties with angiostatic activity. This study explores the therapeutic value of recombinant parvoviruses carrying CXCL4L1 or CXCL10 transgenes (Chi-H1/CXCL4L1 or Chi-H1/CXCL10, respectively) to inhibit the growth of the human Kaposi sarcoma cell line KS-IMM. KS-IMM cells infected by Chi-H1/CXCL4L1 or Chi-H1/CXCL10 released the corresponding chemokine and showed reduced migratory capacity. Therefore, the antitumoral capacity of Chi-H1/CXCL4L1 or Chi-H1/CXCL10 was tested in mice. Either in vitro infected KS-IMM cells were injected or subcutaneously growing KS-IMM xenografts were treated by peritumoral injections of the different viruses. Surprisingly, the transgenes did not increase the antitumoral effect of natural H-1PV. Further experiments indicated that CXCL4L1 and CXCL10 interfered with the expression of the viral NS1 protein in KS-IMM cells. These results indicate that the outcome of parvovirus-based delivery of CXCR3 ligands might be tumor cell type dependent, and hence its application must be considered carefully.

The Chemokine Platelet Factor-4 Variant (PF-4var)/CXCL4L1 Inhibits Diabetes-Induced Blood-Retinal Barrier Breakdown.

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)-1ß 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.